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Sample records for activated chloride channels

  1. Chloride dependence of hyperpolarization-activated chloride channel gates.

    PubMed

    Pusch, M; Jordt, S E; Stein, V; Jentsch, T J

    1999-03-01

    1. 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. 2. 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. 3. With all these channels, reducing [Cl-]i shifted activation to more negative voltages and reduced the maximal activation at most negative voltages. 4. 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. 5. 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. 6. 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-. 7. 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 for the

  2. 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

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

    PubMed

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

    1994-09-13

    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. PMID:7522329

  4. [Polymethoxylated flavonoids activate cystic fibrosis transmembrane conductance regulator chloride channel].

    PubMed

    Cao, Huan-Huan; Fang, Fang; Yu, Bo; Luan, Jian; Jiang, Yu; Yang, Hong

    2015-04-25

    Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel, plays key roles in fluid secretion in serous epithelial cells. Previously, we identified two polymethoxylated flavonoids, 3',4',5,5',6,7-hexamethoxyflavone (HMF) and 5-hydroxy-6,7,3',4'-tetramethoxyflavone (HTF) which could potentiate CFTR chloride channel activities. The present study was aimed to investigate the potentiation effects of HMF and HTF on CFTR Cl(-) channel activities by using a cell-based fluorescence assay and the short circuit Ussing chamber assay. The results of cell-based fluorescence assay showed that both HMF and HTF could dose-dependently potentiate CFTR Cl(-) channel activities in rapid and reversible ways, and the activations could be reversed by the CFTR blocker CFTRinh-172. Notably, HMF showed the highest affinity (EC50 = 2 μmol/L) to CFTR protein among the flavonoid CFTR activators identified so far. The activation of CFTR by HMF or HTF was forskolin (FSK) dependent. Both compounds showed additive effect with FSK and 3-Isobutyl-1-methylx (IBMX) in the activation of CFTR, while had no additive effect with genistein (GEN). In ex vivo studies, HMF and HTF could stimulate transepithelial Cl(-) secretion in rat colonic mucosa and enhance fluid secretion in mouse trachea submucosal glands. These results suggest that HMF and HTF may potentiate CFTR Cl(-) channel activities through both elevation of cAMP level and binding to CFTR protein pathways. The results provide new clues in elucidating structure and activity relationship of flavonoid CFTR activators. HMF might be developed as a new drug in the therapy of CFTR-related diseases such as bronchiectasis and habitual constipation. PMID:25896054

  5. Non-specific activation of the epithelial sodium channel by the CFTR chloride channel

    PubMed Central

    Nagel, Georg; Szellas, Tanjef; Riordan, John R.; Friedrich, Thomas; Hartung, Klaus

    2001-01-01

    The genetic disease cystic fibrosis is caused by mutation of the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Controversial studies reported regulation of the epithelial sodium channel (ENaC) by CFTR. We found that uptake of 22Na+ through ENaC is modulated by activation of CFTR in oocytes, coexpressing CFTR and ENaC, depending on extracellular chloride concentration. Furthermore we found that the effect of CFTR activation could be mimicked by other chloride channels. Voltage– and patch–clamp measurements, however, showed neither stimulation nor inhibition of ENaC-mediated conductance by activated CFTR. We conclude that the observed modulation of 22Na+ uptake by activated CFTR is due to the effect of CFTR-mediated chloride conductance on the membrane potential. These findings argue against the notion of a specific influence of CFTR on ENaC and emphasize the chloride channel function of CFTR. PMID:11266369

  6. Non-specific activation of the epithelial sodium channel by the CFTR chloride channel.

    PubMed

    Nagel, G; Szellas, T; Riordan, J R; Friedrich, T; Hartung, K

    2001-03-01

    The genetic disease cystic fibrosis is caused by mutation of the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Controversial studies reported regulation of the epithelial sodium channel (ENaC) by CFTR. We found that uptake of (22)Na(+) through ENaC is modulated by activation of CFTR in oocytes, coexpressing CFTR and ENaC, depending on extracellular chloride concentration. Furthermore we found that the effect of CFTR activation could be mimicked by other chloride channels. Voltage- and patch-clamp measurements, however, showed neither stimulation nor inhibition of ENaC-mediated conductance by activated CFTR. We conclude that the observed modulation of (22)Na(+) uptake by activated CFTR is due to the effect of CFTR-mediated chloride conductance on the membrane potential. These findings argue against the notion of a specific influence of CFTR on ENaC and emphasize the chloride channel function of CFTR. PMID:11266369

  7. 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. PMID:24489780

  8. Chloride channels in stroke

    PubMed Central

    Zhang, Ya-ping; Zhang, Hao; Duan, Dayue Darrel

    2013-01-01

    Vascular remodeling of cerebral arterioles, including proliferation, migration, and apoptosis of vascular smooth muscle cells (VSMCs), is the major cause of changes in the cross-sectional area and diameter of the arteries and sudden interruption of blood flow or hemorrhage in the brain, ie, stroke. Accumulating evidence strongly supports an important role for chloride (Cl−) channels in vascular remodeling and stroke. At least three Cl− channel genes are expressed in VSMCs: 1) the TMEM16A (or Ano1), which may encode the calcium-activated Cl− channels (CACCs); 2) the CLC-3 Cl− channel and Cl−/H+ antiporter, which is closely related to the volume-regulated Cl− channels (VRCCs); and 3) the cystic fibrosis transmembrane conductance regulator (CFTR), which encodes the PKA- and PKC-activated Cl− channels. Activation of the CACCs by agonist-induced increase in intracellular Ca2+ causes membrane depolarization, vasoconstriction, and inhibition of VSMC proliferation. Activation of VRCCs by cell volume increase or membrane stretch promotes the production of reactive oxygen species, induces proliferation and inhibits apoptosis of VSMCs. Activation of CFTR inhibits oxidative stress and may prevent the development of hypertension. In addition, Cl− current mediated by gamma-aminobutyric acid (GABA) receptor has also been implicated a role in ischemic neuron death. This review focuses on the functional roles of Cl− channels in the development of stroke and provides a perspective on the future directions for research and the potential to develop Cl− channels as new targets for the prevention and treatment of stroke. PMID:23103617

  9. 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. PMID:26558388

  10. 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

  11. 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. PMID:27601995

  12. 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

  13. 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. PMID:22732308

  14. 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

  15. 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. PMID:26794461

  16. 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

  17. Basolateral K+ channel involvement in forskolin-activated chloride secretion in human colon.

    PubMed

    McNamara, B; Winter, D C; Cuffe, J E; O'Sullivan, G C; Harvey, B J

    1999-08-15

    1. In this study we investigated the role of basolateral potassium transport in maintaining cAMP-activated chloride secretion in human colonic epithelium. 2. Ion transport was quantified in isolated human colonic epithelium using the short-circuit current technique. Basolateral potassium transport was studied using nystatin permeabilization. Intracellular calcium measurements were obtained from isolated human colonic crypts using fura-2 spectrofluorescence imaging. 3. In intact isolated colonic strips, forskolin and prostaglandin E2 (PGE2) activated an inward transmembrane current (ISC) consistent with anion secretion (for forskolin DeltaISC = 63.8+/-6.2 microA cm(-2), n = 6; for PGE2 DeltaISC = 34.3+/-5.2 microA cm(-2), n = 6). This current was inhibited in chloride-free Krebs solution or by inhibiting basolateral chloride uptake with bumetanide and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid DIDS). 4. The forskolin- and PGE2-induced chloride secretion was inhibited by basolateral exposure to barium (5 mM), tetrapentylammonium (10 microM) and tetraethylammonium (10 mM). 5. The transepithelial current produced under an apical to serosal K+ gradient in nystatin-perforated colon is generated at the basolateral membrane by K+ transport. Forskolin failed to activate this current under conditions of high or low calcium and failed to increase the levels of intracellular calcium in isolated crypts 6. In conclusion, we propose that potassium recycling through basolateral K+ channels is essential for cAMP-activated chloride secretion. PMID:10432355

  18. 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

  19. 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

  20. Members of the Chloride Intracellular Ion Channel Protein Family Demonstrate Glutaredoxin-Like Enzymatic Activity

    PubMed Central

    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. PMID:25581026

  1. 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

  2. Chloride Channels of Intracellular Membranes

    PubMed Central

    Edwards, John C.; Kahl, Christina R.

    2010-01-01

    Proteins implicated as intracellular chloride channels include the intracellular ClC proteins, the bestrophins, the cystic fibrosis transmembrane conductance regulator, the CLICs, and the recently described Golgi pH regulator. This paper examines current hypotheses regarding roles of intracellular chloride channels and reviews the evidence supporting a role in intracellular chloride transport for each of these proteins. PMID:20100480

  3. Chloride channel activity of ClC-2 is modified by the actin cytoskeleton.

    PubMed Central

    Ahmed, N; Ramjeesingh, M; Wong, S; Varga, A; Garami, E; Bear, C E

    2000-01-01

    The chloride channel ClC-2 has been implicated in essential physiological functions, including cell-volume regulation and fluid secretion by specific epithelial tissues. Although ClC-2 is known to be activated by hyperpolarization and hypo-osmotic shock, the molecular basis for the regulation of this channel remains unclear. Here we show in the Xenopus oocyte expression system that the chloride-channel activity of ClC-2 is enhanced after treatment with the actin-disrupting agents cytochalasin and latrunkulin. These findings suggest that the actin cytoskeleton normally exerts an inhibitory effect on ClC-2 activity. An inhibitory domain was previously defined in the N-terminus of ClC-2, so we sought to determine whether this domain might interact directly with actin in binding assays in vitro. We found that a glutathione S-transferase fusion protein containing the inhibitory domain was capable of binding actin in overlay and co-sedimentation assays. Further, the binding of actin to this relatively basic peptide (pI 8.4) might be mediated through electrostatic interactions because binding was inhibited at high concentrations of NaCl with a half-maximal decrease in signal at 180 mM NaCl. This work suggests that electrostatic interactions between the N-terminus of ClC-2 and the actin cytoskeleton might have a role in the regulation of this channel. PMID:11104687

  4. Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels.

    PubMed

    Wang, F; Zeltwanger, S; Hu, S; Hwang, T C

    2000-05-01

    In cell-attached patches stimulated with cAMP agonists, the single-channel open probability (Po) of the phenylalanine 508-deleted cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) channel, the most common disease-associated mutation in cystic fibrosis, was abnormally low (a functional defect). To investigate the mechanism for the poor response of DeltaF508-CFTR to cAMP stimulation, we examined, in excised inside-out patches, protein kinase A (PKA)-dependent phosphorylation activation and ATP-dependent gating of wild-type (WT) and DeltaF508-CFTR channels expressed in NIH3T3 mouse fibroblasts. For WT-CFTR, the activation time course of CFTR channel current upon addition of PKA and ATP followed a sigmoidal function with time constants that decreased as [PKA] was increased. The curvilinear relationship between [PKA] and the apparent activation rate suggests an incremental phosphorylation-dependent activation of CFTR at multiple phosphorylation sites. The time course of PKA-dependent activation of DeltaF508-CFTR channel current also followed a sigmoidal function, but the rate of activation was at least 7-fold slower than that with WT channels. This result suggests that deletion of phenylalanine 508 causes attenuated PKA-dependent phosphorylation of the CFTR chloride channel. Once DeltaF508-CFTR channels were maximally activated with PKA, the mutant channel and WT channel had indistinguishable steady-state Po values, ATP dose-response relationships and single-channel kinetics, indicating that DeltaF508-CFTR is not defective in ATP-dependent gating. By measuring whole-cell current density, we compared the number of functional channels in WT- and DeltaF508-CFTR cell membrane. Our data showed that the estimated channel density for DeltaF508-CFTR was approximately 10-fold lower than that for WT-CFTR, but the cAMP-dependent whole-cell current density differed by approximately 200-fold. We thus conclude that the functional defect (a decrease in Po) of Delta

  5. Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels

    PubMed Central

    Wang, Fei; Zeltwanger, Shawn; Hu, Shenghui; Hwang, Tzyh-Chang

    2000-01-01

    In cell-attached patches stimulated with cAMP agonists, the single-channel open probability (Po) of the phenylalanine 508-deleted cystic fibrosis transmembrane conductance regulator (ΔF508-CFTR) channel, the most common disease-associated mutation in cystic fibrosis, was abnormally low (a functional defect). To investigate the mechanism for the poor response of ΔF508-CFTR to cAMP stimulation, we examined, in excised inside-out patches, protein kinase A (PKA)-dependent phosphorylation activation and ATP-dependent gating of wild-type (WT) and ΔF508-CFTR channels expressed in NIH3T3 mouse fibroblasts.For WT-CFTR, the activation time course of CFTR channel current upon addition of PKA and ATP followed a sigmoidal function with time constants that decreased as [PKA] was increased. The curvilinear relationship between [PKA] and the apparent activation rate suggests an incremental phosphorylation-dependent activation of CFTR at multiple phosphorylation sites.The time course of PKA-dependent activation of ΔF508-CFTR channel current also followed a sigmoidal function, but the rate of activation was at least 7-fold slower than that with WT channels. This result suggests that deletion of phenylalanine 508 causes attenuated PKA-dependent phosphorylation of the CFTR chloride channel.Once ΔF508-CFTR channels were maximally activated with PKA, the mutant channel and WT channel had indistinguishable steady-state Po values, ATP dose-response relationships and single-channel kinetics, indicating that ΔF508-CFTR is not defective in ATP-dependent gating.By measuring whole-cell current density, we compared the number of functional channels in WT- and ΔF508-CFTR cell membrane. Our data showed that the estimated channel density for ΔF508-CFTR was ∼10-fold lower than that for WT-CFTR, but the cAMP-dependent whole-cell current density differed by ∼200-fold. We thus conclude that the functional defect (a decrease in Po) of ΔF508-CFTR is as important as the trafficking defect (a

  6. 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.

  7. 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. PMID:25853341

  8. 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

  9. 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

  10. Inhibitors of swelling-activated chloride channels increase infarct size and apoptosis in rabbit myocardium.

    PubMed

    Souktani, Rachid; Ghaleh, Bijan; Tissier, Renaud; d'Anglemont de Tassigny, Alexandra; Aouam, Karim; Bedossa, Pierre; Charlemagne, Danièle; Samuel, Janelyse; Henry, Patrick; Berdeaux, Alain

    2003-10-01

    Apoptosis is a significant contributor to myocardial cell death during ischemia-reperfusion and swelling-activated chloride channels (I(Cl,swell)) contribute to apoptosis. However, the relationship between I(Cl,swell) ischemia-reperfusion and apoptosis remains unknown. To further investigate this, New Zealand rabbits underwent a 20-min coronary artery occlusion (CAO) followed by 72 h of coronary artery reperfusion (CAR). Two I(Cl,swell) blockers, 5-nitro-2-[3-phenylpropylamino]benzoic acid (NPPB) and indanyloxyacetic acid 94 (IAA-94) (both 1 mg/kg), were administered prior to CAO and throughout the 72 h CAR. Infarct size (IS) was increased with NPPB and IAA-94 compared with control (vehicle) rabbits (51 +/- 2% and 48 +/- 3% and vs. 35 +/- 2%, respectively, P < 0.05). Similar results were found when NPPB was administered only during the reperfusion period. The percentage of TUNEL-positive nuclei in the border zone of the infarct was increased with NPPB compared with control (37 +/- 2% vs. 25 +/- 31%, P < 0.05) as well as the number of cytoplasmic histone-associated DNA fragments (0.45 +/- 0.06 vs. 0.33 +/- 0.04 absorbance units, P < 0.05). These findings support the concept that I(Cl,swell) channels play an important role in the determination of myocardial infarct size and apoptosis during ischemia-reperfusion. PMID:14703716

  11. Structure-activity and interaction effects of 14 different pyrethroids on voltage-gated chloride ion channels.

    PubMed

    Burr, Steven A; Ray, David E

    2004-02-01

    We have proposed that since the type II pyrethroids deltamethrin and cypermethrin, but not the type I pyrethroid cismethrin act on chloride channels, this could contribute to the bimodal nature of pyrethroid poisoning syndromes. We now examine a wider range of pyrethroid structures on the activity of these calcium-independent voltage-gated maxi-chloride channels. Excised inside-out membrane patches from differentiated mouse neuroblastoma cells were used, and mean channel open probabilities calculated. For single dosing at 10 microM, bioallethrin, beta-cyfluthrin, cypermethrin, deltamethrin, and fenpropathrin were all found to significantly decrease open channel probability (p < 0.05). Bifenthrin, bioresmethrin, cispermethrin, cisresmethrin, cyfluthrin isomers 2 and 4, lambda-cyhalothrin, esfenvalerate, and tefluthrin, did not significantly alter open channel probability (p > 0.05). Since the type II pyrethroids, esfenvalerate, and lambda-cyhalothrin were ineffective, we must conclude that actions at the chloride ion channel target cannot in themselves account for the differences between the two types of poisoning syndrome. Sequential dosing with type II pyrethroids caused no further chloride ion channel closure. The type I pyrethroid cisresmethrin did however prevent a subsequent effect by the mixed type pyrethroid fenpropathrin. In contrast, the type I pyrethroid cispermethrin did not prevent a subsequent effect due to the type II pyrethroid deltamethrin. The difference in effect may be the result of differences in potency, as deltamethrin had a greater effect than fenpropathrin. It therefore appears clear that in some combinations the type I and type II pyrethroids can compete and may bind to the same chloride channel target site. PMID:14657519

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

    PubMed

    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. PMID:24980701

  13. 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. PMID:27138167

  14. Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase

    NASA Astrophysics Data System (ADS)

    Wagner, John A.; Cozens, Alison L.; Schulman, Howard; Gruenert, Dieter C.; Stryer, Lubert; Gardner, Phyllis

    1991-02-01

    CYSTIC fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase1,2 and protein kinase C3,4. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels1-4. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2+-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2+-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.

  15. Calcium-activated chloride channels in cultured embryonic Xenopus spinal neurons.

    PubMed

    Hussy, N

    1992-12-01

    1. Single-channel currents were recorded from Xenopus spinal neurons developing in vitro using the patch-clamp technique, to identify the channels underlying the large and small macroscopic Ca(2+)-activated Cl- currents (ICl(Ca)) present in these cells. 2. Channels of large (maxi-channels; 310 pS) and smaller conductance (mini-channels; 50-60 pS) are activated by elevation of cytoplasmic Ca2+ concentration. Channel activity is not altered by subsequent removal of Ca2+ from the bath, arguing against a direct ligand-type Ca2+ dependence. The much higher incidence of channel activation in cell-attached patches from cells permeabilized with the Ca2+ ionophore A23187 than in excised patches also suggests the involvement of some unidentified intracellular factor. 3. The reversal potential of maxi-Cl- channels is not altered by changes in Na+ concentration, but is shifted in the negative direction by the substitution of Cl- by methanesulfonate on the intracellular side of the patch, indicating their anionic selectivity. 4. Maxi-Cl- channels exhibited the presence of multiple probable subconductance states and showed marked voltage-dependent inactivation above and below +/- 20 mV. 5. Examination of maxi-Cl- channels at early times in culture (6-9 h) and 24 h later did not reveal any developmental change in the characteristics described above. However, the mean open duration of the channel was found to increase twofold during this period of time. 6. The simultaneous presence of maxi- and mini-Cl- channels prevented detailed characterization of the latter. The anionic selectivity of mini-Cl- channels is suggested by their reversal potential that lies close to the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1283407

  16. A synthetic prostone activates apical chloride channels in A6 epithelial cells

    PubMed Central

    Bao, Hui Fang; Liu, Lian; Self, Julie; Duke, Billie Jeanne; Ueno, Ryuji; Eaton, Douglas C.

    2008-01-01

    The bicyclic fatty acid lubiprostone (formerly known as SPI-0211) activates two types of anion channels in A6 cells. Both channel types are rarely, if ever, observed in untreated cells. The first channel type was activated at low concentrations of lubiprostone (<100 nM) in >80% of cell-attached patches and had a unit conductance of ∼3–4 pS. The second channel type required higher concentrations (>100 nM) of lubiprostone to activate, was observed in ∼30% of patches, and had a unit conductance of 8–9 pS. The properties of the first type of channel were consistent with ClC-2 and the second with CFTR. ClC-2's unit current strongly inwardly rectified that could be best fit by models of the channel with multiple energy barrier and multiple anion binding sites in the conductance pore. The open probability and mean open time of ClC-2 was voltage dependent, decreasing dramatically as the patches were depolarized. The order of anion selectivity for ClC-2 was Cl > Br > NO3 > I > SCN, where SCN is thiocyanate. ClC-2 was a “double-barreled” channel favoring even numbers of levels over odd numbers as if the channel protein had two conductance pathways that opened independently of one another. The channel could be, at least, partially blocked by glibenclamide. The properties of the channel in A6 cells were indistinguishable from ClC-2 channels stably transfected in HEK293 cells. CFTR in the patches had a selectivity of Cl > Br ≫ NO3 ≅ SCN ≅ I. It outwardly rectified as expected for a single-site anion channel. Because of its properties, ClC-2 is uniquely suitable to promote anion secretion with little anion reabsorption. CFTR, on the other hand, could promote either reabsorption or secretion depending on the anion driving forces. PMID:18511742

  17. 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

  18. Glutamate-gated Chloride Channels*

    PubMed Central

    Wolstenholme, Adrian J.

    2012-01-01

    Glutamate-gated chloride channels (GluCls) are found only in protostome invertebrate phyla but are closely related to mammalian glycine receptors. They have a number of roles in these animals, controlling locomotion and feeding and mediating sensory inputs into behavior. In nematodes and arthropods, they are targeted by the macrocyclic lactone family of anthelmintics and pesticides, making the GluCls of considerable medical and economic importance. Recently, the three-dimensional structure of a GluCl was solved, the first for any eukaryotic ligand-gated anion channel, revealing a macrocyclic lactone-binding site between the channel domains of adjacent subunits. This minireview will highlight some unique features of the GluCls and illustrate their contribution to our knowledge of the entire Cys loop ligand-gated ion channel superfamily. PMID:23038250

  19. 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. PMID:25253636

  20. CLC chloride channels in Caenorhabditis elegans.

    PubMed

    Schriever, A M; Friedrich, T; Pusch, M; Jentsch, T J

    1999-11-26

    The genome of the nematode Caenorhabditis elegans encodes six putative chloride channels (CeCLC-1 through CeCLC-6) that represent all three known branches of the mammalian CLC gene family. Using promoter fragments to drive the expression of the green fluorescent protein, CeCLC-2, -3, and -4 expression was studied in transgenic C. elegans. CeCLC-4 was specifically expressed in the large H-shaped excretory cell, where it was co-expressed with CeCLC-3, which is also expressed in other cells, including neurons, muscles, and epithelial cells. Also, CeCLC-2 was expressed in several cells of the nervous system, intestinal cells, and vulval muscle cells. Similar to mammalian CLC proteins, only two nematode CLC channels elicited detectable plasma membrane currents in Xenopus oocytes. CeCLC-3 currents were inwardly rectifying and were activated by positive prepulses. Its complex gating behavior can be explained by two gates, at least one of which depends on extracellular anions. In this respect it resembles some mammalian chloride channels with which it also shares a preference of chloride over iodide. C. elegans thus provides new opportunities to understand common mechanisms underlying structure and function in CLC channels and will allow for a genetic dissection of chloride channels in this simple model organism. PMID:10567397

  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. 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. PMID:23959675

  3. 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

    2014-09-01

    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. PMID:25185546

  4. GlialCAM, a CLC-2 Cl- Channel Subunit, Activates the Slow Gate of CLC Chloride Channels

    PubMed Central

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

    2014-01-01

    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. PMID:25185546

  5. 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. PMID:19409890

  6. Caveolin-1 modulates the activity of the volume-regulated chloride channel

    PubMed Central

    Trouet, Dominique; Nilius, Bernd; Jacobs, Axel; Remacle, Claude; Droogmans, Guy; Eggermont, Jan

    1999-01-01

    Caveolae are small invaginations of the plasma membrane that have recently been implicated in signal transduction. In the present study, we have investigated whether caveolins, the principal protein of caveolae, also modulate volume-regulated anion channels (VRACs). ICl,swell, the cell swelling-induced chloride current through VRACs, was studied in three caveolin-1-deficient cell lines: Caco-2, MCF-7 and T47D. Electrophysiological measurements showed that ICl,swell was very small in these cells and that transient expression of caveolin-1 restored ICl,swell. The caveolin-1 effect was isoform specific: caveolin-1β but not caveolin-1α upregulated VRACs. This correlated with a different subcellular distribution of caveolin-1α (perinuclear location) from caveolin-1β (perinuclear and peripheral). To explain the modulation of ICl,swell by caveolin-1 we propose that caveolin increases the availability of VRACs in the plasma membrane or, alternatively, that it plays a crucial role in the signal transduction cascade of VRACs. PMID:10517805

  7. 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. PMID:26530828

  8. Differential effect of calcium-activated potassium and chloride channels on rat basilar artery vasomotion.

    PubMed

    Li, Li; Wang, Rui; Ma, Ke-tao; Li, Xin-zhi; Zhang, Chuan-lin; Liu, Wei-dong; Zhao, Lei; Si, Jun-qiang

    2014-08-01

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

  9. Cytoplasmic loop three of cystic fibrosis transmembrane conductance regulator contributes to regulation of chloride channel activity.

    PubMed

    Seibert, F S; Linsdell, P; Loo, T W; Hanrahan, J W; Riordan, J R; Clarke, D M

    1996-11-01

    To examine the contribution of the large cytoplasmic loops of the cystic fibrosis transmembrane conductance regulator (CFTR) to channel activity, the three point-mutations (S945L, H949Y, G970R) were characterized that have been detected in the third cytoplasmic loop (CL3, residues 933-990) in patients with cystic fibrosis. Chinese hamster ovary cell lines stably expressing wild-type CFTR or mutant G970R-CFTR yielded polypeptides with apparent masses of 170 kDa as the major products, whereas the major products of mutants S945L-CFTR and H949Y-CFTR had apparent masses of 150 kDa. The 150-kDa forms of CFTR were sensitive to endoglycosidase H digestion, indicating that these mutations interfered with maturation of the protein. Increased levels of mature CFTR (170 kDa) could be obtained for mutant H949Y when cells were grown at a lower temperature (26 degrees C) or incubated in the presence of 10% glycerol. For all mutants, the open probability (P0) of the CFTR channels was significantly altered. S945L-CFTR and G970R-CFTR showed a severe reduction in the P0, whereas the H949Y mutation doubled the P0 relative to wild-type. The changes in P0 predominantly resulted from an alteration of the mean burst durations which suggests that CL3 is involved in obtaining and/or maintaining stability of the open state. In addition, mutants S945L and G970R had current-voltage relationships that were not completely linear over the range +/-80 mV, but showed slight outward rectification. The fact that CL3 mutations can have subtle effects on channel conductance indicates that this region may be physically close to the inner mouth of the pore. PMID:8910333

  10. 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.

  11. Assembly of functional CFTR chloride channels.

    PubMed

    Riordan, John R

    2005-01-01

    The assembly of the cystic fibrosis transmembrane regulator (CFTR) chloride channel is of interest from the broad perspective of understanding how ion channels and ABC transporters are formed as well as dealing with the mis-assembly of CFTR in cystic fibrosis. CFTR is functionally distinct from other ABC transporters because it permits bidirectional permeation of anions rather than vectorial transport of solutes. This adaptation of the ABC transporter structure can be rationalized by considering CFTR as a hydrolyzable-ligand-gated channel with cytoplasmic ATP as ligand. Channel gating is initiated by ligand binding when the protein is also phosphorylated by protein kinase A and made reversible by ligand hydrolysis. The two nucleotide-binding sites play different roles in channel activation. CFTR self-associates, possibly as a function of its activation, but most evidence, including the low-resolution three-dimensional structure, indicates that the channel is monomeric. Domain assembly and interaction within the monomer is critical in maturation, stability, and function of the protein. Disease-associated mutations, including the most common, DeltaF508, interfere with domain folding and association, which occur both co- and post-translationally. Intermolecular interactions of mature CFTR have been detected primarily with the N- and C-terminal tails, and these interactions have some impact not only on channel function but also on localization and processing within the cell. The biosynthetic processing of the nascent polypeptide leading to channel assembly involves transient interactions with numerous chaperones and enzymes on both sides of the endoplasmic reticulum membrane. PMID:15709975

  12. Determination of CFTR chloride channel activity and pharmacology using radiotracer flux methods.

    PubMed

    Norez, Caroline; Heda, Ghanshyam D; Jensen, Timothy; Kogan, Ilana; Hughes, Lauren K; Auzanneau, Céline; Dérand, Renaud; Bulteau-Pignoux, Laurence; Li, Canhui; Ramjeesingh, Mohabir; Li, Hongyu; Sheppard, David N; Bear, Christine E; Riordan, John R; Becq, Frédéric

    2004-08-01

    Flux studies using either radioisotopes or ion-selective electrodes are a convenient method to assay the function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Here, we described three different protocols to study the properties, regulation and pharmacology of the CFTR Cl- channel in populations of cells and artificial vesicles. These techniques are widely used to evaluate the function of wild-type and mutant CFTR prior to detailed analyses using the patch-clamp technique. Moreover, they have proved especially valuable in the search for new drugs to treat cystic fibrosis. PMID:15463942

  13. Extracellular ATP inhibits chloride channels in mature mammalian skeletal muscle by activating P2Y1 receptors.

    PubMed

    Voss, Andrew A

    2009-12-01

    ATP is released from skeletal muscle during exercise, a discovery dating back to 1969. Surprisingly, few studies have examined the effects of extracellular ATP on mature mammalian skeletal muscle. This electrophysiological study examined the effects of extracellular ATP on fully innervated rat levator auris longus using two intracellular microelectrodes. The effects of ATP were determined by measuring the relative changes of miniature endplate potentials (mEPPs) and voltage responses to step current pulses in individual muscle fibres. Exposure to ATP (20 microm) prolonged the mEPP falling phase by 31 +/- 7.5% (values +/- s.d., n = 3 fibres). Concurrently, the input resistance increased by 31 +/- 2.0% and the time course of the voltage responses increased by 59 +/- 3.0%. Analogous effects were observed using 2 and 5 microm ATP, and on regions distal from the neuromuscular junction, indicating that physiologically relevant levels of ATP enhanced electrical signalling over the entire muscle fibre. The effects of extracellular ATP were blocked by 200 microm anthracene-9-carboxylic acid, a chloride channel inhibitor, and reduced concentrations of extracellular chloride, indicating that ATP inhibited chloride channels. A high affinity agonist for P2Y receptors, 2-methylthioadenosine-5-O-diphosphate (2MeSADP), induced similar effects to ATP with an EC(50) of 160 +/- 30 nm. The effects of 250 nm2MeSADP were blocked by 500 nmMRS2179, a specific P2Y(1) receptor inhibitor, suggesting that ATP acts on P2Y(1) receptors to inhibit chloride channels. The inhibition of chloride channels by extracellular ATP has implications for muscle excitability and fatigue, and the pathophysiology of myotonias. PMID:19805741

  14. Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension.

    PubMed

    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; Leblanc, Normand

    2012-12-15

    Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca(2+) levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca(2+)-activated Cl- channels (Cl(Ca)), an important excitatory mechanism in PASMCs, are altered in PH is unknown. The potential role of Cl(Ca) 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 Ca(2+)-activated Cl(-) current [I(Cl(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 I(Cl(Ca)) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for Cl(Ca) 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 Ca(2+) channel blocker, and NFA (30 or 100 μM, with or without 10 μM indomethacin to inhibit cyclooxygenases) or T16A(Inh)-A01 (10 μM), TMEM16A/Cl(Ca) channel inhibitors, than that of control animals. In conclusion, augmented Cl(Ca)/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

  15. Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase

    PubMed Central

    Wang, Yong-Xiao; Kotlikoff, Michael I.

    1997-01-01

    To determine the mechanisms responsible for the termination of Ca2+-activated Cl− currents (ICl(Ca)), simultaneous measurements of whole cell currents and intracellular Ca2+ concentration ([Ca2+]i) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with 1 mM ATP, ICl(Ca) decayed before the [Ca2+]i decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca2+]i. Substitution of AMP-PNP or ADP for ATP markedly prolonged the decay of ICl(Ca), resulting in a rate of current decay similar to that of the fall in [Ca2+]i. In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of ICl(Ca) decay was slowed and matched the [Ca2+]i decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect. When a sustained increase in [Ca2+]i was produced in ATP dialyzed cells, the current decayed completely, whereas in cells loaded with 5′-adenylylimidodiphosphate (AMP-PNP), KN93, or the CaMKII inhibitory peptide, ICl(Ca) did not decay. Slowly decaying currents were repeatedly evoked in ADP- or AMP-PNP-loaded cells, but dialysis of adenosine 5′-O-(3-thiotriphosphate) or okadaic acid resulted in a smaller initial ICl(Ca), and little or no current (despite a normal [Ca2+]i transient) with a second stimulation. These data indicate that CaMKII phosphorylation results in the inactivation of calcium-activated chloride channels, and that transition from the inactivated state to the closed state requires protein dephosphorylation. PMID:9405714

  16. 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. PMID:24993131

  17. 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

  18. Blockade of swelling-induced chloride channels by phenol derivatives.

    PubMed Central

    Gschwentner, M.; Jungwirth, A.; Hofer, S.; Wöll, E.; Ritter, M.; Susanna, A.; Schmarda, A.; Reibnegger, G.; Pinggera, G. M.; Leitinger, M.; Frick, J.; Deetjen, P.; Paulmichl, M.

    1996-01-01

    1. In NIH3T3 fibroblasts, the chloride channel involved in regulatory volume decrease (RVD) was identified as ICln, a protein isolated from a cDNA library derived from Madin Darby canine Kidney (MDCK) cells. ICln expressed in Xenopus laevis oocytes gives rise to an outwardly rectifying chloride current, sensitive to the extracellular addition of nucleotides and the known chloride channel blockers, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid). We set out to study whether substances structurally similar to NPPB are able to interfere with RVD. 2. RVD in NIH3T3 fibroblasts and MDCK cells is temperature-dependent. 3. RVD, the swelling-dependent chloride current and the depolarization seen after reducing extracellular osmolarity can be blocked by gossypol and NDGA (nordihydroguaiaretic acid), both structurally related to NPPB. 4. The cyclic AMP-dependent chloride current elicited in CaCo cells is less sensitive to the two substances tested while the calcium-activated chloride current in fibroblasts is insensitive. 5. The binding site for the two phenol derivatives onto ICln seems to be distinct but closely related to the nucleotide binding site identified as G x G x G, a glycine repeat located at the predicted outer mouth of the ICln channel protein. PMID:8733574

  19. Ca2+-induced activation and irreversible inactivation of chloride channels in the perfused plasmalemma of Nitellopsis obtusa.

    PubMed

    Kataev, A A; Zherelova, O M; Berestovsky, G N

    1984-12-01

    Experiments were carried out on the algal cells with removed tonoplast using both continuous intracellular perfusion and voltage clamp on plasmalemma. The transient plasmalemma current induced by depolarization disappeared upon perfusion with the Ca2+-chelating agent, EGTA, since the voltage-dependent calcium channels lost their ability to activate. Subsequent replacement of the perfusion medium containing EGTA by another with Ca2+ for clamped plasmalemma (-100 mV) induced an inward C1- current which showed both activation and inactivation. The maximal amplitude of the current at [C1-]in = 15 mmol/l (which is similar to that in native cells) was approximately twice that in electrically excited cell in vivo. The inactivation of C1 channels in the presence of internal Ca2+ was irreversible and had a time constant of 1-3 min. This supports our earlier suggestion (Lunevsky et al. 1983) that the inactivation of C1 channels in an intact cell (with a time constant of 1-3 s) is due to a decrease in Ca2+ concentration rather than to the activity of their own inactivation mechanism. The C1 channel selectivity sequence was following: C1- much greater than CH3SO-4 approximately equal to K+ much greater than SO2-4 (PK/PSO4 approximately 10). Activation of one half the channels occurs at a Ca2+ concentration of 2 X 10(-5) mol/l. Sr2+ also (though to a lesser extent) activated C1 channels but had to be present in a much more higher concentration than Ca2+. Mg2+ and Ba2+ appeared ineffective. Ca2+ activation did not, apparently, require participation of water-soluble intermediator including ATP. Thus, C1 channel functioning is controlled by Ca2+-, Sr2+-sensitive elements of the subplasmalemma cytoskeleton. PMID:6099298

  20. Calcium-activated chloride channels do not contribute to the odorant transduction current in the marine teleost Isacia conceptionis.

    PubMed

    Osorio, R; Schmachtenberg, O

    2013-11-01

    This study compared the contribution of the Ca²⁺-activated Cl⁻ conductance to the electroolfactogram (EOG) evoked by different odorant classes between the marine Cabinza grunt Isacia conceptionis and rainbow trout Oncorhynchus mykiss. The Ca²⁺-activated Cl⁻ channel blocker niflumic acid significantly diminished odorant responses in O. mykiss, but had no effect on the EOG in I. conceptionis, supporting the notion that Ca²⁺-activated Cl⁻ channels may not operate as odorant transduction current amplifiers in this marine teleost. PMID:24580677

  1. 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. PMID:11001491

  2. 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. PMID:24365472

  3. 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

  4. 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

  5. Regulation of Chloride Channels by Protein Kinase C in Normal and Cystic Fibrosis Airway Epithelia

    NASA Astrophysics Data System (ADS)

    Li, Ming; McCann, John D.; Anderson, Matthew P.; Clancy, John P.; Liedtke, Carole M.; Nairn, Angus C.; Greengard, Paul; Welsh, Michael J.

    1989-06-01

    Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.

  6. 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.

  7. Cell swelling activates ATP-dependent voltage-gated chloride channels in M-1 mouse cortical collecting duct cells.

    PubMed

    Meyer, K; Korbmacher, C

    1996-09-01

    In the present study we used whole-cell patch clamp recordings to investigate swelling-activated Cl-currents (ICl-swell) in M-1 mouse cortical collecting duct (CCD) cells. Hypotonic cell swelling reversibly increased the whole-cell Cl- conductance by about 30-fold. The I-V relationship was outwardly-rectifying and ICl-swell displayed a characteristic voltage-dependence with relatively fast inactivation upon large depolarizing and slow activation upon hyperpolarizing voltage steps. Reversal potential measurements revealed a selectivity sequence SCN- > I- > Br- > Cl- > > gluconate. ICl-swell was inhibited by tamoxifen, NPPB (5-nitro-2(3-phenylpropylamino)-benzoate), DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid), flufenamic acid, niflumic acid, and glibenclamide, in descending order of potency. Extracellular cAMP had no significant effect. ICl-swell was Ca2+ independent, but current activation depended on the presence of a high-energy gamma-phosphate group from intracellular ATP or ATP gamma S. Moreover, it depended on the presence of intracellular Mg2+ and was inhibited by staurosporine, which indicates that a phosphorylation step is involved in channel activation. Increasing the cytosolic Ca2+ concentration by using ionomycin stimulated Cl- currents with a voltage dependence different from that of ICl-swell. Analysis of whole-cell current records during early onset of ICl-swell and during final recovery revealed discontinuous step-like changes of the whole-cell current level which were not observed under nonswelling conditions. A single-channel I-V curve constructed using the smallest resolvable current transitions detected at various holding potentials and revealed a slope conductance of 55, 15, and 8 pS at +120, 0, and -120 mV, respectively. The larger current steps observed in these recordings had about 2, 3, or 4 times the size of the putative single-channel current amplitude, suggesting a coordinated gating of several individual channels or channel

  8. 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

  9. 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. PMID:22685202

  10. Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

    PubMed Central

    Perez-Cornejo, Patricia; Gokhale, Avanti; Duran, Charity; Cui, Yuanyuan; Xiao, Qinghuan; Hartzell, H. Criss; Faundez, Victor

    2012-01-01

    The newly discovered Ca2+-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 Ca2+-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, Ca2+-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. PMID:22685202

  11. 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

  12. 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. PMID:25755701

  13. Altered Regulation of Airway Epithelial Cell Chloride Channels in Cystic Fibrosis

    NASA Astrophysics Data System (ADS)

    Frizzell, Raymond A.; Rechkemmer, Gerhard; Shoemaker, Richard L.

    1986-08-01

    In many epithelial cells the chloride conductance of the apical membrane increases during the stimulation of electrolyte secretion. Single-channel recordings from human airway epithelial cells showed that β -adrenergic stimulation evoked apical membrane chloride channel activity, but this response was absent in cells from patients with cystic fibrosis (CF). However, when membrane patches were excised from CF cells into media containing sufficient free calcium (approximately 180 nanomolar), chloride channels were activated. The chloride channels of CF cells were similar to those of normal cells as judged by their current-voltage relations, ion selectivity, and kinetic behavior. These findings demonstrate the presence of chloride channels in the apical membranes of CF airway cells. Their regulation by calcium appears to be intact, but cyclic adenosine monophosphate (cAMP)-dependent control of their activity is defective.

  14. Recessive mutations in the putative calcium-activated chloride channel Anoctamin 5 cause proximal LGMD2L and distal MMD3 muscular dystrophies.

    PubMed

    Bolduc, Véronique; Marlow, Gareth; Boycott, Kym M; Saleki, Khalil; Inoue, Hiroshi; Kroon, Johan; Itakura, Mitsuo; Robitaille, Yves; Parent, Lucie; Baas, Frank; Mizuta, Kuniko; Kamata, Nobuyuki; Richard, Isabelle; Linssen, Wim H J P; Mahjneh, Ibrahim; de Visser, Marianne; Bashir, Rumaisa; Brais, Bernard

    2010-02-12

    The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies. PMID:20096397

  15. Recessive Mutations in the Putative Calcium-Activated Chloride Channel Anoctamin 5 Cause Proximal LGMD2L and Distal MMD3 Muscular Dystrophies

    PubMed Central

    Bolduc, Véronique; Marlow, Gareth; Boycott, Kym M.; Saleki, Khalil; Inoue, Hiroshi; Kroon, Johan; Itakura, Mitsuo; Robitaille, Yves; Parent, Lucie; Baas, Frank; Mizuta, Kuniko; Kamata, Nobuyuki; Richard, Isabelle; Linssen, Wim H.J.P.; Mahjneh, Ibrahim; de Visser, Marianne; Bashir, Rumaisa; Brais, Bernard

    2010-01-01

    The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies. PMID:20096397

  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. PMID:27044412

  17. 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. PMID:24341413

  18. Mutations within the agonist-binding site convert the homomeric alpha1 glycine receptor into a Zn2+-activated chloride channel.

    PubMed

    Grudzinska, Joanna; Schumann, Tanja; Schemm, Rudolf; Betz, Heinrich; Laube, Bodo

    2008-01-01

    The divalent cation Zn2+ has been shown to regulate inhibitory neurotransmission in the mammalian CNS by affecting the activation of the strychnine-sensitive glycine receptor (GlyR). In spinal neurons and cells expressing recombinant GlyRs, low micromolar (<10 microM) concentrations of Zn2+ enhance glycine currents, whereas higher concentrations (>10 microM) have an inhibitory effect. Mutational studies have localized the Zn2+ binding sites mediating allosteric potentiation and inhibition of GlyRs in distinct regions of the N-terminal extracellular domain of the GlyR alpha-subunits. Here, we examined the Zn2+ sensitivity of different mutations within the agonist binding site of the homomeric alpha(1)-subunit GlyR upon heterologous expression in Xenopus oocytes. This revealed that six substitutions within the ligand-binding pocket result in a total loss of Zn2+ inhibition. Furthermore, substitution of the positively charged residues arginine 65 and arginine 131 by alanine (alpha(1)(R65A), alpha(1)(R131A), or of the aromatic residue phenylalanine 207 by histidine (alpha(1)(F207H)), converted the alpha(1) GlyR into a chloride channel that was activated by Zn2+ alone. Dose-response analysis of the alpha(1)(F207H) GlyR disclosed an EC(50) value of 1.2 microM for Zn2+ activation; concomitantly the apparent glycine affinity was 1000-fold reduced. Thus, single point mutations within the agonist-binding site of the alpha(1) subunit convert the inhibitory GlyR from a glycine-gated into a selectively Zn2+-activated chloride channel. This might be exploited for the design of metal-specific biosensors by modeling-assisted mutagenesis. PMID:18690053

  19. 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.

  20. 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. PMID:25733897

  1. 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. PMID:26305547

  2. 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

  3. The gastric H,K-ATPase blocker lansoprazole is an inhibitor of chloride channels

    PubMed Central

    Schmarda, Andreas; Dinkhauser, Patrick; Gschwentner, Martin; Ritter, Markus; Fürst, Johannes; Scandella, Elke; Wöll, Ewald; Laich, Andreas; Rossmann, Heidi; Seidler, Ursula; Lang, Florian; Paulmichl, Markus

    2000-01-01

    It was postulated that swelling dependent chloride channels are involved in the proton secretion of parietal cells. Since omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are structurally related to phenol derivatives known to block swelling dependent chloride channels, we set out to test, whether these substances – which are known to block the H,K-ATPase – could also lead to an inhibition of swelling-dependent chloride channels. Swelling-dependent chloride channels – characterized in many different cell types – show highly conserved biophysical and pharmacological features, therefore we investigated the effect of omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 on swelling-dependent chloride channels elicited in fibroblasts, after the reduction of the extracellular osmolarity. Omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are able to block swelling-dependent chloride channels (IClswell). Lansoprazole and its protonated metabolite AG2000 act on at least two different sites of the IClswell protein: on an extracellular site which seems to be in a functional proximity to the nucleotide binding site, and on an intracellular site which allows the formation of disulfide-bridges. The inhibition of the proton pump and the simultaneous blocking of chloride channels by omeprazole, lansoprazole and its acid activated sulphenamide form AG2000, as described here could be an effective mode to restrict proton secretion in parietal cells. PMID:10711360

  4. Isolation and Characterization of a High Affinity Peptide Inhibitor of ClC-2 Chloride Channels*

    PubMed Central

    Thompson, Christopher H.; Olivetti, Pedro R.; Fuller, Matthew D.; Freeman, Cody S.; McMaster, Denis; French, Robert J.; Pohl, Jan; Kubanek, Julia; McCarty, Nael A.

    2009-01-01

    The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent KD of ∼20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function. PMID:19574231

  5. Purification and reconstitution of chloride channels from kidney and trachea

    SciTech Connect

    Landry, D.W.; Akabas, M.H.; Redhead, C.; Edelman, A.; Cragoe, E.J. Jr.; Al-Awqati, Q. )

    1989-06-23

    Chloride channels mediate absorption and secretion of fluid in epithelia, and the regulation of these channels is now known to be defective in cystic fibrosis. Indanyl-oxyacetic acid 94 (IAA-94) is a high-affinity ligand for the chloride channel, and an affinity resin based on that structure was developed. Solubilized proteins from kidney and trachea membranes were applied to the affinity matrix, and four proteins with apparent molecular masses of 97, 64, 40, and 27 kilodaltons were eluted from the column by excess IAA-94. A potential-dependent {sup 36}Cl- uptake was observed after reconstituting these proteins into liposomes. Three types of chloride channels with single-channel conductances of 26, 100, and 400 picosiemens were observed after fusion of these liposomes with planar lipid bilayers. Similar types of chloride channels have been observed in epithelia.

  6. Novel Roles for Chloride Channels, Exchangers, and Regulators in Chronic Inflammatory Airway Diseases

    PubMed Central

    Sala-Rabanal, Monica; Yurtsever, Zeynep; Berry, Kayla N.; Brett, Tom J.

    2015-01-01

    Chloride transport proteins play critical roles in inflammatory airway diseases, contributing to the detrimental aspects of mucus overproduction, mucus secretion, and airway constriction. However, they also play crucial roles in contributing to the innate immune properties of mucus and mucociliary clearance. In this review, we focus on the emerging novel roles for a chloride channel regulator (CLCA1), a calcium-activated chloride channel (TMEM16A), and two chloride exchangers (SLC26A4/pendrin and SLC26A9) in chronic inflammatory airway diseases. PMID:26612971

  7. Regulation of the CFTR chloride channel from humans and sharks.

    PubMed

    Hanrahan, J W; Mathews, C J; Grygorczyk, R; Tabcharani, J A; Grzelczak, Z; Chang, X B; Riordan, J R

    1996-07-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) in an ATP-dependent channel which mediates cAMP-stimulated chloride secretion by epithelia, particularly those of the pancreas, airways, and intestine. CFTR homologues have been found in all higher vertebrates examined to date and also in some lower vertebrates, although only the human, shark, and Xenopus genes have been heterologously expressed and shown to generate protein kinase A-activated Cl channels. Once phosphorylated, CFTR channels require hydrolyzable nucleotides to be active, but they can be locked in an open burst state when exposed to mixtures of ATP and its hydrolysis-resistant analogue AMP-PNP. This locking requires low-level phosphorylation at unidentified sites that are not among the ten "strong" (dibasic) PKA consensus sequences on CFTR. Mutagenesis of the dibasic PKA sites, which reduces in vitro phosphorylation by > 98%, reduces open probability (Po) by about 50% whilst having no effect on burst duration. Thus, incremental phosphorylation of these sites under normal conditions does not increase Po by slowing down ATP hydrolysis and stabilizing the open burst state, although locking does strictly require low-level phosphorylation at one or more cryptic sites. In addition to serving as a Cl channel, there is compelling evidence that CFTR inhibits the amiloride-sensitive, epithelial sodium channel (ENaC). The mechanism of coupling is not known but most likely involves physical interactions between the channels, perhaps mediated by an intermediate protein that impinges on other transport proteins. CFTR does not function as a conductive channel for ATP; however, extracellular ATP does regulate epithelial channels through activation of P2U purinergic receptors and, after being hydrolyzed extracellularly, through activation of adenosine receptors which elevate cAMP. PMID:8759925

  8. Cystic fibrosis transmembrane conductance regulator and the outwardly rectifying chloride channel: a relationship between two chloride channels expressed in epithelial cells.

    PubMed

    Hryciw, D H; Guggino, W B

    2000-11-01

    1. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) result in the primary defect observed in patients with cystic fibrosis. 2. The CFTR is a member of the ATPase-binding cassette (ABC) transporter family but, unlike other members of this group, CFTR conducts a chloride current that is activated by cAMP. 3. In epithelial cells, the cAMP-stimulated chloride current is conducted by both CFTR and the outwardly rectifying chloride channel (ORCC). 4. The present review summarizes the current knowledge of the properties of the two channels, as well as their relationship. Because the gene encoding the ORCC has not been identified, a discussion as to possible candidates for this chloride channel is included. PMID:11071305

  9. Cystic Fibrosis Gene Encodes a cAMP-Dependent Chloride Channel in Heart

    NASA Astrophysics Data System (ADS)

    Hart, Padraig; Warth, John D.; Levesque, Paul C.; Collier, Mei Lin; Geary, Yvonne; Horowitz, Burton; Hume, Joseph R.

    1996-06-01

    cAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In this report, a cDNA from rabbit ventricle was isolated and sequenced, which encodes an exon 5 splice variant (exon 5-) of CFTR, with >90% identity to human CFTR cDNA present in epithelial cells. Expression of this cDNA in Xenopus oocytes gave rise to robust cAMP-activated chloride currents that were absent in control water-injected oocytes. Antisense oligodeoxynucleotides directed against CFTR significnatly reduced the density of cAMP-dependent chloride currents in acutely cultured myocytes, thereby establishing a direct functional link between cardiac expression of CFTR protein and an endogenous chloride channel in native cardiac myocytes.

  10. 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

  11. Reversible Silencing of CFTR Chloride Channels by Glutathionylation

    PubMed Central

    Wang, Wei; Oliva, Claudia; Li, Ge; Holmgren, Arne; Lillig, Christopher Horst; Kirk, Kevin L.

    2005-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and ATP-dependent chloride channel that modulates salt and water transport across lung and gut epithelia. The relationship between CFTR and oxidized forms of glutathione is of potential interest because reactive glutathione species are produced in inflamed epithelia where they may be modulators or substrates of CFTR. Here we show that CFTR channel activity in excised membrane patches is markedly inhibited by several oxidized forms of glutathione (i.e., GSSG, GSNO, and glutathione treated with diamide, a strong thiol oxidizer). Three lines of evidence indicate that the likely mechanism for this inhibitory effect is glutathionylation of a CFTR cysteine (i.e., formation of a mixed disulfide with glutathione): (a) channels could be protected from inhibition by pretreating the patch with NEM (a thiol alkylating agent) or by lowering the bath pH; (b) inhibited channels could be rescued by reducing agents (e.g., DTT) or by purified glutaredoxins (Grxs; thiol disulfide oxidoreductases) including a mutant Grx that specifically reduces mixed disulfides between glutathione and cysteines within proteins; and (c) reversible glutathionylation of CFTR polypeptides in microsomes could be detected biochemically under the same conditions. At the single channel level, the primary effect of reactive glutathione species was to markedly inhibit the opening rates of individual CFTR channels. CFTR channel inhibition was not obviously dependent on phosphorylation state but was markedly slowed when channels were first “locked open” by a poorly hydrolyzable ATP analogue (AMP-PNP). Consistent with the latter finding, we show that the major site of inhibition is cys-1344, a poorly conserved cysteine that lies proximal to the signature sequence in the second nucleotide binding domain (NBD2) of human CFTR. This region is predicted to participate in ATP-dependent channel opening and to be occluded in the

  12. 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. PMID:27155485

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

    PubMed Central

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

    2016-01-01

    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 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 IC50 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. PMID:23313619

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

    PubMed

    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

    2015-02-01

    Anoctamin1 (ANO1) encodes a Ca(2+)-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 Ca(2+)-activated Cl(-) current (I(Cl.Ca)) in cardiomyocytes. Myocardial ischemia led to a significant increase in the current density of I(Cl.Ca), which was inhibited by a specific ANO1 inhibitor, T16A(inh)-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 I(Cl.Ca) in the ischemic heart and may contribute, at least in part, to ischemia-induced arrhythmias. PMID:24962810

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

    USGS Publications Warehouse

    Tommasso J.R., Wright, M. I.; 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).

  16. 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.

  17. 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

  18. 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. PMID:26004777

  19. Disease-associated mutations in the fourth cytoplasmic loop of cystic fibrosis transmembrane conductance regulator compromise biosynthetic processing and chloride channel activity.

    PubMed

    Seibert, F S; Linsdell, P; Loo, T W; Hanrahan, J W; Clarke, D M; Riordan, J R

    1996-06-21

    A cluster of 18 point mutations in exon 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been detected in patients with cystic fibrosis. These mutations cause single amino acid substitutions in the most C-terminal cytoplasmic loop (CL4, residues 1035-1102) of the CFTR chloride channel. Heterologous expression of the mutants showed that 12 produced only core-glycosylated CFTR, which was retained in the endoplasmic reticulum; the other six mutants matured and reached the cell surface. In some cases substitution of one member of pairs of adjacent residues resulted in misprocessing, whereas the other did not. Thus, the secondary structure of CL4 may contribute crucially to the proper folding of the entire CFTR molecule. Cyclic AMP-stimulated iodide efflux was not detected from cells expressing the misprocessed variants but was from the other six, indicating that their mutations cause relatively subtle channel defects. Consistent with this, these latter mutations generally are present in patients who are pancreatic-sufficient, while the processing mutants are mostly from patients who are pancreatic-insufficient. Single-channel patch-clamp analysis demonstrated that the processed mutants had the same ohmic conductance as wild-type CFTR, but a lower open probability, generally due to an increase in channel mean closed time and a reduction in mean open time. This suggests that mutations in CL4 do not affect pore properties of CFTR, but disrupt the mechanism of channel gating. PMID:8662892

  20. Blockade of glutamatergic and GABAergic receptor channels by trimethyltin chloride

    PubMed Central

    Krüger, Katharina; Diepgrond, Victoria; Ahnefeld, Maria; Wackerbeck, Christina; Madeja, Michael; Binding, Norbert; Musshoff, Ulrich

    2005-01-01

    Organotin compounds such as trimethyltin chloride (TMT) are among the most toxic of the organometallics. As their main target for toxicity is the central nervous system, the aim of the present study was to investigate the effects of TMT on receptor channels involved in various processes of synaptic transmission. The Xenopus oocyte expression system was chosen for direct assessment of TMT effects on voltage-operated potassium channels and glutamatergic and GABAergic receptors, and hippocampal slices from rat brain for analyzing TMT effects on identified synaptic sites. TMT was found to be ineffective, at 100 μmol l−1, against several potassium- and sodium-operated ion channel functions as well as the metabotropic glutamate receptor. The functions of the ionotropic glutamate and the GABAA receptor channels were inhibited by TMT in micromolar concentrations. Thus, at a maximum concentration of 100 μmol l−1, around 20–30% of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and GABAA receptor-mediated ion currents and 35% of the N-methyl-D-aspartate receptor-mediated ion currents were blocked. In the hippocampal slice model, the inhibitory effects of TMT were much stronger than expected from the results on the ion channels. Bath application of TMT significantly reduced the amplitudes of evoked excitatory postsynaptic field potentials in a concentration-dependent and nonreversible manner.  Induction of long-term potentiation, recorded from the CA1 dendritic region, was inhibited by TMT and failed completely at a concentration of 10 μmol l−1. In general, TMT affects the excitatory and inhibitory synaptic processes in a receptor specific manner and is able to disturb the activity within a neuronal network. PMID:15655511

  1. Permeation through the CFTR chloride channel.

    PubMed

    McCarty, N A

    2000-07-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) protein forms a Cl(-) channel found in the plasma membranes of many epithelial cells, including those of the kidney, gut and conducting airways. Mutation of the gene encoding CFTR is the primary defect in cystic fibrosis, a disease that affects approximately 30 000 individuals in the United States alone. Alteration of CFTR function also plays an important role in the pathophysiology of secretory diarrhea and polycystic kidney disease. The basic mechanisms of permeation in this channel are not well understood. It is not known which portions of the protein contribute to forming the pore or which amino acid residues in those domains are involved in the biophysical processes of ion permeation. In this review, I will discuss (i) the present understanding of ion transport processes in the wild-type CFTR channel, (ii) the experimental approaches currently being applied to investigate the pore, and (iii) a proposed structure that takes into account the present data on mechanisms of ion selectivity in the CFTR channel and on blockade of the pore by open-channel blockers. PMID:10851114

  2. 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." PMID:25546839

  3. 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. PMID:24643479

  4. Changes in accessibility of cytoplasmic substances to the pore associated with activation of the cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed

    El Hiani, Yassine; Linsdell, Paul

    2010-10-15

    Opening of the cystic fibrosis transmembrane conductance regulator Cl(-) channel is dependent both on phosphorylation and on ATP binding and hydrolysis. However, the mechanisms by which these cytoplasmic regulatory factors open the Cl(-) channel pore are not known. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining sixth transmembrane region (TM6) of a cysteine-less variant of cystic fibrosis transmembrane conductance regulator. We find that methanethiosulfonate (MTS) reagents modify irreversibly cysteines substituted for TM6 residues Phe-337, Thr-338, Ser-341, Ile-344, Val-345, Met-348, Ala-349, Arg-352, and Gln-353 when applied to the cytoplasmic side of open channels. However, the apparent rate of modification by internal [2-sulfonatoethyl] methanethiosulfonate (MTSES), a negatively charged MTS reagent, is dependent on the activation state of the channels. In particular, cysteines introduced far along the axis of TM6 from the inside (T338C, S341C, I344C) showed no evidence of significant modification even after prolonged pretreatment of non-activated channels with internal MTSES. In contrast, cysteines introduced closer to the inside of TM6 (V345C, M348C) were readily modified in both activated and non-activated channels. Access of a permeant anion, Au(CN)(2)(-), to T338C was similarly dependent upon channel activation state. The pattern of MTS modification we observe allows us to designate different pore-lining amino acid side chains to distinct functional regions of the channel pore. One logical interpretation of these findings is that cytoplasmic access to residues at the narrowest region of the pore changes concomitant with activation. PMID:20675380

  5. Changes in Accessibility of Cytoplasmic Substances to the Pore Associated with Activation of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel*

    PubMed Central

    El Hiani, Yassine; Linsdell, Paul

    2010-01-01

    Opening of the cystic fibrosis transmembrane conductance regulator Cl− channel is dependent both on phosphorylation and on ATP binding and hydrolysis. However, the mechanisms by which these cytoplasmic regulatory factors open the Cl− channel pore are not known. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining sixth transmembrane region (TM6) of a cysteine-less variant of cystic fibrosis transmembrane conductance regulator. We find that methanethiosulfonate (MTS) reagents modify irreversibly cysteines substituted for TM6 residues Phe-337, Thr-338, Ser-341, Ile-344, Val-345, Met-348, Ala-349, Arg-352, and Gln-353 when applied to the cytoplasmic side of open channels. However, the apparent rate of modification by internal [2-sulfonatoethyl] methanethiosulfonate (MTSES), a negatively charged MTS reagent, is dependent on the activation state of the channels. In particular, cysteines introduced far along the axis of TM6 from the inside (T338C, S341C, I344C) showed no evidence of significant modification even after prolonged pretreatment of non-activated channels with internal MTSES. In contrast, cysteines introduced closer to the inside of TM6 (V345C, M348C) were readily modified in both activated and non-activated channels. Access of a permeant anion, Au(CN)2−, to T338C was similarly dependent upon channel activation state. The pattern of MTS modification we observe allows us to designate different pore-lining amino acid side chains to distinct functional regions of the channel pore. One logical interpretation of these findings is that cytoplasmic access to residues at the narrowest region of the pore changes concomitant with activation. PMID:20675380

  6. 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

  7. 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.

  8. 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

  9. 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. PMID:25367045

  10. Activation of volume-sensitive outwardly rectifying chloride channel by ROS contributes to ER stress and cardiac contractile dysfunction: involvement of CHOP through Wnt.

    PubMed

    Shen, M; Wang, L; Wang, B; Wang, T; Yang, G; Shen, L; Wang, T; Guo, X; Liu, Y; Xia, Y; Jia, L; Wang, X

    2014-01-01

    Endoplasmic reticulum (ER) stress occurring in stringent conditions is critically involved in cardiomyocytes apoptosis and cardiac contractile dysfunction (CCD). However, the molecular machinery that mediates cardiac ER stress and subsequent cell death remains to be fully deciphered, which will hopefully provide novel therapeutic targets for these disorders. Here, we establish tunicamycin-induced model of cardiomyocyte ER stress, which effectively mimicks pathological stimuli to trigger CCD. Tunicamycin activates volume-sensitive outward rectifying Cl(-) currents. Blockade of the volume-sensitive outwardly rectifying (VSOR) Cl(-) channel by 4,4'-diisothiocya-natostilbene-2,2'-disulfonic acid (DIDS), a non-selective Cl(-) channel blocker, and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), a selective VSOR Cl(-) channel blocker, improves cardiac contractility, which correlates with suppressed ER stress through inhibiting the canonical GRP78/eIF2α/ATF4 and XBP1 pathways, and promotes survival of cardiomyocytes by inverting tunicamycin-induced decrease of Wnt through the CHOP pathway. VSOR activation of tunicamycin-treated cardiomyocytes is attributed to increased intracellular levels of reactive oxygen species (ROS). Our study demonstrates a pivotal role of ROS/VSOR in mediating ER stress and functional impairment of cardiomyocytes via the CHOP-Wnt pathway, and suggests the therapeutic values of VSOR Cl(-) channel blockers against ER stress-associated cardiac anomalies. PMID:25412307

  11. Activation of volume-sensitive outwardly rectifying chloride channel by ROS contributes to ER stress and cardiac contractile dysfunction: involvement of CHOP through Wnt

    PubMed Central

    Shen, M; Wang, L; Wang, B; Wang, T; Yang, G; Shen, L; Wang, T; Guo, X; Liu, Y; Xia, Y; Jia, L; Wang, X

    2014-01-01

    Endoplasmic reticulum (ER) stress occurring in stringent conditions is critically involved in cardiomyocytes apoptosis and cardiac contractile dysfunction (CCD). However, the molecular machinery that mediates cardiac ER stress and subsequent cell death remains to be fully deciphered, which will hopefully provide novel therapeutic targets for these disorders. Here, we establish tunicamycin-induced model of cardiomyocyte ER stress, which effectively mimicks pathological stimuli to trigger CCD. Tunicamycin activates volume-sensitive outward rectifying Cl− currents. Blockade of the volume-sensitive outwardly rectifying (VSOR) Cl− channel by 4,4'-diisothiocya-natostilbene-2,2'-disulfonic acid (DIDS), a non-selective Cl− channel blocker, and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), a selective VSOR Cl− channel blocker, improves cardiac contractility, which correlates with suppressed ER stress through inhibiting the canonical GRP78/eIF2α/ATF4 and XBP1 pathways, and promotes survival of cardiomyocytes by inverting tunicamycin-induced decrease of Wnt through the CHOP pathway. VSOR activation of tunicamycin-treated cardiomyocytes is attributed to increased intracellular levels of reactive oxygen species (ROS). Our study demonstrates a pivotal role of ROS/VSOR in mediating ER stress and functional impairment of cardiomyocytes via the CHOP-Wnt pathway, and suggests the therapeutic values of VSOR Cl− channel blockers against ER stress-associated cardiac anomalies. PMID:25412307

  12. Inhibition of a neuronal voltage-dependent chloride channel by the type II pyrethroid, deltamethrin.

    PubMed

    Forshaw, P J; Lister, T; Ray, D E

    1993-02-01

    Following the previous finding that the Type II pyrethroid, deltamethrin, increased membrane resistance in peripheral nerve and muscle in a chloride-dependent manner, the action of deltamethrin on neuronal voltage-dependent chloride channels was assessed using inside-out patches from NIE-115 neuroblastoma cells. These were bathed in symmetrical solutions, containing 149 mM chloride and the membrane potential stepped from 0 mV to voltages ranging from +/- 10 to 80 mV for 2 or 5 sec. Active patches contained large conductance channels (343 +/- 11 pS, n = 8), which inactivated relatively slowly during the voltage step and could be resolved into a number of substates. The channels were confirmed as being chloride specific on the basis of substitution experiments with isethionate and pharmacological blockade by 9-anthracene carboxylic acid (9-ACA). Within 20 min of adding deltamethrin (2 microM) to the bath solution, open channel probability (Po) fell from 0.50 +/- 0.06 to 0.24 +/- 0.04 (n = 11) a highly significant result. Glycerinformal solvent alone (0.1% v/v) caused a non-significant rise to 0.65 +/- 0.09 (n = 4). The decreased open channel probability after deltamethrin was due to an increased incidence of both the closed channel state and low conductance substates. In addition, deltamethrin frequently caused flickering between substrates similar to that seen after 9-ACA. Deltamethrin did not change single channel conductance, current-voltage relationship or time-dependent channel inactivation, but decreased open channel probability over the complete range of membrane voltage tested.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8383811

  13. Cystic fibrosis transmembrane conductance regulator chloride channel blockers: Pharmacological, biophysical and physiological relevance

    PubMed Central

    Linsdell, Paul

    2014-01-01

    Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel causes cystic fibrosis, while inappropriate activity of this channel occurs in secretory diarrhea and polycystic kidney disease. Drugs that interact directly with CFTR are therefore of interest in the treatment of a number of disease states. This review focuses on one class of small molecules that interacts directly with CFTR, namely inhibitors that act by directly blocking chloride movement through the open channel pore. In theory such compounds could be of use in the treatment of diarrhea and polycystic kidney disease, however in practice all known substances acting by this mechanism to inhibit CFTR function lack either the potency or specificity for in vivo use. Nevertheless, this theoretical pharmacological usefulness set the scene for the development of more potent, specific CFTR inhibitors. Biophysically, open channel blockers have proven most useful as experimental probes of the structure and function of the CFTR chloride channel pore. Most importantly, the use of these blockers has been fundamental in developing a functional model of the pore that includes a wide inner vestibule that uses positively charged amino acid side chains to attract both permeant and blocking anions from the cell cytoplasm. CFTR channels are also subject to this kind of blocking action by endogenous anions present in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physiological control of CFTR channel function, in particular as a novel mechanism linking CFTR function dynamically to the composition of epithelial cell secretions. It has also been suggested that future drugs could target this same pathway as a way of pharmacologically increasing CFTR activity in cystic fibrosis. Studying open channel blockers and their mechanisms of action has resulted in significant advances in our understanding of CFTR as a pharmacological target in disease

  14. Cystic fibrosis transmembrane conductance regulator chloride channel blockers: Pharmacological, biophysical and physiological relevance.

    PubMed

    Linsdell, Paul

    2014-02-26

    Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel causes cystic fibrosis, while inappropriate activity of this channel occurs in secretory diarrhea and polycystic kidney disease. Drugs that interact directly with CFTR are therefore of interest in the treatment of a number of disease states. This review focuses on one class of small molecules that interacts directly with CFTR, namely inhibitors that act by directly blocking chloride movement through the open channel pore. In theory such compounds could be of use in the treatment of diarrhea and polycystic kidney disease, however in practice all known substances acting by this mechanism to inhibit CFTR function lack either the potency or specificity for in vivo use. Nevertheless, this theoretical pharmacological usefulness set the scene for the development of more potent, specific CFTR inhibitors. Biophysically, open channel blockers have proven most useful as experimental probes of the structure and function of the CFTR chloride channel pore. Most importantly, the use of these blockers has been fundamental in developing a functional model of the pore that includes a wide inner vestibule that uses positively charged amino acid side chains to attract both permeant and blocking anions from the cell cytoplasm. CFTR channels are also subject to this kind of blocking action by endogenous anions present in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physiological control of CFTR channel function, in particular as a novel mechanism linking CFTR function dynamically to the composition of epithelial cell secretions. It has also been suggested that future drugs could target this same pathway as a way of pharmacologically increasing CFTR activity in cystic fibrosis. Studying open channel blockers and their mechanisms of action has resulted in significant advances in our understanding of CFTR as a pharmacological target in disease

  15. Dimeric structure of single chloride channels from Torpedo electroplax.

    PubMed Central

    Miller, C; White, M M

    1984-01-01

    The inhibition by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) of Cl- channels from Torpedo electroplax incorporated in planar phospholipid bilayer membranes is studied. DIDS irreversibly and rapidly inhibits the macroscopic conductance of membranes containing many channels. At the single-channel level, the effect of DIDS is more complicated. The uninhibited single channel displays three "substates" of conductances 20, 10, and 0 pS. Short exposure (5-30 s) to 10 microM DIDS converts this three-level active channel into a "conventional" channel of 10-pS conductance. Longer exposure eliminates all channel fluctuations. The results are taken as strong evidence that the Cl- channel is constructed as a functional dimer of identical protein subunits. PMID:6326143

  16. A Synthetic Chloride Channel Relaxes Airway Smooth Muscle of the Rat

    PubMed Central

    Yau, Kwok-hei; Mak, Judith Choi-wo; Leung, Susan Wai-sum; Yang, Dan; Vanhoutte, Paul M.

    2012-01-01

    Synthetic ion channels may have potential therapeutic applications, provided they possess appropriate biological activities. The present study was designed to examine the ability of small molecule-based synthetic Cl– channels to modulate airway smooth muscle responsiveness. Changes in isometric tension were measured in rat tracheal rings. Relaxations to the synthetic chloride channel SCC-1 were obtained during sustained contractions to KCl. The anion dependency of the effect of SCC-1 was evaluated by ion substitution experiments. The sensitivity to conventional Cl– transport inhibitors was also tested. SCC-1 caused concentration-dependent relaxations during sustained contractions to potassium chloride. This relaxing effect was dependent on the presence of extracellular Cl– and HCO3−. It was insensitive to conventional Cl– channels/transport inhibitors that blocked the cystic fibrosis transmembrane conductance regulator and calcium-activated Cl– channels. SCC-1 did not inhibit contractions induced by carbachol, endothelin-1, 5-hydroxytryptamine or the calcium ionophore A23187. SCC-1 relaxes airway smooth muscle during contractions evoked by depolarizing solutions. The Cl– conductance conferred by this synthetic compound is distinct from the endogenous transport systems for chloride anions. PMID:23049786

  17. 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

  18. Multi-ion pore behaviour in the CFTR chloride channel.

    PubMed

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

    1993-11-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a non-rectifying, low-conductance channel regulated by ATP and phosphorylation, which mediates apical chloride conductance in secretory epithelia and malfunctions in cystic fibrosis (CF). Mutations at Lys 335 and Arg 347 in the sixth predicted transmembrane helix of CFTR alter its halide selectivity in whole-cell studies and its single channel conductance, but the physical basis of these alterations is unknown and permeation in CFTR is poorly understood. Here we present evidence that wild-type CFTR can contain more than one anion simultaneously. The conductance of CFTR passes through a minimum when channels are bathed in mixtures of two permeant anions. This anomalous mole fraction effect can be abolished by replacing Arg 347 with an aspartate and can be toggled on or off by varying the pH after the same residue is replaced with a histidine. Thus the CFTR channel should provide a convenient model in which to study multi-ion pore behaviour and conduction. The loss of multiple occupancy may explain how naturally occurring CF mutations at this site cause disease. PMID:7694154

  19. CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex

    PubMed Central

    Cormet-Boyaka, Estelle; Di, Anke; Chang, Steven Y.; Naren, Anjaparavanda P.; Tousson, Albert; Nelson, Deborah J.; Kirk, Kevin L.

    2002-01-01

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion reactions in eukaryotic cells by assembling into complexes that link vesicle-associated SNAREs with SNAREs on target membranes (t-SNAREs). Many SNARE complexes contain two t-SNAREs that form a heterodimer, a putative intermediate in SNARE assembly. Individual t-SNAREs (e.g., syntaxin 1A) also regulate synaptic calcium channels and cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial chloride channel that is defective in cystic fibrosis. Whether the regulation of ion channels by individual t-SNAREs is related to SNARE complex assembly and membrane fusion is unknown. Here we show that CFTR channels are coordinately regulated by two cognate t-SNAREs, SNAP-23 (synaptosome-associated protein of 23 kDa) and syntaxin 1A. SNAP-23 physically associates with CFTR by binding to its amino-terminal tail, a region that modulates channel gating. CFTR-mediated chloride currents are inhibited by introducing excess SNAP-23 into HT29-Cl.19A epithelial cells. Conversely, CFTR activity is stimulated by a SNAP-23 antibody that blocks the binding of this t-SNARE to the CFTR amino-terminal tail. The physical and functional interactions between SNAP-23 and CFTR depend on syntaxin 1A, which binds to both proteins. We conclude that CFTR channels are regulated by a t-SNARE complex that may tune CFTR activity to rates of membrane traffic in epithelial cells. PMID:12209004

  20. Capsaicin potentiates wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride-channel currents.

    PubMed

    Ai, Tomohiko; Bompadre, Silvia G; Wang, Xiaohui; Hu, Shenghui; Li, Min; Hwang, Tzyh-Chang

    2004-06-01

    To examine the effects of capsaicin on cystic fibrosis transmembrane conductance regulator (CFTR), we recorded wild-type and mutant CFTR chloride-channel currents using patch-clamp methods. The effects of capsaicin were compared with those of genistein, a well-characterized CFTR activator. In whole-cell experiments, capsaicin potentiates cAMP-stimulated wild-type CFTR currents expressed in NIH 3T3 cells or Chinese hamster ovary cells in a dose-dependent manner with a maximal response approximately 60% of that with genistein and an apparent Kd of 48.4 +/- 6.8 microM. In cell-attached recordings, capsaicin alone fails to activate CFTR in cells that show negligible basal CFTR activity, indicating that capsaicin does not stimulate the cAMP cascade. The magnitude of potentiation with capsaicin depends on the channel activity before drug application; the lower the prestimulated Po, the higher the potentiation. Single-channel kinetic analysis shows that capsaicin potentiates CFTR by increasing the opening rate and decreasing the closing rate of the channel. Capsaicin may act as a partial agonist of genistein because the maximally enhanced wild-type CFTR currents with genistein are partially inhibited by capsaicin. Capsaicin increases DeltaR-CFTR, a protein kinase A (PKA)-independent, constitutively active channel, in cell-attached patches. In excised inside-out patches, capsaicin potentiates the PKA-phosphorylated, ATP-dependent CFTR activity. Both capsaicin and genistein potentiate the cAMP-stimulated G551D-CFTR, DeltaF508-CFTR, and 8SA mutant channel currents. The binding site for capsaicin is probably located at the cytoplasmic domain of CFTR, because pipette application of capsaicin fails to potentiate CFTR activity. In conclusion, capsaicin is a partial agonist of genistein in activation of the CFTR chloride channel. Both compounds affect ATP-dependent gating of CFTR. PMID:15155835

  1. 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.

  2. Mechanically Activated Ion Channels.

    PubMed

    Ranade, Sanjeev S; Syeda, Ruhma; Patapoutian, Ardem

    2015-09-23

    Mechanotransduction, the conversion of physical forces into biochemical signals, is essential for various physiological processes such as the conscious sensations of touch and hearing, and the unconscious sensation of blood flow. Mechanically activated (MA) ion channels have been proposed as sensors of physical force, but the identity of these channels and an understanding of how mechanical force is transduced has remained elusive. A number of recent studies on previously known ion channels along with the identification of novel MA ion channels have greatly transformed our understanding of touch and hearing in both vertebrates and invertebrates. Here, we present an updated review of eukaryotic ion channel families that have been implicated in mechanotransduction processes and evaluate the qualifications of the candidate genes according to specified criteria. We then discuss the proposed gating models for MA ion channels and highlight recent structural studies of mechanosensitive potassium channels. PMID:26402601

  3. Mechanism of chloride permeation in the cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed

    Linsdell, Paul

    2006-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel important in transepithelial salt and water transport. While there is a paucity of direct structural information on CFTR, much has been learned about the molecular determinants of the CFTR Cl- channel pore region and the mechanism of Cl- permeation through the pore from indirect structure-function studies. The first and sixth transmembrane regions of the CFTR protein play major roles in forming the channel pore and determining its functional properties by interacting with permeating Cl- ions. Positively charged amino acid side-chains are involved in attracting negatively charged Cl- ions into the pore region, where they interact briefly with a number of discrete sites on the pore walls. The pore appears able to accommodate more than one Cl- ion at a time, and Cl- ions bound inside the pore are probably sensitive to one another's presence. Repulsive interactions between Cl- ions bound concurrently within the pore may be important in ensuring rapid movement of Cl- ions through the pore. Chloride ion binding sites also interact with larger anions that can occlude the pore and block Cl- permeation, thus inhibiting CFTR function. Other ions besides Cl- are capable of passing through the pore, and specific amino acid residues that may be important in allowing the channel to discriminate between different anions have been identified. This brief review summarizes these mechanistic insights and tries to incorporate them into a simple cartoon model depicting the interactions between the channel and Cl- ions that are important for ion translocation. PMID:16157656

  4. Fatty Acids Inhibit Apical Membrane Chloride Channels in Airway Epithelia

    NASA Astrophysics Data System (ADS)

    Anderson, Matthew P.; Welsh, Michael J.

    1990-09-01

    Apical membrane Cl^- channels control the rate of transepithelial Cl^- secretion in airway epithelia. cAMP-dependent protein kinase and protein kinase C regulate Cl^- channels by phosphorylation; in cystic fibrosis cells, phosphorylation-dependent activation of Cl^- channels is defective. Another important signaling system involves arachidonic acid, which is released from cell membranes during receptor-mediated stimulation. Here we report that arachidonic acid reversibly inhibited apical membrane Cl^- channels in cell-free patches of membrane. Arachidonic acid itself inhibited the channel and not a cyclooxygenase or lipoxygenase metabolite because (i) inhibitors of these enzymes did not block the response, (ii) fatty acids that are not substrates for the enzymes had the same effect as arachidonic acid, and (iii) metabolites of arachidonic acid did not inhibit the channel. Inhibition occurred only when fatty acids were added to the cytosolic surface of the membrane patch. Unsaturated fatty acids were more potent than saturated fatty acids. Arachidonic acid inhibited Cl^- channels from both normal and cystic fibrosis cells. These results suggest that fatty acids directly inhibit apical membrane Cl^- channels in airway epithelial cells.

  5. 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.

  6. Bongkrekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart.

    PubMed

    Malekova, Lubica; Kominkova, Viera; Ferko, Miroslav; Stefanik, Peter; Krizanova, Olga; Ziegelhöffer, Attila; Szewczyk, Adam; Ondrias, Karol

    2007-01-01

    The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 microM), ATR and CAT (5-100 microM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects. PMID:17123460

  7. Enhancement of an outwardly rectifying chloride channel in hippocampal pyramidal neurons after cerebral ischemia.

    PubMed

    Li, Jianguo; Chang, Quanzhong; Li, Xiaoming; Li, Xiawen; Qiao, Jiantian; Gao, Tianming

    2016-08-01

    Cerebral ischemia induces delayed, selective neuronal death in the CA1 region of the hippocampus. The underlying molecular mechanisms remain unclear, but it is known that apoptosis is involved in this process. Chloride efflux has been implicated in the progression of apoptosis in various cell types. Using both the inside-out and whole-cell configurations of the patch-clamp technique, the present study characterized an outwardly rectifying chloride channel (ORCC) in acutely dissociated pyramid neurons in the hippocampus of adult rats. The channel had a nonlinear current-voltage relationship with a conductance of 42.26±1.2pS in the positive voltage range and 18.23±0.96pS in the negative voltage range, indicating an outward rectification pattern. The channel is Cl(-) selective, and the open probability is voltage-dependent. It can be blocked by the classical Cl(-) channel blockers DIDS, SITS, NPPB and glibenclamide. We examined the different changes in ORCC activity in CA1 and CA3 pyramidal neurons at 6, 24 and 48h after transient forebrain ischemia. In the vulnerable CA1 neurons, ORCC activity was persistently enhanced after ischemic insult, whereas in the invulnerable CA3 neurons, no significant changes occurred. Further analysis of channel kinetics suggested that multiple openings are a major contributor to the increase in channel activity after ischemia. Pharmacological blockade of the ORCC partly attenuated cell death in the hippocampal neurons. We propose that the enhanced activity of ORCC might contribute to selective neuronal damage in the CA1 region after cerebral ischemia, and that ORCC may be a therapeutic target against ischemia-induced cell death. PMID:27181516

  8. c-Src Control of Chloride Channel Support for Osteoclast HCl Transport and Bone Resorption*

    PubMed Central

    Edwards, John C.; Cohen, Christopher; Xu, Weibing; Schlesinger, Paul H.

    2006-01-01

    Bone degradation by osteoclasts depends upon active transport of hydrogen ions to solubilize bone mineral. This transport is supported by the parallel actions of a proton ATPase and a chloride channel located in the osteoclast ruffled membrane. We have previously identified a novel chloride channel, p62, which appears to be the avian counterpart to CLIC-5b and is expressed coincident with the appearance of acid secretion as avian osteoclasts differentiate in culture. In this article, we show that suppression of CLIC-5b in differentiating avian osteoclasts results in decreased acidification by vesicles derived from these cells and decreased ability of the cells to resorb bone. Acidification is rescued by the presence of valinomycin, consistent with a selective loss of chloride channel but not proton pump activity. Osteoclast bone resorption is known to be dependent on the expression of the tyrosine kinase, c-Src. We show that CLIC-5b from osteoclasts has affinity for both Src SH2 and SH3 domains. We find that suppression of expression of Src in developing osteoclasts results in decreased vesicular acidification, which is rescued by valinomycin, consistent with the loss of chloride conductance in the proton pump-containing vesicles. Suppression of c-Src causes no change in the steady state level of CLIC-5b expression, but does result in failure of proton pump and CLIC-5b to colocalize in cultured osteoclast precursors. We conclude that suppression of c-Src interferes with osteoclast bone resorption by disrupting functional co-localization of proton pump and CLIC-5b. PMID:16831863

  9. A cAMP-Regulated Chloride Channel in Lymphocytes that is Affected in Cystic Fibrosis

    NASA Astrophysics Data System (ADS)

    Chen, Jennifer H.; Schulman, Howard; Gardner, Phyllis

    1989-02-01

    A defect in regulation of a chloride channel appears to be the molecular basis for cystic fibrosis (CF), a common lethal genetic disease. It is shown here that a chloride channel with kinetic and regulatory properties similar to those described for secretory epithelial cells is present in both T and B lymphocyte cell lines. The regulation of the channels by adenosine 3',5'-monophosphate (cAMP)--dependent protein kinase in transformed B cells from CF patients is defective. Thus, lymphocytes may be an accessible source of CF tissue for study of this defect, for cloning of the chloride channel complex, and for diagnosis of the disease.

  10. CLC-0 and CFTR: chloride channels evolved from transporters.

    PubMed

    Chen, Tsung-Yu; Hwang, Tzyh-Chang

    2008-04-01

    CLC-0 and cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels play important roles in Cl(-) transport across cell membranes. These two proteins belong to, respectively, the CLC and ABC transport protein families whose members encompass both ion channels and transporters. Defective function of members in these two protein families causes various hereditary human diseases. Ion channels and transporters were traditionally viewed as distinct entities in membrane transport physiology, but recent discoveries have blurred the line between these two classes of membrane transport proteins. CLC-0 and CFTR can be considered operationally as ligand-gated channels, though binding of the activating ligands appears to be coupled to an irreversible gating cycle driven by an input of free energy. High-resolution crystallographic structures of bacterial CLC proteins and ABC transporters have led us to a better understanding of the gating properties for CLC and CFTR Cl(-) channels. Furthermore, the joined force between structural and functional studies of these two protein families has offered a unique opportunity to peek into the evolutionary link between ion channels and transporters. A promising byproduct of this exercise is a deeper mechanistic insight into how different transport proteins work at a fundamental level. PMID:18391167

  11. 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. PMID:24671572

  12. Modulation of CFTR chloride channels by calyculin A and genistein.

    PubMed

    Yang, I C; Cheng, T H; Wang, F; Price, E M; Hwang, T C

    1997-01-01

    Modulation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel by calyculin A and genistein was studied in Hi-5 insect cells infected with baculovirus containing the wild-type CFTR cDNA. In cell-attached patches, CFTR channel activity was not observed until stimulated by forskolin in 90% of the cells, suggesting a low level of basal adenosine 3',5'-cyclic monophosphate activity. Calyculin A, a specific inhibitor of phosphatases 1 and 2A, increased forskolin-induced CFTR activity by 17.2-fold. CFTR channel currents did not deactivate completely after forskolin was withdrawn in the continued presence of calyculin A. Genistein enhanced forskolin-induced CFTR activity by 44.9-fold but could neither activate the CFTR by itself nor prevent complete deactivation on removal of forskolin. Genistein together with calyculin A could adequately prevent deactivation of CFTR currents. Noise analysis of the macroscopic CFTR currents revealed significant differences in the mean current-variance-relationship and the corner frequency of the noise spectra between currents activated by forskolin plus genistein and those activated by forskolin plus calyculin A. Furthermore, genistein enhanced CFTR activity induced by saturating concentrations of forskolin and calyculin A. Our results suggest that genistein and calyculin A modulate the CFTR by different mechanisms and that genistein might inhibit calyculin A-insensitive dephosphorylation of the CFTR. PMID:9038820

  13. Cys-loop ligand-gated chloride channels in dorsal unpaired median neurons of Locusta migratoria.

    PubMed

    Janssen, Daniel; Derst, Christian; Rigo, Jean-Michel; Van Kerkhove, Emmy

    2010-05-01

    In insects, inhibitory neurotransmission is generally associated with members of the cys-loop ligand-gated anion channels, such as the glutamate-gated chloride channel (GluCl), the GABA-gated chloride channels (GABACl), and the histamine-gated chloride channels (HisCl). These ionotropic receptors are considered established target sites for the development of insecticides, and therefore it is necessary to obtain a better insight in their distribution, structure, and functional properties. Here, by combining electrophysiology and molecular biology techniques, we identified and characterized GluCl, GABACl, and HisCl in dorsal unpaired median (DUM) neurons of Locust migratoria. In whole cell patch-clamp recordings, application of glutamate, GABA, or histamine induced rapidly activating ionic currents. GluCls were sensitive to ibotenic acid and blocked by picrotoxin and fipronil. The pharmacological profile of the L. migratoria GABACl fitted neither the vertebrate GABA(A) nor GABA(C) receptor and was similar to the properties of the cloned Drosophila melanogaster GABA receptor subunit (Rdl). The expression of Rdl-like subunit-containing GABA receptors was shown at the molecular level using RT-PCR. Sequencing analysis indicated that the orthologous GABACl of D. melanogaster CG10357-A is expressed in DUM neurons of L. migratoria. Histamine-induced currents exhibited a fast onset and desensitized completely on continuous application of histamine. In conclusion, within the DUM neurons of L. migratoria, we identified three different cys-loop ligand-gated anion channels that use GABA, glutamate, or histamine as their neurotransmitter. PMID:20200125

  14. 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

  15. Intracellular chloride channel protein CLIC1 regulates macrophage function through modulation of phagosomal acidification

    PubMed Central

    Jiang, Lele; Salao, Kanin; Li, Hui; Rybicka, Joanna M.; Yates, Robin M.; Luo, Xu Wei; Shi, Xin Xin; Kuffner, Tamara; Tsai, Vicky Wang-Wei; Husaini, Yasmin; Wu, Liyun; Brown, David A.; Grewal, Thomas; Brown, Louise J.; Curmi, Paul M. G.; Breit, Samuel N.

    2012-01-01

    Summary Intracellular chloride channel protein 1 (CLIC1) is a 241 amino acid protein of the glutathione S transferase fold family with redox- and pH-dependent membrane association and chloride ion channel activity. Whilst CLIC proteins are evolutionarily conserved in Metazoa, indicating an important role, little is known about their biology. CLIC1 was first cloned on the basis of increased expression in activated macrophages. We therefore examined its subcellular localisation in murine peritoneal macrophages by immunofluorescence confocal microscopy. In resting cells, CLIC1 is observed in punctate cytoplasmic structures that do not colocalise with markers for endosomes or secretory vesicles. However, when these macrophages phagocytose serum-opsonised zymosan, CLIC1 translocates onto the phagosomal membrane. Macrophages from CLIC1−/− mice display a defect in phagosome acidification as determined by imaging live cells phagocytosing zymosan tagged with the pH-sensitive fluorophore Oregon Green. This altered phagosomal acidification was not accompanied by a detectable impairment in phagosomal-lysosomal fusion. However, consistent with a defect in acidification, CLIC1−/− macrophages also displayed impaired phagosomal proteolytic capacity and reduced reactive oxygen species production. Further, CLIC1−/− mice were protected from development of serum transfer induced K/BxN arthritis. These data all point to an important role for CLIC1 in regulating macrophage function through its ion channel activity and suggest it is a suitable target for the development of anti-inflammatory drugs. PMID:22956539

  16. Molecular characterisation of a pH-gated chloride channel from Sarcoptes scabiei.

    PubMed

    Mounsey, Kate E; Dent, Joseph A; Holt, Deborah C; McCarthy, James; Currie, Bart J; Walton, Shelley F

    2007-09-01

    Reports of ivermectin resistance in scabies mites raise concerns regarding the sustainability of mass intervention programs for scabies worldwide and for the treatment of crusted scabies. Ligand gated ion channels (LGICs) are the primary targets of ivermectin in invertebrates. We report the molecular characterisation of SsCl--a novel LGIC from Sarcoptes scabiei var. hominis. While SsCl shows sequence similarity to other LGICs, phylogenetic analysis does not suggest strong homology to conventional glutamate, histamine or GABA gated channels. Instead, it is most similar to Drosophila pH-sensitive and group 1 clades. When expressed in Xenopus oocytes, SsCl forms a homomeric, pH-gated chloride channel that is irreversibly activated by ivermectin. These results provide the first confirmation that this group of LGIC exists in arachnids, and suggest that SsCl may be an in vivo target of ivermectin in S. scabiei. PMID:17602250

  17. 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. PMID:26779604

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

    PubMed Central

    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. PMID:24026363

  19. 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. PMID:20926782

  20. High glucose inhibits ClC-2 chloride channels and attenuates cell migration of rat keratinocytes

    PubMed Central

    Pan, Fuqiang; Guo, Rui; Cheng, Wenguang; Chai, Linlin; Wang, Wenping; Cao, Chuan; Li, Shirong

    2015-01-01

    Background Accumulating evidence has demonstrated that migration of keratinocytes is critical to wound epithelialization, and defects of this function result in chronic delayed-healing wounds in diabetes mellitus patients, and the migration has been proved to be associated with volume-activated chloride channels. The aim of the study is to investigate the effects of high glucose (HG, 25 mM) on ClC-2 chloride channels and cell migration of keratinocytes. Methods Newborn Sprague Dawley rats were used to isolate and culture the keratinocyte in this study. Immunofluorescence assay, real-time polymerase chain reaction, and Western blot assay were used to examine the expression of ClC-2 protein or mRNA. Scratch wound assay was used to measure the migratory ability of keratinocytes. Transwell cell migration assay was used to measure the invasion and migration of keratinocytes. Recombinant lentivirus vectors were established and transducted to keratinocytes. Whole-cell patch clamp was used to perform the electrophysiological studies. Results We found that the expression of ClC-2 was significantly inhibited when keratinocytes were exposed to a HG (25 mM) medium, accompanied by the decline of volume-activated Cl− current (ICl,vol), migration potential, and phosphorylated PI3K as compared to control group. When knockdown of ClC-2 by RNAi or pretreatment with wortmannin, similar results were observed, including ICl,vol and migration keratinocytes were inhibited. Conclusion Our study proved that HG inhibited ClC-2 chloride channels and attenuated cell migration of rat keratinocytes via inhibiting PI3K signaling. PMID:26355894

  1. Isotonic contractile impairment due to genetic CLC-1 chloride channel deficiency in myotonic mouse diaphragm muscle.

    PubMed

    van Lunteren, Erik; Pollarine, Jennifer; Moyer, Michelle

    2007-07-01

    The hallmark of genetic CLC-1 chloride channel deficiency in myotonic humans, goats and mice is delayed muscle relaxation resulting from persistent electrical discharges. In addition to the ion channel defect, muscles from myotonic humans and mice also have major changes in fibre type and myosin isoform composition, but the extent to which this affects isometric contractions remains controversial. Many muscles, including the diaphragm, shorten considerably during normal activities, but shortening contractions have never been assessed in myotonic muscle. The present study tested the hypothesis that CLC-1 deficiency leads to an impairment of muscle isotonic contractile performance. This was tested in vitro on diaphragm muscle from SWR/J-Clcn1(adr-mto)/J myotonic mice. The CLC-1-deficient muscle demonstrated delayed relaxation, as expected. During the contractile phase, there were significant reductions in power and work across a number of stimulation frequencies and loads in CLC-1-deficient compared with normal muscle, the magnitude of which in many instances exceeded 50%. Reductions in shortening and velocity of shortening occurred, and were more pronounced when calculated as a function of absolute than relative load. However, the maximal unloaded shortening velocity calculated from Hill's equation was not altered significantly. The impaired isotonic contractile performance of CLC-1-deficient muscle persisted during fatigue-inducing stimulation. These data indicate that genetic CLC-1 chloride channel deficiency in mice not only produces myotonia but also substantially worsens the isotonic contractile performance of diaphragm muscle. PMID:17483199

  2. Functional Characterization of a Novel Family of Acetylcholine-Gated Chloride Channels in Schistosoma mansoni

    PubMed Central

    MacDonald, Kevin; Buxton, Samuel; Kimber, Michael J.; Day, Tim A.; Robertson, Alan P.; Ribeiro, Paula

    2014-01-01

    Acetylcholine is the canonical excitatory neurotransmitter of the mammalian neuromuscular system. However, in the trematode parasite Schistosoma mansoni, cholinergic stimulation leads to muscle relaxation and a flaccid paralysis, suggesting an inhibitory mode of action. Information about the pharmacological mechanism of this inhibition is lacking. Here, we used a combination of techniques to assess the role of cholinergic receptors in schistosome motor function. The neuromuscular effects of acetylcholine are typically mediated by gated cation channels of the nicotinic receptor (nAChR) family. Bioinformatics analyses identified numerous nAChR subunits in the S. mansoni genome but, interestingly, nearly half of these subunits carried a motif normally associated with chloride-selectivity. These putative schistosome acetylcholine-gated chloride channels (SmACCs) are evolutionarily divergent from those of nematodes and form a unique clade within the larger family of nAChRs. Pharmacological and RNA interference (RNAi) behavioral screens were used to assess the role of the SmACCs in larval motor function. Treatment with antagonists produced the same effect as RNAi suppression of SmACCs; both led to a hypermotile phenotype consistent with abrogation of an inhibitory neuromuscular mediator. Antibodies were then generated against two of the SmACCs for use in immunolocalization studies. SmACC-1 and SmACC-2 localize to regions of the peripheral nervous system that innervate the body wall muscles, yet neither appears to be expressed directly on the musculature. One gene, SmACC-1, was expressed in HEK-293 cells and characterized using an iodide flux assay. The results indicate that SmACC-1 formed a functional homomeric chloride channel and was activated selectively by a panel of cholinergic agonists. The results described in this study identify a novel clade of nicotinic chloride channels that act as inhibitory modulators of schistosome neuromuscular function. Additionally, the

  3. Chloride channels in the small intestinal cell line IEC-18.

    PubMed

    Basavappa, Srisaila; Vulapalli, Sreesatya Raju; Zhang, Hui; Yule, David; Coon, Steven; Sundaram, Uma

    2005-01-01

    Small intestinal crypt cells play a critical role in modulating Cl- secretion during digestion. The types of Cl- channels mediating Cl- secretion in the small intestine was investigated using the intestinal epithelial cell line, IEC-18, which was derived from rat small intestine crypt cells. In initial radioisotope efflux studies, exposure to forskolin, ionomycin or a decrease in extracellular osmolarity significantly increased 36Cl efflux as compared to control cells. Whole cell patch clamp techniques were subsequently used to examine in more detail the swelling-, Ca2+-, and cAMP-activated Cl- conductance. Decreasing the extracellular osmolarity from 290 to 200 mOsm activated a large outwardly rectifying Cl- current that was voltage-independent and had an anion selectivity of I- > Cl-. Increasing cytosolic Ca2+ by ionomycin activated whole cell Cl- currents, which were also outwardly rectifying but were voltage-dependent. The increase in intracellular Ca2+ levels with ionomycin was confirmed with fura-2 loaded IEC-18 cells. A third type of whole cell Cl- current was observed after increases in intracellular cAMP induced by forskolin. These cAMP-activated Cl- currents have properties consistent with cystic fibrosis transmembrane regulator (CFTR) Cl- channels, as the currents were blocked by glibenclamide or NPPB but insensitive to DIDS. In addition, the current-voltage relationship was linear and had an anion selectivity of Cl- > I-. Confocal immunofluorescence studies and Western blots with two different anti-CFTR antibodies confirmed the expression of CFTR. These results suggest that small intestinal crypt cells express multiple types of Cl- channels, which may all contribute to net Cl- secretion. PMID:15389550

  4. Generation of cAMP-Activated Chloride Currents by Expression of CFTR

    NASA Astrophysics Data System (ADS)

    Anderson, Matthew P.; Rich, Devra P.; Gregory, Richard J.; Smith, Alan E.; Welsh, Michael J.

    1991-02-01

    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. In order to evaluate its function, CFTR was expressed in HeLa, Chinese hamster ovary (CHO), and NIH 3T3 fibroblast cells, and anion permeability was assessed with a fluorescence microscopic assay and the whole-cell patch-clamp technique. Adenosine 3',5'-monophosphate (cAMP) increased anion permeability and chloride currents in cells expressing CFTR, but not in cells expressing a mutant CFTR (ΔF508) or in nontransfected cells. The simplest interpretation of these observations is that CFTR is itself a cAMP-activated chloride channel. The alternative interpretation, that CFTR directly or indirectly regulates chloride channels, requires that these cells have endogenous cryptic, chloride channels that are stimulated by cAMP only in the presence of CFTR.

  5. Chloride channel protein 2 prevents glutamate-induced apoptosis in retinal ganglion cells

    PubMed Central

    Bi, Miao-Miao; Hong, Sen; Ma, Ling-Jun; Zhou, Hong-Yan; Lu, Jia; Zhao, Jing; Zheng, Ya-Juan

    2016-01-01

    Objective(s): The purpose of this study was to investigate the role of chloride channel protein 2 (ClC-2) in glutamate-induced apoptosis in the retinal ganglion cell line (RGC-5). Materials and Methods: RGC-5 cells were treated with 1 mM glutamate for 24 hr. The expression of ClC-2, Bax, and Bcl-2 was detected by western blot analysis. Cell survival and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Caspase-3 and -9 activities were determined by a colorimetric assay. The roles of ClC-2 in glutamate-induced apoptosis were examined by using ClC-2 complementary deoxyribonucleic acid (cDNA) and small inference ribonucleic acid (RNA) transfection technology. Results: Overexpression of ClC-2 in RGC-5 cells significantly decreased glutamate-induced apoptosis and increased cell viability, whereas silencing of ClC-2 with short hairpin (sh) RNA produced opposite effects. ClC-2 overexpression increased the expression of Bcl-2, decreased the expression of Bax, and decreased caspase-3 and -9 activation in RGC-5 cells treated with glutamate, but silencing of ClC-2 produced opposite effects. Conclusion: Our data suggest that ClC-2 chloride channels might play a protective role in glutamate-induced apoptosis in retinal ganglion cells via the mitochondria-dependent apoptosis pathway.

  6. Functional expression of chloride channels and their roles in the cell cycle and cell proliferation in highly differentiated nasopharyngeal carcinoma cells

    PubMed Central

    Huang, Weiyuan; Liu, Mei; Zhu, Linyan; Liu, Shanwen; Luo, Hai; Ma, Lianshun; Wang, Haibo; Lu, Ruiling; Sun, Xiaoxue; Chen, Lixin; Wang, Liwei

    2014-01-01

    Abstract We previously demonstrated that the growth of the poorly differentiated nasopharyngeal carcinoma cells (CNE‐2Z) was more dependent on the activities of volume‐activated chloride channels than that of the normal nasopharyngeal epithelial cells (NP69‐SV40T). However, the activities and roles of such volume‐activated chloride channels in highly differentiated nasopharyngeal carcinoma cells (CNE‐1) are not clarified. In this study, it was found that a volume‐activated chloride current and a regulatory volume decrease (RVD) were induced by 47% hypotonic challenges. The current density and the capacity of RVD in the highly differentiated CNE‐1 cells were lower than those in the poorly differentiated CNE‐2Z cells, and higher than those in the normal cells (NP69‐SV40T). The chloride channel blockers, 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB) and tamoxifen inhibited the current and RVD. Depletion of intracellular Cl− abolished the RVD. The chloride channel blockers reversibly inhibited cell proliferation in a concentration‐ and time‐dependent manner, and arrested cells at the G0/G1 phases, but did not change cell viability. The sensitivity of the three cell lines to the chloride channel blockers was different, with the highest in poorly differentiated cells (CNE‐2Z) and the lowest in the normal cells (NP69‐SV40T). ClC‐3 proteins were expressed in the three cells and distributed inside the cells as well as on the cell membrane. In conclusion, the highly differentiated nasopharyngeal carcinoma CNE‐1 cells functionally expressed the volume‐activated chloride channels, which may play important roles in controlling cell proliferation through modulating the cell cycle, and may be associated with cell differentiation. Chloride channels may be a potential target of anticancer therapy. PMID:25214521

  7. 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. PMID:16432888

  8. 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

  9. 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

  10. Flickery block of single CFTR chloride channels by intracellular anions and osmolytes.

    PubMed

    Linsdell, P; Hanrahan, J W

    1996-08-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and nucleotide-dependent chloride channel. Single CFTR currents recorded on cell show slight outward rectification, which has previously been suggested to be due to an asymmetrical chloride ion gradient or to a specific interaction between permeant intracellular anions and the channel. Using a single-channel recording from Chinese hamster ovary cells stably expressing CFTR, we have found that both the sparingly permeant anion glutamate and the impermeant anion gluconate cause a rapid, voltage-dependent block of CFTR channels when applied to the intracellular, but not the extracellular, face of excised patches. Both the affinity and the voltage dependence of block were affected by the extracellular chloride concentration in a manner consistent with chloride ions being able to repel these blocking ions from the pore. These results are discussed in terms of previous models of CFTR current outward rectification, and it is suggested that this rectification may result from a combination of asymmetrical chloride concentrations and voltage-dependent block of the channel by large cytoplasmic anions. In addition, we find that CFTR conductance is decreased by high concentrations of intracellular sucrose, sorbitol, and urea in a manner consistent with a rapid block of the channel by these molecules. PMID:8770004

  11. Luminal non-selective cation and outwardly rectifying chloride channels in cultured strial marginal cells from gerbil.

    PubMed

    Yeh, T; Van den Abbeele, T; Marianovski, R; Herman, P; Tran Ba Huy, P

    1995-10-01

    Ionic channels located on the luminal side of strial marginal cells (MCs) of gerbil in culture were investigated using the patch-clamp technique. Two types of channels were identified. The most frequently recorded single-channel activity corresponded to a non-selective cation (NSC) channel with a conductance of 23.7 +/- 0.2 pS (n = 18) in symmetrical NaCl conditions. The channel was activated by internal Ca2+ and inhibited by internal adenine nucleotides and flufenamic acid. Spontaneous activity of NSC channels was found in 16% of the cell-attached patches and with a very high density (9 +/- 2 levels/patch, n = 28) in 100% of the excised patches. An outwardly rectifying chloride (ORC) channel was also identified in 14% of the patches but only after excision. The channel exhibited at 0 mV a unit conductance of 26.8 +/- 1.3 pS (n = 8) and a strong outward rectification in symmetrical NaCl conditions, and the open probability increased with depolarization. The luminal NSC channel and the ORC channel evidenced in this study might participate in the production of endolymph. Although extrapolation of the presents results to the in vivo situation should be made with caution, this study suggests that culture of strial MCs may be a suitable model for investigation of endolymph physiology. PMID:8975008

  12. 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. PMID:25762128

  13. Mechanism of lonidamine inhibition of the CFTR chloride channel

    PubMed Central

    Gong, Xiandi; Burbridge, Susan M; Lewis, Angie C; Wong, Patrick Y D; Linsdell, Paul

    2002-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is blocked by a broad range of organic anionic compounds. Here we investigate the effects of the indazole compound lonidamine on CFTR channels expressed in mammalian cell lines using patch clamp recording. Application of lonidamine to the intracellular face of excised membrane patches caused a voltage-dependent block of CFTR currents, with an apparent Kd of 58 μM at −100 mV. Block by lonidamine was apparently independent of channel gating but weakly sensitive to the extracellular Cl− concentration. Intracellular lonidamine led to the introduction of brief interruptions in the single channel current at hyperpolarized voltages, leading to a reduction in channel mean open time. Lonidamine also introduced a new component of macroscopic current variance. Spectral analysis of this variance suggested a blocker on rate of 1.79 μM−1 s−1 and an off-rate of 143 s−1. Several point mutations within the sixth transmembrane region of CFTR (R334C, F337S, T338A and S341A) significantly weakened block of macroscopic CFTR current, suggesting that lonidamine enters deeply into the channel pore from its intracellular end. These results identify and characterize lonidamine as a novel CFTR open channel blocker and provide important information concerning its molecular mechanism of action. PMID:12411425

  14. Mechanism of lonidamine inhibition of the CFTR chloride channel.

    PubMed

    Gong, Xiandi; Burbridge, Susan M; Lewis, Angie C; Wong, Patrick Y D; Linsdell, Paul

    2002-11-01

    1. The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is blocked by a broad range of organic anionic compounds. Here we investigate the effects of the indazole compound lonidamine on CFTR channels expressed in mammalian cell lines using patch clamp recording. 2. Application of lonidamine to the intracellular face of excised membrane patches caused a voltage-dependent block of CFTR currents, with an apparent K(d) of 58 micro M at -100 mV. 3. Block by lonidamine was apparently independent of channel gating but weakly sensitive to the extracellular Cl(-) concentration. 4. Intracellular lonidamine led to the introduction of brief interruptions in the single channel current at hyperpolarized voltages, leading to a reduction in channel mean open time. Lonidamine also introduced a new component of macroscopic current variance. Spectral analysis of this variance suggested a blocker on rate of 1.79 micro M(-1) s(-1) and an off-rate of 143 s(-1). 5. Several point mutations within the sixth transmembrane region of CFTR (R334C, F337S, T338A and S341A) significantly weakened block of macroscopic CFTR current, suggesting that lonidamine enters deeply into the channel pore from its intracellular end. 6. These results identify and characterize lonidamine as a novel CFTR open channel blocker and provide important information concerning its molecular mechanism of action. PMID:12411425

  15. Molecular dissection of gating in the ClC-2 chloride channel.

    PubMed

    Jordt, S E; Jentsch, T J

    1997-04-01

    The ClC-2 chloride channel is probably involved in the regulation of cell volume and of neuronal excitability. Site-directed mutagenesis was used to understand ClC-2 activation in response to cell swelling, hyperpolarization and acidic extracellular pH. Similar to equivalent mutations in ClC-0, neutralizing Lys566 at the end of the transmembrane domains results in outward rectification and a shift in voltage dependence, but leaves the basic gating mechanism, including swelling activation, intact. In contrast, mutations in the cytoplasmic loop between transmembrane domains D7 and D8 abolish all three modes of activation by constitutively opening the channel without changing its pore properties. These effects resemble those observed with deletions of an amino-terminal inactivation domain, and suggest that it may act as its receptor. Such a 'ball-and-chain' type mechanism may act as a final pathway in the activation of ClC-2 elicited by several stimuli. PMID:9130703

  16. Inhibition of ClC-2 chloride channels by a peptide component or components of scorpion venom.

    PubMed

    Thompson, C H; Fields, D M; Olivetti, P R; Fuller, M D; Zhang, Z R; Kubanek, J; McCarty, N A

    2005-11-01

    ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide. PMID:16596447

  17. 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. PMID:19114635

  18. Mutations at the Signature Sequence of CFTR Create a Cd2+-gated Chloride Channel

    PubMed Central

    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 [Cd2+] can dramatically increase the activity of G551D-CFTR in the absence of ATP. This effect of Cd2+ 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 Cd2+ activation, suggesting an involvement of endogenous cysteine residue(s) in mediating this effect of Cd2+. The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR's NBD1, show robust response to Cd2+. On the other hand, negligible effects of Cd2+ were seen with T547C, Q552C, and R553C, indicating that a specific region of the signature sequence is involved in transmitting the signal of Cd2+ binding to the gate. Collectively, these results suggest that the effect of Cd2+ 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. PMID:19114635

  19. 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. PMID:26711895

  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. Syntaxin 1A inhibits CFTR chloride channels by means of domain-specific protein–protein interactions

    PubMed Central

    Naren, Anjaparavanda P.; Quick, Michael W.; Collawn, James F.; Nelson, Deborah J.; Kirk, Kevin L.

    1998-01-01

    Previously we showed that the functional activity of the epithelial chloride channel that is encoded by the cystic fibrosis gene (CFTR) is reciprocally modulated by two components of the vesicle fusion machinery, syntaxin 1A and Munc-18. Here we report that syntaxin 1A inhibits CFTR chloride channels by means of direct and domain-specific protein–protein interactions. Syntaxin 1A stoichiometrically binds to the N-terminal cytoplasmic tail of CFTR, and this binding is blocked by Munc-18. The modulation of CFTR currents by syntaxin 1A is eliminated either by deletion of this tail or by injecting this tail as a blocking peptide into coexpressing Xenopus oocytes. The CFTR binding site on syntaxin 1A maps to the third predicted helical domain (H3) of this membrane protein. Moreover, CFTR Cl− currents are effectively inhibited by a minimal syntaxin 1A construct (i.e., the membrane-anchored H3 domain) that cannot fully substitute for wild-type syntaxin 1A in membrane fusion reactions. We also show that syntaxin 1A binds to and inhibits the activities of disease-associated mutants of CFTR, and that the chloride current activity of recombinant ΔF508 CFTR (i.e., the most common cystic fibrosis mutant) can be potentiated by disrupting its interaction with syntaxin 1A in cultured epithelial cells. Our results provide evidence for a direct physical interaction between CFTR and syntaxin 1A that limits the functional activities of normal and disease-associated forms of this chloride channel. PMID:9724814

  2. The Validation of Nematode-Specific Acetylcholine-Gated Chloride Channels as Potential Anthelmintic Drug Targets

    PubMed Central

    Wever, Claudia M.; Farrington, Danielle; Dent, Joseph A.

    2015-01-01

    New compounds are needed to treat parasitic nematode infections in humans, livestock and plants. Small molecule anthelmintics are the primary means of nematode parasite control in animals; however, widespread resistance to the currently available drug classes means control will be impossible without the introduction of new compounds. Adverse environmental effects associated with nematocides used to control plant parasitic species are also motivating the search for safer, more effective compounds. Discovery of new anthelmintic drugs in particular has been a serious challenge due to the difficulty of obtaining and culturing target parasites for high-throughput screens and the lack of functional genomic techniques to validate potential drug targets in these pathogens. We present here a novel strategy for target validation that employs the free-living nematode Caenorhabditis elegans to demonstrate the value of new ligand-gated ion channels as targets for anthelmintic discovery. Many successful anthelmintics, including ivermectin, levamisole and monepantel, are agonists of pentameric ligand-gated ion channels, suggesting that the unexploited pentameric ion channels encoded in parasite genomes may be suitable drug targets. We validated five members of the nematode-specific family of acetylcholine-gated chloride channels as targets of agonists with anthelmintic properties by ectopically expressing an ivermectin-gated chloride channel, AVR-15, in tissues that endogenously express the acetylcholine-gated chloride channels and using the effects of ivermectin to predict the effects of an acetylcholine-gated chloride channel agonist. In principle, our strategy can be applied to validate any ion channel as a putative anti-parasitic drug target. PMID:26393923

  3. 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.

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

    PubMed Central

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

    2009-01-01

    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. PMID:16554808

  5. Expression of the chloride channel CLC-K in human airway epithelial cells.

    PubMed

    Mummery, Jennifer L; Killey, Jennifer; Linsdell, Paul

    2005-12-01

    Airway submucosal gland function is severely disrupted in cystic fibrosis (CF), as a result of genetic mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), an apical membrane Cl(-) channel. To identify other Cl(-) channel types that could potentially substitute for lost CFTR function in these cells, we investigated the functional and molecular expression of Cl(-) channels in Calu-3 cells, a human cell line model of the submucosal gland serous cell. Whole cell patch clamp recording from these cells identified outwardly rectified, pH- and calcium-sensitive Cl(-) currents that resemble those previously ascribed to ClC-K type chloride channels. Using reverse transcription polymerase chain reaction, we identified expression of mRNA for ClC-2, ClC-3, ClC-4, ClC-5, ClC-6, ClC-7, ClC-Ka, and ClC-Kb, as well as the common ClC-K channel beta subunit barttin. Western blotting confirmed that Calu-3 cells express both ClC-K and barttin protein. Thus, Calu-3 cells express multiple members of the ClC family of Cl(-) channels that, if also expressed in native submucosal gland serous cells within the CF lung, could perhaps act to partially substitute lost CFTR function. Furthermore, this work represents the first evidence for functional ClC-K chloride channel expression within the lung. PMID:16462912

  6. Chloride inhibition of nitrite-induced methemoglobinemia in channel catfish (Ictalurus punctatus)

    USGS Publications Warehouse

    Tomasso, J.R.; Simco, B.A.; Davis, K.B.

    1979-01-01

    Exposure of channel catfish (Ictalurus punctatus) fingerlings for 24?h to 1.0, 2.5, and 5.0?mg/L nitrite (pH?=?7; hardness?=?40?mg/L; temperature?=?22–25 °C) produced methemoglobin levels of 20.7?±?1.9%, 59.8?±?1.9%, and 77.4?±?1.4% (SE), respectively. However, methemoglobin levels were not elevated when fish were simultaneously exposed to 1.0, 2.5, and 5.0?mg/L nitrite and 25, 50, and 100?mg/L sodium chloride, respectively. Acclimation to sodium chloride for 24?h before exposure to nitrite did not enhance the inhibitory action of sodium chloride. Fish exposed to 5?mg/L nitrite for 5?h developed 42.5?±?3.8% methemoglobin. When transferred to water containing 5?mg/L nitrite and 250?mg/L sodium chloride, methemoglobin levels returned to normal within 24?h. Environmental chloride probably inhibits methemoglobin formation by competing with nitrite for entrance into the gills of the fish. An ionic ratio of 16 Cl- to 1 NO2- is capable of complete suppression of nitrite-induced methemoglobin formation. Bicarbonate ion present in the test water (1?meq/L) may also have contributed to the inhibitive action of chloride.

  7. Fate and effects of methylene chloride in activated sludge.

    PubMed

    Klecka, G M

    1982-09-01

    Activated sludge obtained from a municipal wastewater treatment plant was acclimated to methylene chloride at concentrations between 1 and 100 mg/liter by continuous exposure to the compound for 9 to 11 days. Acclimated cultures were shown to mineralize methylene chloride to carbon dioxide and chloride. Rates of methylene chloride degradation were 0.14, 2.3, and 7.4 mg of CH2Cl2 consumed per h per g of mixed-liquor suspended solids for cultures incubated in the presence of 1, 10, and 100 mg/liter, respectively. Concentrations of methylene chloride between 10 and 1,000 mg/liter had no significant effect on O2 consumption or glucose metabolism by activated sludge. A hypothetical model was developed to examine the significance of volatilization and biodegradation for the removal of methylene chloride from an activated sludge reactor. Application of the model indicated that the rate of biodegradation was approximately 12 times greater than the rate of volatilization. Thus, biodegradation may be the predominant process determining the fate of methylene chloride in activated sludge systems continuously exposed to the compound. PMID:7138008

  8. Fate and effects of methylene chloride in activated sludge.

    PubMed Central

    Klecka, G M

    1982-01-01

    Activated sludge obtained from a municipal wastewater treatment plant was acclimated to methylene chloride at concentrations between 1 and 100 mg/liter by continuous exposure to the compound for 9 to 11 days. Acclimated cultures were shown to mineralize methylene chloride to carbon dioxide and chloride. Rates of methylene chloride degradation were 0.14, 2.3, and 7.4 mg of CH2Cl2 consumed per h per g of mixed-liquor suspended solids for cultures incubated in the presence of 1, 10, and 100 mg/liter, respectively. Concentrations of methylene chloride between 10 and 1,000 mg/liter had no significant effect on O2 consumption or glucose metabolism by activated sludge. A hypothetical model was developed to examine the significance of volatilization and biodegradation for the removal of methylene chloride from an activated sludge reactor. Application of the model indicated that the rate of biodegradation was approximately 12 times greater than the rate of volatilization. Thus, biodegradation may be the predominant process determining the fate of methylene chloride in activated sludge systems continuously exposed to the compound. PMID:7138008

  9. Interaction of Human Chloride Intracellular Channel Protein 1 (CLIC1) with Lipid Bilayers: A Fluorescence Study.

    PubMed

    Hare, Joanna E; Goodchild, Sophia C; Breit, Samuel N; Curmi, Paul M G; Brown, Louise J

    2016-07-12

    Chloride intracellular channel protein 1 (CLIC1) is very unusual as it adopts a soluble glutathione S-transferase-like canonical fold but can also autoinsert into lipid bilayers to form an ion channel. The conversion between these forms involves a large, but reversible, structural rearrangement of the CLIC1 module. The only identified environmental triggers controlling the metamorphic transition of CLIC1 are pH and oxidation. Until now, there have been no high-resolution structural data available for the CLIC1 integral membrane state, and consequently, a limited understanding of how CLIC1 unfolds and refolds across the bilayer to form a membrane protein with ion channel activity exists. Here we show that fluorescence spectroscopy can be used to establish the interaction and position of CLIC1 in a lipid bilayer. Our method employs a fluorescence energy transfer (FRET) approach between CLIC1 and a dansyl-labeled lipid analogue to probe the CLIC1-lipid interface. Under oxidizing conditions, a strong FRET signal between the single tryptophan residue of CLIC1 (Trp35) and the dansyl-lipid analogue was detected. When considering the proportion of CLIC1 interacting with the lipid bilayer, as estimated by fluorescence quenching experiments, the FRET distance between Trp35 and the dansyl moiety on the membrane surface was determined to be ∼15 Å. This FRET-detected interaction provides direct structural evidence that CLIC1 associates with membranes. The results presented support the current model of an oxidation-driven interaction of CLIC1 with lipid bilayers and also propose a membrane anchoring role for Trp35. PMID:27299171

  10. 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. PMID:27397895

  11. Expression pattern and function of alternative splice variants of glutamate-gated chloride channel in the housefly Musca domestica.

    PubMed

    Kita, Tomo; Ozoe, Fumiyo; Ozoe, Yoshihisa

    2014-02-01

    Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems. cDNAs encoding two alternative splice variants (MdGluClB and C) of the GluCl subunit were cloned from the housefly Musca domestica. The expression patterns of three variants, including the previously reported MdGluClA, differed among the body parts (head, thorax, abdomen, and leg) of the adult housefly and among developmental stages (embryo, larva, pupa, and adult). The MdGluClA and B transcripts were abundant in the central nervous system of the adult, whereas the MdGluClC transcript was expressed in the central nervous system and as the predominant variant in the peripheral tissues. The sensitivities to the agonist glutamate and the allosteric activator ivermectin B1a did not differ between channels containing MdGluCl variants when they were singly or co-expressed in Xenopus oocytes. By contrast, MdGluClA and B channels were more sensitive to the channel blockers fipronil and picrotoxinin than was MdGluClC channels. Heteromeric channels containing different subunit variants were more sensitive to picrotoxinin than were homomeric channels. Heteromeric channels were more sensitive to fipronil than were homomeric MdGluClC channels but not than homomeric MdGluClA and B channels. These results suggest that functionally indistinguishable but pharmacologically distinct GluCls are expressed in a spatially and temporally distinct manner in the housefly. PMID:24291284

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

    PubMed

    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

  13. Contribution of chloride channel permease to fluoride resistance in Streptococcus mutans.

    PubMed

    Murata, Takatoshi; Hanada, Nobuhiro

    2016-06-01

    Genes encoding fluoride transporters have been identified in bacterial and archaeal species. The genome sequence of the cariogenic Streptococcus mutans bacteria suggests the presence of a putative fluoride transporter, which is referred to as a chloride channel permease. Two homologues of this gene (GenBank locus tags SMU_1290c and SMU_1289c) reside in tandem in the genome of S. mutans The aim of this study was to determine whether the chloride channel permeases contribute to fluoride resistance. We constructed SMU_1290c- and SMU_1289c-knockout S. mutans UA159 strains. We also constructed a double-knockout strain lacking both genes. SMU_1290c or SMU_1289c was transformed into a fluoride transporter- disrupted Escherichia coli strain. All bacterial strains were cultured under appropriate conditions with or without sodium fluoride, and fluoride resistance was evaluated. All three gene-knockout S. mutans strains showed lower resistance to sodium fluoride than did the wild-type strain. No significant changes in resistance to other sodium halides were recognized between the wild-type and double-knockout strains. Both SMU_1290c and SMU_1289c transformation rescued fluoride transporter-disrupted E. coli cell from fluoride toxicity. We conclude that the chloride channel permeases contribute to fluoride resistance in S. mutans. PMID:27190286

  14. 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

  15. State-dependent inhibition of cystic fibrosis transmembrane conductance regulator chloride channels by a novel peptide toxin.

    PubMed

    Fuller, Matthew D; Thompson, Christopher H; Zhang, Zhi-Ren; Freeman, Cody S; Schay, Eszter; Szakács, Gergely; Bakos, Eva; Sarkadi, Balázs; McMaster, Denis; French, Robert J; Pohl, Jan; Kubanek, Julia; McCarty, Nael A

    2007-12-28

    Peptide toxins from animal venom have been used for many years for the identification and study of cation-permeable ion channels. However, no peptide toxins have been identified that interact with known anion-selective channels, including cystic fibrosis transmembrane conductance regulator (CFTR), the protein defective in cystic fibrosis and a member of the ABC transporter superfamily. Here, we describe the identification and initial characterization of a novel 3.7-kDa peptide toxin, GaTx1, which is a potent and reversible inhibitor of CFTR, acting from the cytoplasmic side of the membrane. Thus, GaTx1 is the first peptide toxin identified that inhibits a chloride channel of known molecular identity. GaTx1 exhibited high specificity, showing no effect on a panel of nine transport proteins, including Cl(-) and K(+) channels, and ABC transporters. GaTx1-mediated inhibition of CFTR channel activity is strongly state-dependent; both potency and efficacy are reduced under conditions of elevated [ATP], suggesting that GaTx1 may function as a non-competitive inhibitor of ATP-dependent channel gating. This tool will allow the application of new quantitative approaches to study CFTR structure and function, particularly with respect to the conformational changes that underlie transitions between open and closed states. PMID:17951250

  16. 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

  17. 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

  18. 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

    2016-01-01

    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

  19. 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

  20. Calcium-activated chloride currents prolongs the duration of contractions in pregnant rat myometrial tissue.

    PubMed

    Young, Roger C; Bemis, Adam

    2009-08-01

    We investigated the importance of pharmacologically blocking calcium-activated chloride (I(Cl(Ca))) and L-type calcium currents on isometric contractions of strips of D21 pregnant rat myometrial tissue, while simultaneously measuring the electrical activity of the tissue strips with extracellular contact electrodes. When measured with contact electrodes, the duration of the spiking activity directly reflects the duration of the tissue-level plateau potential. We correlated the number of spikes, durations of spiking activity, and the spiking frequencies with changes of the area under the force curves as a function of exposure to low doses of anthracene-9-carboxylate (9-AC, a non-specific Cl channel blocker), chlorotoxin (a specific I(Cl(Ca)) blocker) and nifedipine (an L-type calcium channel blocker). The area under the force curve was measured only during spiking electrical activity, thereby separating pharmacological effects on tissue relaxation from those that modulate force production. Blocking chloride channels reduced impulse, shortened the duration of spiking activity, and reduced the number of spikes generated in each contraction. This was observed without a change in the frequency of spike production or a reduction of peak force. Nifedipine reduced impulse, shortened the duration of spiking activity, and reduced the number of spikes. In contrast to chloride channel blockade, nifedipine reduced maximum spike frequency and peak force. Taken together, our data suggest that blocking L-type calcium channels reduces impulse directly by reducing peak force, and indirectly by reducing activation of I(Cl(Ca)) , which shortens the duration of the contraction. PMID:19380901

  1. Inhibition of ANO1/TMEM16A Chloride Channel by Idebenone and Its Cytotoxicity to Cancer Cell Lines

    PubMed Central

    Kim, Minseo; Lee, Ho K.; Kim, Jin-Hee; Jeong, Jin-Hyun; Namkung, Wan

    2015-01-01

    The expression levels of anoctamin 1 (ANO1, TMEM16A), a calcium-activated chloride channel (CaCC), are significantly increased in several tumors, and inhibition of ANO1 is known to reduce cell proliferation and migration. Here, we performed cell-based screening of a collection of natural products and drug-like compounds to identify inhibitors of ANO1. As a result of the screening, idebenone, miconazole and plumbagin were identified as novel ANO1 inhibitors. Electrophysiological studies showed that idebenone, a synthetic analog of coenzyme Q10, completely blocked ANO1 activity in FRT cells expressing ANO1 without any effect on intracellular calcium signaling and CFTR, a cAMP-regulated chloride channel. The CaCC activities in PC-3 and CFPAC-1 cells expressing abundant endogenous ANO1 were strongly blocked by idebenone. Idebenone inhibited cell proliferation and induced apoptosis in PC-3 and CFPAC-1 cells, but not in A549 cells, which do not express ANO1. These data suggest that idebenone, a novel ANO1 inhibitor, has potential for use in cancer therapy. PMID:26196390

  2. Multistep Mechanism of Chloride Translocation in a Strongly Anion-Selective Porin Channel

    PubMed Central

    Zachariae, Ulrich; Helms, Volkhard; Engelhardt, Harald

    2003-01-01

    The strongly anion-selective porin channel Omp32 from the bacterium Delftia acidovorans differs from other unspecific porins by its pronounced selectivity for anions and its particularly small channel cross-section. Multinanosecond molecular dynamics simulations of chloride ion movement in this pore protein suggest that translocated anions interact intimately with the charges of a “basic ladder”, whose dynamics lead the anions in a stepwise manner through the constriction zone of the channel. The ladder-steps comprise the central clustered arginine groups and flanking basic residues at its exoplasmic and periplasmic sides. The computed free energy profile of ion movement in and around the constriction zone shows a corresponding succession of free energy minima and barriers. A number of polar atoms from other amino acids contribute to the coordination of Cl− at certain sites and to its temporary immobilization in the channel. A special binding site occurs at the transition of the constriction zone to the periplasmic funnel, binding the chloride ion over significant lengths of time. The results from our MD study offer a possible explanation for the nonlinear conductance properties and unusual salt-dependent characteristics of Omp32 observed earlier in experimental measurements. PMID:12885642

  3. Comparative pharmacology of flatworm and roundworm glutamate-gated chloride channels: Implications for potential anthelmintics

    PubMed Central

    Lynagh, Timothy; Cromer, Brett A.; Dufour, Vanessa; Laube, Bodo

    2014-01-01

    Pharmacological targeting of glutamate-gated chloride channels (GluCls) is a potent anthelmintic strategy, evidenced by macrocyclic lactones that eliminate numerous roundworm infections by activating roundworm GluCls. Given the recent identification of flatworm GluCls and the urgent need for drugs against schistosomiasis, flatworm GluCls should be evaluated as potential anthelmintic targets. This study sought to identify agonists or modulators of one such GluCl, SmGluCl-2 from the parasitic flatworm Schistosoma mansoni. The effects of nine glutamate-like compounds and three monoterpenoid ion channel modulators were measured by electrophysiology at SmGluCl-2 recombinantly expressed in Xenopus laevis oocytes. For comparison with an established anthelmintic target, experiments were also performed on the AVR-14B GluCl from the parasitic roundworm Haemonchus contortus. l-Glutamate was the most potent agonist at both GluCls, but l-2-aminoadipate, d-glutamate and d-2-aminoadipate activated SmGluCl-2 (EC50 1.0 ± 0.1 mM, 2.4 ± 0.4 mM, 3.6 ± 0.7 mM, respectively) more potently than AVR-14B. Quisqualate activated only SmGluCl-2 whereas l-aspartate activated only AVR-14B GluCls. Regarding the monoterpenoids, both GluCls were inhibited by propofol, thymol and menthol, SmGluCl-2 most potently by thymol (IC50 484 ± 85 μM) and least potently by menthol (IC50 > 3 mM). Computational docking suggested that agonist and inhibitor potency is attributable to particular interactions with extracellular or membrane-spanning amino acid residues. These results reveal that flatworm GluCls are pharmacologically susceptible to numerous agonists and modulators and indicate that changes to the glutamate γ-carboxyl or to the propofol 6-isopropyl group can alter the differential pharmacology at flatworm and roundworm GluCls. This should inform the development of more potent compounds and in turn lead to novel anthelmintics. PMID:25516835

  4. Synthesis of photoreactive ivermectin B1a derivatives and their actions on Haemonchus and Bombyx glutamate-gated chloride channels.

    PubMed

    Fuse, Toshinori; Ikeda, Izumi; Kita, Tomo; Furutani, Shogo; Nakajima, Hiromitsu; Matsuda, Kazuhiko; Ozoe, Fumiyo; Ozoe, Yoshihisa

    2015-05-01

    Glutamate-gated chloride channels (GluCls) are inhibitory neurotransmitter receptors that are present only in invertebrates such as nematodes and insects. These channels are important targets of insecticidal, acaricidal, and anthelmintic macrolides such as avermectins, ivermectin (IVM), and milbemycins. To identify the amino acid residues that interact with IVM in GluCls, three IVM B1a derivatives with different photoreactive substitutions at C-13 were synthesized in the present study. These derivatives displayed low- or subnanomolar affinity for parasitic nematode (Haemonchus contortus) and silkworm (Bombyx mori) GluCls expressed in COS-1 cells. The derivatives also activated homomeric H. contortus GluCls expressed in Xenopus oocytes. The results indicate that synthesized photoreactive IVM B1a derivatives have superior affinity and functionality for chemically labeling the macrolide-binding site in GluCls. . PMID:25987225

  5. 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

  6. 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

  7. The contribution of raised intraneuronal chloride to epileptic network activity.

    PubMed

    Alfonsa, Hannah; Merricks, Edward M; Codadu, Neela K; Cunningham, Mark O; Deisseroth, Karl; Racca, Claudia; Trevelyan, Andrew J

    2015-05-20

    Altered inhibitory function is an important facet of epileptic pathology. A key concept is that GABAergic activity can become excitatory if intraneuronal chloride rises. However, it has proved difficult to separate the role of raised chloride from other contributory factors in complex network phenomena, such as epileptic pathology. Therefore, we asked what patterns of activity are associated with chloride dysregulation by making novel use of Halorhodopsin to load clusters of mouse pyramidal cells artificially with Cl(-). Brief (1-10 s) activation of Halorhodopsin caused substantial positive shifts in the GABAergic reversal potential that were proportional to the charge transfer during the illumination and in adult neocortical pyramidal neurons decayed with a time constant of τ = 8.0 ± 2.8s. At the network level, these positive shifts in EGABA produced a transient rise in network excitability, with many distinctive features of epileptic foci, including high-frequency oscillations with evidence of out-of-phase firing (Ibarz et al., 2010). We show how such firing patterns can arise from quite small shifts in the mean intracellular Cl(-) level, within heterogeneous neuronal populations. Notably, however, chloride loading by itself did not trigger full ictal events, even with additional electrical stimulation to the underlying white matter. In contrast, when performed in combination with low, subepileptic levels of 4-aminopyridine, Halorhodopsin activation rapidly induced full ictal activity. These results suggest that chloride loading has at most an adjunctive role in ictogenesis. Our simulations also show how chloride loading can affect the jitter of action potential timing associated with imminent recruitment to an ictal event (Netoff and Schiff, 2002). PMID:25995461

  8. The Contribution of Raised Intraneuronal Chloride to Epileptic Network Activity

    PubMed Central

    Alfonsa, Hannah; Merricks, Edward M.; Codadu, Neela K.; Cunningham, Mark O.; Deisseroth, Karl; Racca, Claudia

    2015-01-01

    Altered inhibitory function is an important facet of epileptic pathology. A key concept is that GABAergic activity can become excitatory if intraneuronal chloride rises. However, it has proved difficult to separate the role of raised chloride from other contributory factors in complex network phenomena, such as epileptic pathology. Therefore, we asked what patterns of activity are associated with chloride dysregulation by making novel use of Halorhodopsin to load clusters of mouse pyramidal cells artificially with Cl−. Brief (1–10 s) activation of Halorhodopsin caused substantial positive shifts in the GABAergic reversal potential that were proportional to the charge transfer during the illumination and in adult neocortical pyramidal neurons decayed with a time constant of τ = 8.0 ± 2.8s. At the network level, these positive shifts in EGABA produced a transient rise in network excitability, with many distinctive features of epileptic foci, including high-frequency oscillations with evidence of out-of-phase firing (Ibarz et al., 2010). We show how such firing patterns can arise from quite small shifts in the mean intracellular Cl− level, within heterogeneous neuronal populations. Notably, however, chloride loading by itself did not trigger full ictal events, even with additional electrical stimulation to the underlying white matter. In contrast, when performed in combination with low, subepileptic levels of 4-aminopyridine, Halorhodopsin activation rapidly induced full ictal activity. These results suggest that chloride loading has at most an adjunctive role in ictogenesis. Our simulations also show how chloride loading can affect the jitter of action potential timing associated with imminent recruitment to an ictal event (Netoff and Schiff, 2002). PMID:25995461

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

    PubMed

    Fatehi, Mohammad; Linsdell, Paul

    2008-03-01

    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. PMID:18167343

  10. Conduction Mechanisms of Chloride Ions in ClC-Type Channels

    PubMed Central

    Corry, Ben; O'Mara, Megan; Chung, Shin-Ho

    2004-01-01

    The conduction properties of ClC-0 and ClC-1 chloride channels are examined using electrostatic calculations and three-dimensional Brownian dynamics simulations. We create an open-state configuration of the prokaryotic ClC Cl− channel using its known crystallographic structure as a basis. Two residues that are occluding the channel are slowly pushed outward with molecular dynamics to create a continuous ion-conducting path with the minimum radius of 2.5 Å. Then, retaining the same pore shape, the prokaryotic ClC channel is converted to either ClC-0 or ClC-1 by replacing all the nonconserved dipole-containing and charged amino acid residues. Employing open-state ClC-0 and ClC-1 channel models, current-voltage curves consistent with experimental measurements are obtained. We find that conduction in these pores involves three ions. We locate the binding sites, as well as pinpointing the rate-limiting steps in conduction, and make testable predictions about how the single channel current across ClC-0 and ClC-1 will vary as the ionic concentrations are increased. Finally, we demonstrate that a ClC-0 homology model created from an alternative sequence alignment fails to replicate any of the experimental observations. PMID:14747320

  11. Antiangiogenic activity of 2-formyl-8-hydroxy-quinolinium chloride.

    PubMed

    Lam, K-H; Lee, K K-H; Kok, S H-L; Wong, R S-M; Lau, F-Y; Cheng, G Y-M; Wong, W-Y; Tong, S-W; Chan, K-W; Chan, R Y-K; Tang, J C-O; Cheng, C-H; Hau, D K-P; Bian, Z-X; Gambari, R; Chui, C-H

    2016-05-01

    Tumour growth is closely related to the development of new blood vessels to supply oxygen and nutrients to cancer cells. Without the neovascular formation, tumour volumes cannot increase and undergo metastasis. Antiangiogenesis is one of the most promising approaches for antitumour therapy. The exploration of new antiangiogenic agents would be helpful in antitumour therapy. Quinoline is an aromatic nitrogen compound characterized by a double-ring structure which exhibits a benzene ring fused to pyridine at two adjacent carbon atoms. The high stability of quinoline makes it preferable in a variety of therapeutic and pharmaceutical applications, including antitumour treatment. This work is to examine the potential antiangiogenic activity of the synthetic compound 2-Formyl-8-hydroxy-quinolinium chloride. We found that 2-Formyl-8-hydroxy-quinolinium chloride could inhibit the growth of human umbilical vein endothelial cells in vitro. Using the diethylnitrosamine-induced hepatocarcinogenesis model, 2-Formyl-8-hydroxy-quinolinium chloride showed strong antiangiogenic activity. Furthermore, 2-Formyl-8-hydroxy-quinolinium chloride could inhibit the growth of large Hep3B xenografted tumour from the nude mice. We assume that 2-Formyl-8-hydroxy-quinolinium chloride could be a potential antiangiogenic and antitumour agent and it is worthwhile to further study its underlying working mechanism. PMID:27133051

  12. Evidence for a channel for the electrogenic transport of chloride ion in the rat hepatocyte.

    PubMed

    Bear, C E; Petrunka, C N; Strasberg, S M

    1985-01-01

    Chloride is the major inorganic anion in bile but its mechanism of passage from blood to bile is uncertain. Specific membrane channels account for most net inorganic anion flux in other cell types such as the proximal tubular cell and red blood cell; disulfonic stilbenes inhibit anion movement through these channels. Therefore, we have sought the presence of similar channels in the hepatocyte. Net inorganic anion flux or conductance was initiated in isolated rat hepatocytes by valinomycin in the presence of an outward potassium gradient. Potassium concentration in the extracellular medium increased from 2.75 +/- 0.02 in control cell suspensions to 3.15 +/- 0.04 in valinomycin-treated cell suspensions. Membrane potential difference (Em) (mV), determined as the distribution of [14C]tetraphenyl phosphonium ion was -28 mV in control cells and -42 mV in valinomycin-treated cells (p less than 0.05). Intracellular chloride concentration (36Cl-) (mEq per liter of cell water) decreased significantly from 38.6 in control cells to 32.0 in valinomycin-treated cells. The observed intracellular concentrations (36Cl-) in both control and valinomycin-treated cell suspensions closely approximates values predicted on the basis of the Nernst equation: 41 and 29 (mEq per liter of cell water), respectively, suggesting that the chloride ion is passively distributed on the basis of the membrane potential difference. Furthermore, net rate-limited cell water loss of approximately 15% of control values was associated with the above valinomycin-stimulated changes in ion distribution, as assessed using three methods of cell water volume determination.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2581880

  13. 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. PMID:17700963

  14. Age-dependent chloride channel expression in skeletal muscle fibres of normal and HSALR myotonic mice

    PubMed Central

    DiFranco, Marino; Yu, Carl; Quiñonez, Marbella; Vergara, Julio L

    2013-01-01

    We combine electrophysiological and optical techniques to investigate the role that the expression of chloride channels (ClC-1) plays on the age-dependent electrical properties of mammalian muscle fibres. To this end, we comparatively evaluate the magnitude and voltage dependence of chloride currents (ICl), as well as the resting resistance, in fibres isolated from control and human skeletal actin (HSA)LR mice (a model of myotonic dystrophy) of various ages. In control mice, the maximal peak chloride current ([peak-ICl]max) increases from −583 ± 126 to −956 ± 260 μA cm−2 (mean ± SD) between 3 and 6 weeks old. Instead, in 3-week-old HSALR mice, ICl are significantly smaller (−153 ± 33 μA cm−2) than in control mice, but after a long period of ∼14 weeks they reach statistically comparable values. Thus, the severe ClC-1 channelopathy in young HSALR animals is slowly reversed with aging. Frequency histograms of the maximal chloride conductance (gCl,max) in fibres of young HSALR animals are narrow and centred in low values; alternatively, those from older animals show broad distributions, centred at larger gCl,max values, compatible with mosaic expressions of ClC-1 channels. In fibres of both animal strains, optical data confirm the age-dependent increase in gCl, and additionally suggest that ClC-1 channels are evenly distributed between the sarcolemma and transverse tubular system membranes. Although gCl is significantly depressed in fibres of young HSALR mice, the resting membrane resistance (Rm) at −90 mV is only slightly larger than in control mice due to upregulation of a Rb-sensitive resting conductance (gK,IR). In adult animals, differences in Rm are negligible between fibres of both strains, and the contributions of gCl and gK,IR are less altered in HSALR animals. We surmise that while hyperexcitability in young HSALR mice can be readily explained on the basis of reduced gCl, myotonia in adult HSALR animals may be explained on the basis of a

  15. 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.

  16. 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

  17. Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation.

    PubMed

    Bai, Yonghong; Li, Min; Hwang, Tzyh-Chang

    2010-09-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily that functions as a chloride channel. Previous work has suggested that the external side of the sixth transmembrane segment (TM6) plays an important role in governing chloride permeation, but the function of the internal side remains relatively obscure. Here, on a cysless background, we performed cysteine-scanning mutagenesis and modification to screen the entire TM6 with intracellularly applied thiol-specific methanethiosulfonate reagents. Single-channel amplitude was reduced in seven cysteine-substituted mutants, suggesting a role of these residues in maintaining the pore structure for normal ion permeation. The reactivity pattern of differently charged reagents suggests that the cytoplasmic part of TM6 assumes a secondary structure of an alpha helix, and that reactive sites (341, 344, 345, 348, 352, and 353) reside in two neighboring faces of the helix. Although, as expected, modification by negatively charged reagents inhibits anion permeation, interestingly, modification by positively charged reagents of cysteine thiolates on one face (344, 348, and 352) of the helix affects gating. For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. For R352C, which exhibited reduced single-channel amplitude, modifications by two positively charged reagents with different chemical properties completely restored the single-channel amplitude but had distinct effects on both the open time and the closed time. These results corroborate the idea that a helix rotation of TM6, which has been proposed to be part of the molecular motions during transport cycles in other ABC transporters, is associated with gating of the CFTR pore. PMID:20805575

  18. Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation

    PubMed Central

    Bai, Yonghong; Li, Min

    2010-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the adenosine triphosphate–binding cassette (ABC) transporter superfamily that functions as a chloride channel. Previous work has suggested that the external side of the sixth transmembrane segment (TM6) plays an important role in governing chloride permeation, but the function of the internal side remains relatively obscure. Here, on a cysless background, we performed cysteine-scanning mutagenesis and modification to screen the entire TM6 with intracellularly applied thiol-specific methanethiosulfonate reagents. Single-channel amplitude was reduced in seven cysteine-substituted mutants, suggesting a role of these residues in maintaining the pore structure for normal ion permeation. The reactivity pattern of differently charged reagents suggests that the cytoplasmic part of TM6 assumes a secondary structure of an α helix, and that reactive sites (341, 344, 345, 348, 352, and 353) reside in two neighboring faces of the helix. Although, as expected, modification by negatively charged reagents inhibits anion permeation, interestingly, modification by positively charged reagents of cysteine thiolates on one face (344, 348, and 352) of the helix affects gating. For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. For R352C, which exhibited reduced single-channel amplitude, modifications by two positively charged reagents with different chemical properties completely restored the single-channel amplitude but had distinct effects on both the open time and the closed time. These results corroborate the idea that a helix rotation of TM6, which has been proposed to be part of the molecular motions during transport cycles in other ABC transporters, is associated with gating of the CFTR pore. PMID:20805575

  19. Calcium-activated chloride currents in olfactory sensory neurons from mice lacking bestrophin-2

    PubMed Central

    Pifferi, Simone; Dibattista, Michele; Sagheddu, Claudia; Boccaccio, Anna; Al Qteishat, Ahmed; Ghirardi, Filippo; Tirindelli, Roberto; Menini, Anna

    2009-01-01

    Olfactory sensory neurons use a chloride-based signal amplification mechanism to detect odorants. The binding of odorants to receptors in the cilia of olfactory sensory neurons activates a transduction cascade that involves the opening of cyclic nucleotide-gated channels and the entry of Ca2+ into the cilia. Ca2+ activates a Cl− current that produces an efflux of Cl− ions and amplifies the depolarization. The molecular identity of Ca2+-activated Cl− channels is still elusive, although some bestrophins have been shown to function as Ca2+-activated Cl− channels when expressed in heterologous systems. In the olfactory epithelium, bestrophin-2 (Best2) has been indicated as a candidate for being a molecular component of the olfactory Ca2+-activated Cl− channel. In this study, we have analysed mice lacking Best2. We compared the electrophysiological responses of the olfactory epithelium to odorant stimulation, as well as the properties of Ca2+-activated Cl− currents in wild-type (WT) and knockout (KO) mice for Best2. Our results confirm that Best2 is expressed in the cilia of olfactory sensory neurons, while odorant responses and Ca2+-activated Cl− currents were not significantly different between WT and KO mice. Thus, Best2 does not appear to be the main molecular component of the olfactory channel. Further studies are required to determine the function of Best2 in the cilia of olfactory sensory neurons. PMID:19622610

  20. 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

  1. Differential distribution of glutamate- and GABA-gated chloride channels in the housefly Musca domestica.

    PubMed

    Kita, Tomo; Ozoe, Fumiyo; Azuma, Masaaki; Ozoe, Yoshihisa

    2013-09-01

    l-Glutamic acid (glutamate) mediates fast inhibitory neurotransmission by affecting glutamate-gated chloride channels (GluCls) in invertebrates. The molecular function and pharmacological properties of GluCls have been well studied, but not much is known about their physiological role and localization in the insect body. The distribution of GluCls in the housefly (Musca domestica L.) was thus compared with the distribution of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls). Quantitative PCR and ligand-binding experiments indicate that the GluCl and GABACl transcripts and proteins are predominantly expressed in the adult head. Intense GluCl immunostaining was detected in the lamina, leg motor neurons, and legs of adult houseflies. The GABACl (Rdl) immunostaining was more widely distributed, and was found in the medulla, lobula, lobula plate, mushroom body, antennal lobe, and ellipsoid body. The present findings suggest that GluCls have physiological roles in different tissues than GABACls. PMID:23806605

  2. The functional, oxygen-linked chloride binding sites of hemoglobin are contiguous within a channel in the central cavity.

    PubMed

    Ueno, H; Manning, J M

    1992-04-01

    Chloride ion is a major allosteric regulator for many hemoglobins and particularly for bovine hemoglobin. A site-directed reagent for amino groups, methyl acetyl phosphate, when used for global rather than selective modification of R (oxy) and T (deoxy) state bovine hemoglobin, can acetylate those functional amino groups involved in binding of chloride; the extensively acetylated hemoglobin tetramer retains nearly full cooperativity. The chloride-induced decrease in the oxygen affinity parallels the acetylation of bovine hemoglobin (i.e., their effects are mutually exclusive), suggesting that methyl acetyl phosphate is a good probe for the functional chloride binding sites in hemoglobins. Studies on the overall alkaline Bohr effect indicates that the part of the contribution dependent on chloride and reduced by 60% after acetylation is due to amino groups, Val-1(alpha) and Lys-81(beta); the remaining 40% is contributed by the imidazole side chain of His-146(beta), which is not acetylated by methyl acetyl phosphate, and is not dependent on chloride. The five amino groups--Val-1(alpha), Lys-99(alpha), Met-1(beta), Lys-81(beta), and Lys-103(beta)--of bovine hemoglobin that are acetylated in an oxygen-linked fashion are considered functional chloride binding sites. Molecular modeling indicates that these functional chloride binding sites are contiguous from one end of the central cavity of hemoglobin to the other; some of them are aligned within a chloride channel connecting each end of the dyad axis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1326985

  3. A GABA-activated chloride-conductance not blocked by picrotoxin on spiny lobster neuromuscular preparations.

    PubMed Central

    Albert, J.; Lingle, C. J.; Marder, E.; O'Neil, M. B.

    1986-01-01

    Conductance increases to gamma-aminobutyric acid (GABA) were recorded in the gm6b and opener muscle of the spiny lobsters, Panulirus interruptus and P. argus. GABA-evoked responses were insensitive to picrotoxin at concentrations as high as 5 X 10(-5) M. Some blockade by picrotoxin was observed at higher concentrations. In normal physiological saline, the reversal potential of the Panulirus GABA-induced response was near the resting potential. The reversal potential was unaffected by reductions in sodium and calcium. Reduction of chloride by 50% resulted in a greater than 10 mV shift in the reversal potential of the GABA-induced response. Muscimol was able to mimic the action of GABA while baclofen was without effect. Bicuculline was a weak blocker. Avermectin B1a irreversibly increased the chloride permeability of the gm6b membrane. This conductance increase was blocked by picrotoxin over a range of concentrations similar to those required for blockade of the GABA-induced response. GABA-induced responses of the gm6b muscle of Homarus americanus were blocked almost completely by picrotoxin 10(-6) M. Sensitivity to picrotoxin is not invariably associated with GABA-activated chloride-mediated conductance increases. It is suggested that alteration in the binding-site for picrotoxin on the GABA-activated chloride-ion channel does not change other functional characteristics of the GABA-induced response. PMID:3708210

  4. Chloride channel inhibition by the venom of the scorpion Leiurus quinquestriatus.

    PubMed

    DeBin, J A; Strichartz, G R

    1991-01-01

    The venom of the scorpion Leiurus quinquestriatus produced a significant, reversible inhibition of reconstituted Cl- channels of the small conductance type found in rat colonic epithelial cells. The kinetics of single-channel block by this venom were consistent with a first-order binding reaction in which the binding of one ligand molecule is sufficient to induce channel block. Single-channel mean block times were c.6 sec at -20 mV, and a KI in the submicromolar range is predicted. The active component has a mol. wt of roughly 5000 as judged by molecular sieve chromatography. PMID:1726031

  5. Cl- Channels in CF: Lack of Activation by Protein Kinase C and cAMP-Dependent Protein Kinase

    NASA Astrophysics Data System (ADS)

    Hwang, Tzyh-Chang; Lu, Luo; Zeitlin, Pamela L.; Gruenert, Dieter C.; Huganir, Richard; Guggino, William B.

    1989-06-01

    Secretory chloride channels can be activated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase in normal airway epithelial cells but not in cells from individuals with cystic fibrosis (CF). In excised, inside-out patches of apical membrane of normal human airway cells and airway cells from three patients with CF, the chloride channels exhibited a characteristic outwardly rectifying current-voltage relation and depolarization-induced activation. Channels from normal tissues were activated by both cAMP-dependent protein kinase and protein kinase C. However, chloride channels from CF patients could not be activated by either kinase. Thus, gating of normal epithelial chloride channels is regulated by both cAMP-dependent protein kinase and protein kinase C, and regulation by both kinases is defective in CF.

  6. Chloride channel inhibition by a red wine extract and a synthetic small molecule prevents rotaviral secretory diarrhoea in neonatal mice

    PubMed Central

    Ko, Eun-A; Jin, Byung-Ju; Namkung, Wan; Ma, Tonghui; Thiagarajah, Jay R.; Verkman, A. S.

    2014-01-01

    Background Rotavirus is the most common cause of severe secretory diarrhoea in infants and young children globally. The rotaviral enterotoxin, NSP4, has been proposed to stimulate calcium-activated chloride channels (CaCC) on the apical plasma membrane of intestinal epithelial cells. We previously identified red wine and small molecule CaCC inhibitors. Objective To investigate the efficacy of a red wine extract and a synthetic small molecule, CaCCinh-A01, in inhibiting intestinal CaCCs and rotaviral diarrhoea. Design Inhibition of CaCC-dependent current was measured in T84 cells and mouse ileum. The effectiveness of an orally administered wine extract and CaCCinh-A01 in inhibiting diarrhoea in vivo was determined in a neonatal mouse model of rotaviral infection. Results Screening of ~150 red wines revealed a Cabernet Sauvignon that inhibited CaCC current in T84 cells with IC50 at a ~1:200 dilution, and higher concentrations producing 100% inhibition. A >1 kdalton wine extract prepared by dialysis, which retained full inhibition activity, blocked CaCC current in T84 cells and mouse intestine. In rotavirus-inoculated mice, oral administration of the wine extract prevented diarrhoea by inhibition of intestinal fluid secretion without affecting rotaviral infection. The wine extract did not inhibit the cystic fibrosis chloride channel (CFTR) in cell cultures, nor did it prevent watery stools in neonatal mice administered cholera toxin, which activates CFTR-dependent fluid secretion. CaCCinh-A01 also inhibited rotaviral diarrhoea. Conclusions Our results support a pathogenic role for enterocyte CaCCs in rotaviral diarrhoea and demonstrate the antidiarrhoeal action of CaCC inhibition by an alcohol-free, red wine extract and by a synthetic small molecule. PMID:24052273

  7. Electrophysiological evidence for 4-isobutyl-3-isopropylbicyclophosphorothionate as a selective blocker of insect GABA-gated chloride channels.

    PubMed

    Akiyoshi, Yuki; Ju, Xiu-Lian; Furutani, Shogo; Matsuda, Kazuhiko; Ozoe, Yoshihisa

    2013-06-01

    Invertebrate γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and glutamate-gated chloride channels (GluCls), which function as inhibitory neurotransmitter receptors, are important targets of insecticides and antiparasitic agents. The antagonism of GABACls and GluCls by 4-isobutyl-3-isopropylbicyclophosphorothionate (PS-14) was examined in cultured cockroach and rat neurons using a whole-cell patch-clamp method. The results indicated that PS-14 selectively blocks cockroach GABACls relative to cockroach GluCls and rat GABACls. PS-14 represents a useful probe for the study of insect GABA receptors. PMID:23591113

  8. Inhibition of cystic fibrosis transmembrane conductance regulator chloride channel currents by arachidonic acid.

    PubMed

    Linsdell, P

    2000-06-01

    Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is inhibited by a number of different classes of organic anions which are able to enter and block the channel pore from its cytoplasmic end. Here I show, using patch clamp recording from CFTR-transfected baby hamster kidney cell lines, that the cis-unsaturated fatty acid arachidonic acid also inhibits CFTR Cl- currents when applied to the cytoplasmic face of excised membrane patches. This inhibition was of a relatively high affinity compared with other known CFTR inhibitors, with an apparent Kd of 6.5 +/- 0.9 microM. However, in contrast with known CFTR pore blockers, inhibition by arachidonic acid was only very weakly voltage dependent, and was insensitive to the extracellular Cl- concentration. Arachidonic acid-mediated inhibition of CFTR Cl- currents was not abrogated by inhibitors of lipoxygenases, cyclooxygenases or cytochrome P450, suggesting that arachidonic acid itself, rather than some metabolite, directly affects CFTR. Similar inhibition of CFTR Cl- currents was seen with other fatty acids, with the rank order of potency linoleic > or = arachidonic > or = oleic > elaidic > or = palmitic > or = myristic. These results identify fatty acids as novel high affinity modulators of the CFTR Cl- channel. PMID:10914639

  9. Cell volume-sensitive chloride channels: phenotypic properties and molecular identity.

    PubMed

    Okada, Yasunobu

    2006-01-01

    Cell volume regulation is essential for the survival of cells. After osmotic swelling, animal cells show a regulatory volume decrease by releasing intracellular K(+), Cl (-)and water. In most cell types, volume-regulatory Cl(-) efflux is induced by activation of electroconductive anion pathways. Among these volume-activated Cl(-) channels, the most important and specific is a volume-sensitive outwardly rectifying (VSOR) Cl(-) channel. The phonotypical properties have been well described. Extracellular application of anionic forms of ATP and glibenclamide give rise to voltage-dependent open-channel block of this channel, the fact suggesting that its outer vestibule and pore are larger and smaller, respectively, than the sizes of ATP and glibenclamide. Consistent with this prediction, the pore radius of VSOR Cl(-) channel (0.63 nm) which has been recently determined is slightly smaller than the radii of ATP and glibenclamide. The activities of VSOR Cl(-) channels are implicated not only in regulatory volume decrease but also in many other physiological or pathophysiological cell events including cell death induction. Despite their ubiquitous expression and physiological/ pathophysiological significance, there is still a paucity of the molecular information of the VSOR Cl(-) channel. PMID:17065805

  10. Thermally activated TRPV3 channels.

    PubMed

    Luo, Jialie; Hu, Hongzhen

    2014-01-01

    TRPV3 is a temperature-sensitive transient receptor potential (TRP) ion channel. The TRPV3 protein functions as a Ca(2+)-permeable nonselective cation channel with six transmembrane domains forming a tetrameric complex. TRPV3 is known to be activated by warm temperatures, synthetic small-molecule chemicals, and natural compounds from plants. Its function is regulated by a variety of physiological factors including extracellular divalent cations and acidic pH, intracellular adenosine triphosphate, membrane voltage, and arachidonic acid. TRPV3 shows a broad expression pattern in both neuronal and non-neuronal tissues including epidermal keratinocytes, epithelial cells in the gut, endothelial cells in blood vessels, and neurons in dorsal root ganglia and CNS. TRPV3 null mice exhibit abnormal hair morphogenesis and compromised skin barrier function. Recent advances suggest that TRPV3 may play critical roles in inflammatory skin disorders, itch, and pain sensation. Thus, identification of selective TRPV3 activators and inhibitors could potentially lead to beneficial pharmacological interventions in several diseases. The intent of this review is to summarize our current knowledge of the tissue expression, structure, function, and mechanisms of activation of TRPV3. PMID:25366242

  11. Novel muscle chloride channel mutations and their effects on heterozygous carriers

    SciTech Connect

    Mailaender, V.; Heine, R.; Deymeer, F.

    1996-02-01

    Mutations within CLCN1, the gene encoding the major skeletal muscle chloride channel, cause either dominant Thomsen disease or recessive Becker-type myotonia, which are sometimes difficult to discriminate, because of reduced penetrance or lower clinical expressivity in females. We screened DNA of six unrelated Becker patients and found four novel CLCN1 mutations (Gln-74-Stop, Tyr-150-Cys, Tyr-261-Cys, and Ala-415-Val) and a previously reported 14-bp deletion. Five patients were homozygous for the changes (Gln-74-Stop, Ala-41 5-Val, and 14-bp deletion), four of them due to parental consanguinity. The sixth patient revealed compound heterozygosity for Tyr-150-Cys and Tyr-261-Cys. Heterozygous carriers of the Becker mutations did not display any clinical symptoms of myotonia. However, all heterozygous males, but none of the heterozygous females, exhibited myotonic discharges in the electromyogram suggesting (1) a gene dosage effect of the mutations on the chloride conductance and (2) male predominance of subclinical myotonia. Furthermore, we report a novel Gly-200-Arg mutation resulting in a dominant phenotype in a male and a partially dominant phenotype in his mother. We discuss potential causes of the gender preference and the molecular mechanisms that may determine the mode of inheritance. 31 refs., 4 figs., 1 tab.

  12. Emerging role of cystic fibrosis transmembrane conductance regulator - an epithelial chloride channel in gastrointestinal cancers.

    PubMed

    Hou, Yuning; Guan, Xiaoqing; Yang, Zhe; Li, Chunying

    2016-03-15

    Cystic fibrosis transmembrane conductance regulator (CFTR), a glycoprotein with 1480 amino acids, has been well established as a chloride channel mainly expressed in the epithelial cells of various tissues and organs such as lungs, sweat glands, gastrointestinal system, and reproductive organs. Although defective CFTR leads to cystic fibrosis, a common genetic disorder in the Caucasian population, there is accumulating evidence that suggests a novel role of CFTR in various cancers, especially in gastroenterological cancers, such as pancreatic cancer and colon cancer. In this review, we summarize the emerging findings that link CFTR with various cancers, with focus on the association between CFTR defects and gastrointestinal cancers as well as the underlying mechanisms. Further study of CFTR in cancer biology may help pave a new way for the diagnosis and treatment of gastrointestinal cancers. PMID:26989463

  13. Disease-associated mutations in the extracytoplasmic loops of cystic fibrosis transmembrane conductance regulator do not impede biosynthetic processing but impair chloride channel stability.

    PubMed

    Hämmerle, M M; Aleksandrov, A A; Riordan, J R

    2001-05-01

    Consistent with its function as a chloride channel regulated entirely from the cytoplasmic side of the plasma membrane, the cystic fibrosis transmembrane conductance regulator (CFTR) glycoprotein exposes little of its mass on the exterior surface of cells. The first and fourth extracytoplasmic loops (ELs) contain approximately 15 and 30 residues, respectively; the other four ELs are extremely short. To examine the influence of missense mutants in ELs detected in patients with cystic fibrosis, we have expressed them in mammalian (baby hamster kidney (BHK21)) cells and assessed their biosynthetic processing and chloride channel activity. In contrast to previous findings that 18 of 30 disease-associated missense mutations in cytoplasmic loops caused retention of the nascent polypeptides in the endoplasmic reticulum, all the EL mutants studied matured and were transported to the cell surface. This pronounced asymmetry is consistent with the notion that endoplasmic reticulum quality control of nascent CFTR is exerted primarily on the cytoplasmic side of the membrane. Although this set of EL mutations has little effect on CFTR maturation, most of them seriously compromise its chloride channel activity. Substitutions at six different positions in EL1 and single positions in EL2 and EL4 all destabilized the open state, some of them severely, indicating that the ELs contribute to the stability of the CFTR ion pore. PMID:11278813

  14. 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. PMID:22234285

  15. Low-conductance chloride channels in IEC-6 and CF nasal cells expressing CFTR.

    PubMed

    Bijman, J; Dalemans, W; Kansen, M; Keulemans, J; Verbeek, E; Hoogeveen, A; De Jonge, H; Wilke, M; Dreyer, D; Lecocq, J P

    1993-03-01

    The properties of the cystic fibrosis gene product (CFTR) were studied by expression of cloned cDNA in different cell systems. Infection of both simian fibroblast (Vero) cells and immortalized CF nasal polyp cells (NCF3A) with a vaccinia virus encoding CFTR induced forskolin-induced Cl- permeability and low-conductance (8 pS) Cl- channels. By stable transfection of the rat intestinal crypt-derived cell line IEC-6 we have isolated a clone, IEC-CF7, which expresses CFTR mRNA and antigen. IEC-CF7 cells, but not IEC-6, display forskolin-induced Cl- permeability and multiple linear low-conductance (+/- 8 pS) Cl- channels in cell-attached membrane patches. In excised patches of IEC-CF7 cells, low-conductance Cl- channels could be activated by addition of the catalytic subunit of the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) plus ATP. During bath fluid replacement studies, the activated low-conductance channel remained active in the absence of ATP at room temperature and showed saturation kinetics. Rectifying (32 pS) Cl- channels were not observed in either IEC-6 cells or IEC-CF7 cells, indicating that there is no relation between CFTR expression and the incidence of this channel. Our data strongly support the conclusion that CFTR can act as a low-conductance Cl- channel, gated by PKA. The IEC-6-derived cell line IEC-CF7 may prove to be a useful model in the study of CFTR function because of the absence of 32-pS Cl- channel activity and its potential for differentiation. PMID:7681632

  16. Molecular Pharmacology of Kidney and Inner Ear CLC-K Chloride Channels

    PubMed Central

    Gradogna, Antonella; Pusch, Michael

    2010-01-01

    CLC-K channels belong to the CLC gene family, which comprises both Cl− channels and Cl−/H+ antiporters. They form homodimers which additionally co-assemble with the small protein barttin. In the kidney, they are involved in NaCl reabsorption; in the inner ear they are important for endolymph production. Mutations in CLC-Kb lead to renal salt loss (Bartter's syndrome); mutations in barttin lead additionally to deafness. CLC-K channels are interesting potential drug targets. CLC-K channel blockers have potential as alternative diuretics, whereas CLC-K activators could be used for the treatment of patients with Bartter's syndrome. Several small organic acids inhibit CLC-K channels from the outside by binding to a site in the external vestibule of the ion conducting pore. Benzofuran derivatives with affinities better than 10 μM have been discovered. Niflumic acid (NFA) exhibits a complex interaction with CLC-K channels. Below ∼1 mM, NFA activates CLC-Ka, whereas at higher concentrations NFA inhibits channel activity. The co-planarity of the rings of the NFA molecule is essential for its activating action. Mutagenesis has led to the identification of potential regions of the channel that interact with NFA. CLC-K channels are also modulated by pH and [Ca2+]ext. The inhibition at low pH has been shown to be mediated by a His-residue at the beginning of helix Q, the penultimate transmembrane helix. Two acidic residues from opposite subunits form two symmetrically related intersubunit Ca2+ binding sites, whose occupation increases channel activity. The relatively high affinity CLC-K blockers may already serve as leads for the development of useful drugs. On the other hand, the CLC-K potentiator NFA has a quite low affinity, and, being a non-steroidal anti-inflammatory drug, can be expected to exert significant side effects. More specific and more potent activators will be needed and it will be important to understand the molecular mechanisms that underlie NFA

  17. [Activity of Ca(2+)-dependent neutral proteinases in rat organs under cobalt and mercury chloride injection].

    PubMed

    Kaliman, P A; Samokhin, A A; Samokhina, L M

    2003-01-01

    The activity of Ca(2+)-dependent neutral proteinases in rats under cobalt and mercury chloride injection was investigated. The calpains activity increase in the lungs, heart, liver and kidneys was revealed after 2 h cobalt chloride action. The mercury chloride gives a reliable increase of calcium-dependent neutral proteinases only in the kidneys. PMID:14574747

  18. Regulation of mesangial chloride channels by insulin and glucose: role in diabetic nephropathy.

    PubMed

    Ling, B N

    1996-01-01

    1. In response to vasoactive peptides (e.g. angiotensin II (AngII), vasopressin, endothelin-1, platelet-activating factor), glomerular mesangial cell contraction is mediated through activation of a Ca2+-dependent Cl- conductance that, in turn, promotes membrane depolarization and voltage-activated Ca2+ entry. 2. Using patch clamp technology, our laboratory was the first to characterize a candidate Ca2+-dependent, 4 pS Cl- channel that is stimulated by vasoactive peptides in cultured rat mesangial cells. In the absence of extracellular insulin, the activation of Cl- channels by AngII is abolished. We find that Cl- channel sensitivity to intracellular Ca2+ and the membrane density of AngII receptors is also dependent on the presence of insulin. 3. Our studies also show that high extracellular glucose interferes with mesangial cell IP3 generation and Cl- channel stimulation. Importantly, we find that the insulin-dependency of Cl- channels occurs within the range of plasma insulin concentrations observed in normal, obese, hypertensive and diabetic humans (i.e. 1-100 mu U/mL). Similarly, normal regulation of Cl- channel activity is also modulated by glucose concentrations commonly observed in the plasma of diabetic humans (5-30 mmol/L). 4. There is substantial evidence, both in diabetic humans and animal models, that the provision of insulin and improved glycaemic control corrects or prevents glomerular hyperfiltration. The requirement for normal insulin and glucose levels, for the proper regulation of the 4 pS Cl- channel, provides a mechanism for impaired Ca2+ uptake and contraction observed in glomerular mesangial cells in association with insulin deficiency and hyperglocaemia. PMID:8713502

  19. Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel.

    PubMed

    Tsai, Ming-Feng; Li, Min; Hwang, Tzyh-Chang

    2010-05-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR's opening-closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N(3)-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N(3)-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1-NBD2 interface. The open state of CFTR has been shown to represent a two-ATP-bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a "partial NBD dimer" state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and "partial" separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR's NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed. PMID:20421370

  20. Functional Role of CLC-2 Chloride Inward Rectifier Channels in Cardiac Sinoatrial Nodal Pacemaker Cells

    PubMed Central

    Huang, Z. Maggie; Prasad, Chaithra; Britton, Fiona C.; Ye, Linda L.; Hatton, William J.; Duan, Dayue

    2009-01-01

    A novel Cl− inward rectifier channel (Cl,ir) encoded by ClC-2, a member of the ClC voltage-gated Cl− channel gene superfamily, has been recently discovered in cardiac myocytes of several species. However, the physiological role of Cl,ir channels in the heart remains unknown. In this study we tested the hypothesis that Cl,ir channels may play an important role in cardiac pacemaker activity. In isolated guinea-pig sinoatrial node (SAN) cells, Cl,ir current was activated by hyperpolarization and hypotonic cell swelling. RT-PCR and immunohistological analyses confirmed the molecular expression of ClC-2 in guinea-pig SAN cells. Hypotonic stress increased the diastolic depolarization slope and decreased the maximum diastolic potential, action potential amplitude, APD50, APD90, and the cycle-length of the SAN cells. These effects were largely reversed by intracellular dialysis of anti-ClC-2 antibody, which significantly inhibited Cl,ir current but not other pacemaker currents, including the hyperpolarization-activated non-selective cationic “funny” current (If), the L-type Ca2+ currents (ICa,L), the slowly-activating delayed rectifier IKs and the volume-regulated outwardly-rectifying Cl− current (ICl,vol). Telemetry electrocardiograph studies in conscious ClC-2 knockout (Clcn2−/−) mice revealed a decreased chronotropic response to acute exercise stress when compared to their age-matched Clcn2+/+ and Clcn2+/− littermates. Targeted inactivation of ClC-2 does not alter intrinsic heart rate but prevented the positive chronotropic effect of acute exercise stress through a sympathetic regulation of ClC-2 channels. These results provide compelling evidence that ClC-2-encoded endogenous Cl,ir channels may play an important role in the regulation of cardiac pacemaker activity, which may become more prominent under stressed or pathological conditions. PMID:19376127

  1. Ion channel modifying agents influence the electrical activity generated by canine intrinsic cardiac neurons in situ.

    PubMed

    Thompson, G W; Horackova, M; Armour, J A

    2000-04-01

    This study was designed to establish whether agents known to modify neuronal ion channels influence the behavior of mammalian intrinsic cardiac neurons in situ and, if so, in a manner consistent with that found previously in vitro. The activity generated by right atrial neurons was recorded extracellularly in varying numbers of anesthetized dogs before and during continuous local arterial infusion of several neuronal ion channel modifying agents. Veratridine (7.5 microM), the specific modifier of Na+-selective channels, increased neuronal activity (95% above control) in 80% of dogs tested (n = 25). The membrane depolarizing agent potassium chloride (40 mM) reduced neuronal activity (43% below control) in 84% of dogs tested (n = 19). The inhibitor of voltage-sensitive K+ channels, tetraethylammonium (10 mM), decreased neuronal activity (42% below control) in 73% of dogs tested (n = 11). The nonspecific potassium channel inhibitor barium chloride (5 mM) excited neurons (47% above control) in 13 of 19 animals tested. Cadmium chloride (200 microM), which inhibits Ca2+-selective channels and Ca2+-dependent K+ channels, increased neuronal activity (65% above control) in 79% of dogs tested (n = 14). The specific L-type Ca2+ channel blocking agent nifedipine (5 microM) reduced neuronal activity (52% blow control in 72% of 11 dogs tested), as did the nonspecific inhibitor of L-type Ca2+ channels, nickel chloride (5 mM) (36% below control in 69% of 13 dogs tested). Each agent induced either excitatory or inhibitory responses, depending on the agent tested. It is concluded that specific ion channels (I(Na), I(CaL), I(Kv), and I(KCa)) that have been associated with intrinsic cardiac neurons in vitro are involved in their capacity to generate action potentials in situ. PMID:10772056

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

    PubMed

    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

  3. 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

  4. Dual roles of plasmalemmal chloride channels in induction of cell death.

    PubMed

    Okada, Yasunobu; Maeno, Emi; Shimizu, Takahiro; Manabe, Kenichi; Mori, Shin-Ichiro; Nabekura, Takashi

    2004-06-01

    Even under anisotonic conditions, most cells can regulate their volume by mechanisms called regulatory volume decrease (RVD) and increase (RVI) after osmotic swelling or shrinkage, respectively. In contrast, the initial processes of necrosis and apoptosis are associated with persistent swelling and shrinkage. Necrotic volume increase (NVI) is initiated by uptake of osmolytes, such as Na+, Cl- and lactate, under conditions of injury, hypoxia, ischaemia, acidosis or lactacidosis. Persistence of NVI is caused by dysfunction of RVD due to impairment of volume-sensitive Cl- channels under conditions of ATP deficiency or lactacidosis. Both lactacidosis-induced RVD dysfunction and necrotic cell death are prevented by pretreatment of cells with the vacuolating cytotoxin-A (VacA) toxin protein purified from Helicobacter pylori, which forms a lactacidosis-resistant anion channel. Apoptotic volume decrease (AVD) is triggered by activation of K+ and Cl- conductances following stimulation with a mitochondrion-mediated or death receptor-mediated apoptosis inducer. Apoptotic cell death can be prevented by blocking the Cl- channels but not the K+-Cl- cotransporters. Thus, the volume regulatory anion channel plays, unless impaired, a cell-rescuing role in the necrotic process by ensuring RVD after swelling induced by necrotic insults, whereas normotonic activation of the anion channel plays a cell-killing role in the apoptotic process by triggering AVD following stimulation with apoptosis inducers. PMID:15103464

  5. Oscillatory chloride current evoked by temperature jumps during muscarinic and serotonergic activation in Xenopus oocyte.

    PubMed Central

    Miledi, R; Parker, I; Sumikawa, K

    1987-01-01

    the calcium-chelating agent EGTA. 7. Intracellular injection of inositol 1,4,5-trisphosphate evoked an oscillatory membrane current, during which Tjump responses developed similar to those after muscarinic activation. Intracellular injection of calcium evoked a chloride current, but this was not accompanied by Tjump responses. 8. We conclude that the oscillatory currents evoked by temperature jumps arise from chloride channels activated by intracellular calcium. This calcium is probably mobilized from intracellular stores by inositol trisphosphate which is liberated as a result of activation of muscarinic receptors, and also receptors for serotonin and glutamate.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2821235

  6. Cyclic nucleotide-activated channels in carp olfactory receptor cells.

    PubMed

    Kolesnikov, S S; Kosolapov, A V

    1993-07-25

    When applied from the cytoplasmic side, cyclic 3',5'-adenosine and guanosine monophosphates reversibly increased the ion permeability of inside-out patches of carp olfactory neuron plasma membrane. The cAMP (cGMP)-induced permeability via cAMP (cGMP) concentration was fitted by Hill's equation with the exponents of 1.07 +/- 0.15 (1.12 +/- 0.05) and EC50 = 1.3 +/- 0.6 microM (0.9 +/- 0.3 microM). Substitution of NaCl in the bathing solution by chlorides of other alkali metals resulted in a slight shift of reversal potential of the cyclic nucleotide-dependent (CN) current, which indicates a weak selectivity of the channels. Permeability coefficients calculated by Goldman-Hodgkin-Katz's equation corresponded to the following relation: PNa/PK/PLi/PRb/PCs = 1:0.98:0.94:0.70:0.61. Ca2+ and Mg2+ in physiological concentrations blocked the channels activated by cyclic nucleotides (CN-channels). In the absence of divalent cations the conductance of single CN-channels was equal to 51 +/- 9 pS in 100 mM NaCl solution. Channel density did not exceed 1 micron-2. The maximal open state probability of the channel (Po) tended towards 1.0 at a high concentration of cAMP or cGMP. Dichlorobenzamil decreased Po without changing the single CN-channel' conductance. CN-channels exhibited burst activity. Mean open and closed times as well as the burst duration depended on agonist concentration. A kinetic model with four states (an inactivated, a closed and two open ones) is suggested to explain the regularities of CN-channel gating and dose-response relations. PMID:8334139

  7. Molecular mechanism of arachidonic acid inhibition of the CFTR chloride channel.

    PubMed

    Zhou, Jing-Jun; Linsdell, Paul

    2007-06-01

    Arachidonic acid inhibits the activity of a number of different Cl- channels, however its molecular mechanism of action is not known. Here we show that inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by arachidonic acid is weakened following mutagenesis of two positively charged pore-lining amino acids. Charge-neutralizing mutants K95Q and R303Q both increased the Kd for inhibition from approximately 3.5 microM in wild type to approximately 17 microM. At both sites, the effects of mutagenesis were dependent of the charge of the substituted side chain. We suggest that arachidonic acid interacts electrostatically with positively charged amino acid side chains in the cytoplasmic vestibule of the CFTR channel pore to block Cl- permeation. PMID:17397825

  8. A provisional transport mechanism for a chloride channel-type Cl-/H+ exchanger.

    PubMed

    Miller, Christopher; Nguitragool, Wang

    2009-01-27

    Chloride channel (CLC)-type Cl-/H+ exchangers are widespread throughout the biological world, and one of these, CLC-ec1 from Escherichia coli, has been extensively studied. The structure of this protein is known, and several of its mechanistic hot spots have been identified, but a mechanism for Cl-/H+ exchange has not previously been offered. We herein confirm by direct measurements of Cl- and H+ fluxes a Cl--to-H+ exchange stoichiometry of 2, and summarize experimental facts pertinent to the exchange mechanism. While the mechanism must involve a conformational cycle of alternating exposure of substrate-binding sites to the two sides of the membrane, CLC transporters do not adhere to a familiar ping-pong scheme in which the two ions bind in a mutually exclusive fashion. Instead, Cl- and H+ occupy the ion-binding region simultaneously. A conformational cycle is proposed that accounts for the exchange stoichiometry, several key mutants and the tendency of the protein to become uncoupled and allow 'slippage' of Cl-. PMID:18977737

  9. Functional identification of a sarcolemmal chloride channel from bovine tracheal smooth muscle.

    PubMed

    Salvail, D; Alioua, A; Rousseau, E

    1996-11-01

    The biophysical and pharmacological characteristics of unitary Cl- currents from bovine tracheal smooth muscle cells were studied after reconstitution of microsomal vesicles into planar lipid bilayers. Two types of currents were recorded simultaneously in KCl buffer: the well-defined Ca(2+)-dependent K+ conductance [GK(Ca)] and a much smaller Cl- current, indicating that the Cl- channels under scrutiny originate from the same membrane as the GK(Ca)-type channels, the plasma membrane of airway smooth muscle (ASM) cells. The GK(Ca) activities were eliminated by the use of CsCl buffer. The average unitary Cl- conductance measured in 50 mM trans-250 mM cis CsCl was 77 +/- 6 pS (n = 21), and the reversal potential measured in various CsCl gradients followed the Cl- equilibrium potential as determined from the Nernst equation. In contrast with the previous reports describing the Ca2+ sensitivity of macroscopic ASM Cl- currents, this channel was found to be insensitive to cytoplasmic and extracellular Ca2+ levels. Phosphorylation cocktails, including protein kinases A, G, or C, did not alter the activity of the channel nor did changes in pH. Among a series of Cl- channel inhibitors, 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid [50% effective concentration (EC50) = 30 microM] and 5-nitro-2-(3-phenylpropylamino) benzoic acid (EC50 = 130 microM) were the most potent blockers of the current examined. The exact role of this surface Cl- conductance remains unclear, and its involvement in cellular activity needs further investigation. PMID:8944656

  10. Dibasic protein kinase A sites regulate bursting rate and nucleotide sensitivity of the cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed

    Mathews, C J; Tabcharani, J A; Chang, X B; Jensen, T J; Riordan, J R; Hanrahan, J W

    1998-04-15

    1. The relationship between phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and its gating by nucleotides was examined using the patch clamp technique by comparing strongly phosphorylated wild-type (WT) channels with weakly phosphorylated mutant channels lacking four (4SA) or all ten (10SA) dibasic consensus sequences for phosphorylation by protein kinase A (PKA). 2. The open probability (Po) of strongly phosphorylated WT channels in excised patches was about twice that of 4SA and 10SA channels, after correcting for the number of functional channels per patch by addition of adenylylimidodiphosphate (AMP-PNP). The mean burst durations of WT and mutant channels were similar, and therefore the elevated Po of WT was due to its higher bursting rate. 3. The ATP dependence of the 10SA mutant was shifted to higher nucleotide concentrations compared with WT channels. The relationship between Po and [ATP] was noticeably sigmoid for 10SA channels (Hill coefficient, 1.8), consistent with positive co-operativity between two sites. Increasing ATP concentration to 10 mM caused the Po of both WT and 10SA channels to decline. 4. Wild-type and mutant CFTR channels became locked in open bursts when exposed to mixtures of ATP and the non-hydrolysable analogue AMP-PNP. The rate at which the low phosphorylation mutants became locked open was about half that of WT channels, consistent with Po being the principal determinant of locking rate in WT and mutant channels. 5. We conclude that phosphorylation at 'weak' PKA sites is sufficient to sustain the interactions between the ATP binding domains that mediate locking by AMP-PNP. Phosphorylation of the strong dibasic PKA sites controls the bursting rate and Po of WT channels by increasing the apparent affinity of CFTR for ATP. PMID:9508802

  11. Dibasic protein kinase A sites regulate bursting rate and nucleotide sensitivity of the cystic fibrosis transmembrane conductance regulator chloride channel

    PubMed Central

    Mathews, Ceri J; Tabcharani, Joseph A; Chang, Xiu-Bao; Jensen, Timothy J; Riordan, John R; Hanrahan, John W

    1998-01-01

    The relationship between phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and its gating by nucleotides was examined using the patch clamp technique by comparing strongly phosphorylated wild-type (WT) channels with weakly phosphorylated mutant channels lacking four (4SA) or all ten (10SA) dibasic consensus sequences for phosphorylation by protein kinase A (PKA). The open probability (Po) of strongly phosphorylated WT channels in excised patches was about twice that of 4SA and 10SA channels, after correcting for the number of functional channels per patch by addition of adenylylimidodiphosphate (AMP-PNP). The mean burst durations of WT and mutant channels were similar, and therefore the elevated Po of WT was due to its higher bursting rate. The ATP dependence of the 10SA mutant was shifted to higher nucleotide concentrations compared with WT channels. The relationship between Po and [ATP] was noticeably sigmoid for 10SA channels (Hill coefficient, 1.8), consistent with positive co-operativity between two sites. Increasing ATP concentration to 10 mM caused the Po of both WT and 10SA channels to decline. Wild-type and mutant CFTR channels became locked in open bursts when exposed to mixtures of ATP and the non-hydrolysable analogue AMP-PNP. The rate at which the low phosphorylation mutants became locked open was about half that of WT channels, consistent with Po being the principal determinant of locking rate in WT and mutant channels. We conclude that phosphorylation at ‘weak’ PKA sites is sufficient to sustain the interactions between the ATP binding domains that mediate locking by AMP-PNP. Phosphorylation of the strong dibasic PKA sites controls the bursting rate and Po of WT channels by increasing the apparent affinity of CFTR for ATP. PMID:9508802

  12. Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel

    PubMed Central

    Tsai, Ming-Feng; Li, Min

    2010-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N3-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N3-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1–NBD2 interface. The open state of CFTR has been shown to represent a two-ATP–bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a “partial NBD dimer” state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and “partial” separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR’s NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed. PMID:20421370

  13. Impaired surface membrane insertion of homo- and heterodimeric human muscle chloride channels carrying amino-terminal myotonia-causing mutations

    PubMed Central

    Ronstedt, Katharina; Sternberg, Damien; Detro-Dassen, Silvia; Gramkow, Thomas; Begemann, Birgit; Becher, Toni; Kilian, Petra; Grieschat, Matthias; Machtens, Jan-Philipp; Schmalzing, Günther; Fischer, Martin; Fahlke, Christoph

    2015-01-01

    Mutations in the muscle chloride channel gene (CLCN1) cause myotonia congenita, an inherited condition characterized by muscle stiffness upon sudden forceful movement. We here studied the functional consequences of four disease-causing mutations that predict amino acid substitutions Q43R, S70L, Y137D and Q160H. Wild-type (WT) and mutant hClC-1 channels were heterologously expressed as YFP or CFP fusion protein in HEK293T cells and analyzed by whole-cell patch clamp and fluorescence recordings on individual cells. Q43R, Y137D and Q160H, but not S70L reduced macroscopic current amplitudes, but left channel gating and unitary current amplitudes unaffected. We developed a novel assay combining electrophysiological and fluorescence measurements at the single-cell level in order to measure the probability of ion channel surface membrane insertion. With the exception of S70L, all tested mutations significantly reduced the relative number of homodimeric hClC-1 channels in the surface membrane. The strongest effect was seen for Q43R that reduced the surface insertion probability by more than 99% in Q43R homodimeric channels and by 92 ± 3% in heterodimeric WT/Q43R channels compared to homodimeric WT channels. The new method offers a sensitive approach to investigate mutations that were reported to cause channelopathies, but display only minor changes in ion channel function. PMID:26502825

  14. BK channel activation: structural and functional insights

    PubMed Central

    Lee, Urvi S.; Cui, Jianmin

    2010-01-01

    The voltage and Ca2+ activated K+ (BK) channels are involved in the regulation of neurotransmitter release and neuronal excitability. Structurally, BK channels are homologous to voltage- and ligand-gated K+ channels, having a voltage sensor and pore as the membrane-spanning domain and a cytosolic domain containing metal binding sites. Recently published electron cryomicroscopy (cryo-EM) and X-ray crystallographic structures of the BK channel provided the first look into the assembly of these domains, corroborating the close interactions among these domains during channel gating that have been suggested by functional studies. This review discusses these latest findings and an emerging new understanding about BK channel gating and implications for diseases such as epilepsy, in which mutations in BK channel genes have been associated. PMID:20663573

  15. Elevated expression of chloride intracellular channel 1 is correlated with poor prognosis in human gliomas

    PubMed Central

    2012-01-01

    Background Chloride intracellular channel 1 (CLIC1) is expressed ubiquitously in human tissues and is involved in the regulation of cell cycle, cell proliferation and differentiation. Recent studies have shown that CLIC1 is highly expressed in several human malignant tumors. However, its roles in human gliomas are still unclear. The aim of this study was to investigate the clinicopathological significance and prognostic value of CLIC1 expression in human gliomas. Methods CLIC1 expression in human gliomas and nonneoplastic brain tissues was measured by real-time quantitative RT-PCR assay and immunohistochemistry. Its association with clinicopathological factors or prognosis in patients with gliomas was statistically analyzed. Results The expression of CLIC1 at both mRNA and protein levels was significantly increased in high-grade (Grade III~IV) glioma tissues compared with that in low-grade (Grade I~II) and nonneoplastic brain tissues, and was up-regulated with ascending tumor World Health Organization (WHO) grades. The elevated expression of CLIC1 protein was also significantly correlated with low Karnofsky performance score (KPS) (P=0.008). Moreover, both univariate and multivariate analysis shown that high CLIC1 expression was significantly associated with poor prognosis in patients with gliomas (P<0.001 and P=0.01, respectively). In particular, the elevated CLIC1 expression also correlated with shorter overall survival in different glioma subgroups stratified according to the WHO grading. Conclusions Our data provide the first evidence that CLIC1 expression might play an important role in the regulation of aggressiveness in human gliomas. The elevated expression of CLIC1 might represent a valuable prognostic marker for this disease. PMID:22578365

  16. Mutation-induced Blocker Permeability and Multiion Block of the CFTR Chloride Channel Pore

    PubMed Central

    Gong, Xiandi; Linsdell, Paul

    2003-01-01

    Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is blocked by a broad range of anions that bind tightly within the pore. Here we show that the divalent anion Pt(NO2)42− acts as an impermeant voltage-dependent blocker of the CFTR pore when added to the intracellular face of excised membrane patches. Block was of modest affinity (apparent Kd 556 μM), kinetically fast, and weakened by extracellular Cl− ions. A mutation in the pore region that alters anion selectivity, F337A, but not another mutation at the same site that has no effect on selectivity (F337Y), had a complex effect on channel block by intracellular Pt(NO2)42− ions. Relative to wild-type, block of F337A-CFTR was weakened at depolarized voltages but strengthened at hyperpolarized voltages. Current in the presence of Pt(NO2)42− increased at very negative voltages in F337A but not wild-type or F337Y, apparently due to relief of block by permeation of Pt(NO2)42− ions to the extracellular solution. This “punchthrough” was prevented by extracellular Cl− ions, reminiscent of a “lock-in” effect. Relief of block in F337A by Pt(NO2)42− permeation was only observed for blocker concentrations above 300 μM; as a result, block at very negative voltages showed an anomalous concentration dependence, with an increase in blocker concentration causing a significant weakening of block and an increase in Cl− current. We interpret this effect as reflecting concentration-dependent permeability of Pt(NO2)42− in F337A, an apparent manifestation of an anomalous mole fraction effect. We suggest that the F337A mutation allows intracellular Pt(NO2)42− to enter deeply into the CFTR pore where it interacts with multiple binding sites, and that simultaneous binding of multiple Pt(NO2)42− ions within the pore promotes their permeation to the extracellular solution. PMID:14610019

  17. Mutation-induced blocker permeability and multiion block of the CFTR chloride channel pore.

    PubMed

    Gong, Xiandi; Linsdell, Paul

    2003-12-01

    Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is blocked by a broad range of anions that bind tightly within the pore. Here we show that the divalent anion Pt(NO2)42- acts as an impermeant voltage-dependent blocker of the CFTR pore when added to the intracellular face of excised membrane patches. Block was of modest affinity (apparent Kd 556 microM), kinetically fast, and weakened by extracellular Cl- ions. A mutation in the pore region that alters anion selectivity, F337A, but not another mutation at the same site that has no effect on selectivity (F337Y), had a complex effect on channel block by intracellular Pt(NO2)42- ions. Relative to wild-type, block of F337A-CFTR was weakened at depolarized voltages but strengthened at hyperpolarized voltages. Current in the presence of Pt(NO2)42- increased at very negative voltages in F337A but not wild-type or F337Y, apparently due to relief of block by permeation of Pt(NO2)42- ions to the extracellular solution. This "punchthrough" was prevented by extracellular Cl- ions, reminiscent of a "lock-in" effect. Relief of block in F337A by Pt(NO2)42- permeation was only observed for blocker concentrations above 300 microM; as a result, block at very negative voltages showed an anomalous concentration dependence, with an increase in blocker concentration causing a significant weakening of block and an increase in Cl- current. We interpret this effect as reflecting concentration-dependent permeability of Pt(NO2)42- in F337A, an apparent manifestation of an anomalous mole fraction effect. We suggest that the F337A mutation allows intracellular Pt(NO2)42- to enter deeply into the CFTR pore where it interacts with multiple binding sites, and that simultaneous binding of multiple Pt(NO2)42- ions within the pore promotes their permeation to the extracellular solution. PMID:14610019

  18. Disruption of vascular Ca2+-activated chloride currents lowers blood pressure

    PubMed Central

    Heinze, Christoph; Seniuk, Anika; Sokolov, Maxim V.; Huebner, Antje K.; Klementowicz, Agnieszka E.; Szijártó, István A.; Schleifenbaum, Johanna; Vitzthum, Helga; Gollasch, Maik; Ehmke, Heimo; Schroeder, Björn C.; Hübner, Christian A.

    2014-01-01

    High blood pressure is the leading risk factor for death worldwide. One of the hallmarks is a rise of peripheral vascular resistance, which largely depends on arteriole tone. Ca2+-activated chloride currents (CaCCs) in vascular smooth muscle cells (VSMCs) are candidates for increasing vascular contractility. We analyzed the vascular tree and identified substantial CaCCs in VSMCs of the aorta and carotid arteries. CaCCs were small or absent in VSMCs of medium-sized vessels such as mesenteric arteries and larger retinal arterioles. In small vessels of the retina, brain, and skeletal muscle, where contractile intermediate cells or pericytes gradually replace VSMCs, CaCCs were particularly large. Targeted disruption of the calcium-activated chloride channel TMEM16A, also known as ANO1, in VSMCs, intermediate cells, and pericytes eliminated CaCCs in all vessels studied. Mice lacking vascular TMEM16A had lower systemic blood pressure and a decreased hypertensive response following vasoconstrictor treatment. There was no difference in contractility of medium-sized mesenteric arteries; however, responsiveness of the aorta and small retinal arterioles to the vasoconstriction-inducing drug U46619 was reduced. TMEM16A also was required for peripheral blood vessel contractility, as the response to U46619 was attenuated in isolated perfused hind limbs from mutant mice. Out data suggest that TMEM16A plays a general role in arteriolar and capillary blood flow and is a promising target for the treatment of hypertension. PMID:24401273

  19. BK channels: multiple sensors, one activation gate.

    PubMed

    Yang, Huanghe; Zhang, Guohui; Cui, Jianmin

    2015-01-01

    Ion transport across cell membranes is essential to cell communication and signaling. Passive ion transport is mediated by ion channels, membrane proteins that create ion conducting pores across cell membrane to allow ion flux down electrochemical gradient. Under physiological conditions, majority of ion channel pores are not constitutively open. Instead, structural region(s) within these pores breaks the continuity of the aqueous ion pathway, thereby serves as activation gate(s) to control ions flow in and out. To achieve spatially and temporally regulated ion flux in cells, many ion channels have evolved sensors to detect various environmental stimuli or the metabolic states of the cell and trigger global conformational changes, thereby dynamically operate the opening and closing of their activation gate. The sensors of ion channels can be broadly categorized as chemical sensors and physical sensors to respond to chemical (such as neural transmitters, nucleotides and ions) and physical (such as voltage, mechanical force and temperature) signals, respectively. With the rapidly growing structural and functional information of different types of ion channels, it is now critical to understand how ion channel sensors dynamically control their gates at molecular and atomic level. The voltage and Ca(2+) activated BK channels, a K(+) channel with an electrical sensor and multiple chemical sensors, provide a unique model system for us to understand how physical and chemical energy synergistically operate its activation gate. PMID:25705194

  20. BK channels: multiple sensors, one activation gate

    PubMed Central

    Yang, Huanghe; Zhang, Guohui; Cui, Jianmin

    2015-01-01

    Ion transport across cell membranes is essential to cell communication and signaling. Passive ion transport is mediated by ion channels, membrane proteins that create ion conducting pores across cell membrane to allow ion flux down electrochemical gradient. Under physiological conditions, majority of ion channel pores are not constitutively open. Instead, structural region(s) within these pores breaks the continuity of the aqueous ion pathway, thereby serves as activation gate(s) to control ions flow in and out. To achieve spatially and temporally regulated ion flux in cells, many ion channels have evolved sensors to detect various environmental stimuli or the metabolic states of the cell and trigger global conformational changes, thereby dynamically operate the opening and closing of their activation gate. The sensors of ion channels can be broadly categorized as chemical sensors and physical sensors to respond to chemical (such as neural transmitters, nucleotides and ions) and physical (such as voltage, mechanical force and temperature) signals, respectively. With the rapidly growing structural and functional information of different types of ion channels, it is now critical to understand how ion channel sensors dynamically control their gates at molecular and atomic level. The voltage and Ca2+ activated BK channels, a K+ channel with an electrical sensor and multiple chemical sensors, provide a unique model system for us to understand how physical and chemical energy synergistically operate its activation gate. PMID:25705194

  1. Physiological roles and diseases of tmem16/anoctamin proteins: are they all chloride channels?

    PubMed Central

    Duran, Charity; Hartzell, H Criss

    2011-01-01

    The Tmem16 gene family was first identified by bioinformatic analysis in 2004. In 2008, it was shown independently by 3 laboratories that the first two members (Tmem16A and Tmem16B) of this 10-gene family are Ca2+-activated Cl− channels. Because these proteins are thought to have 8 transmembrane domains and be anion-selective channels, the alternative name, Anoctamin (anion and octa=8), has been proposed. However, it remains unclear whether all members of this family are, in fact, anion channels or have the same 8-transmembrane domain topology. Since 2008, there have been nearly 100 papers published on this gene family. The excitement about Tmem16 proteins has been enhanced by the finding that Ano1 has been linked to cancer, mutations in Ano5 are linked to several forms of muscular dystrophy (LGMDL2 and MMD-3), mutations in Ano10 are linked to autosomal recessive spinocerebellar ataxia, and mutations in Ano6 are linked to Scott syndrome, a rare bleeding disorder. Here we review some of the recent developments in understanding the physiology and structure-function of the Tmem16 gene family. PMID:21642943

  2. Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel

    PubMed Central

    Qu, Zhiqiang; Hartzell, Criss

    2004-01-01

    Bestrophins have been proposed to constitute a new family of Cl channels that are activated by cytosolic Ca. We showed previously that mutation of serine-79 to cysteine in mouse bestrophin-2 (mBest2) altered the relative permeability and conductance to SCN. In this paper, we have overexpressed various mutant constructs of mBest2 in HEK-293 cells to explore the contributions to anion selectivity of serine-79 and other amino acids (V78, F80, G83, F84, V86, and T87) located in the putative second transmembrane domain (TMD2). Residues selected for mutagenesis were distributed throughout TMD2, but mutations at all positions changed the selectivity. The effects on selectivity were rather modest. Replacement of residues 78, 79, 80, 83, 84, 86, or 87 with cysteine had similar effects: the permeability of the channel to SCN relative to Cl (PSCN/PCl) was decreased three- to fourfold and the relative SCN conductance (GSCN/GCl) was increased five- to tenfold. Side chains at positions 78 and 80 appeared to be situated close to the permeant anion, because the electrostatic charge at these positions affected permeation in specific ways. The effects of charged sulfhydryl-reactive MTS reagents were the opposite in the V78C and F80C mutants and the effects were partially mimicked by substitution of F80 with charged amino acids. In S79T, switching from Cl to SCN caused slow changes in GSCN/GCl (τ = 16.6 s), suggesting that SCN binding to the channel altered channel gating as well as conductance. The data in this paper and other data support a model in which TMD2 plays an important role in forming the bestrophin pore. We suggest that the major determinant in anion permeation involves partitioning of the permeant anion into an aqueous pore whose structural features are rather flexible. Furthermore, anion permeation and gating may be linked. PMID:15452198

  3. Rapid recycling of ClC-2 chloride channels between plasma membrane and endosomes: role of a tyrosine endocytosis motif in surface retrieval.

    PubMed

    Cornejo, Isabel; Niemeyer, María Isabel; Zúñiga, Leandro; Yusef, Yamil R; Sepúlveda, Francisco V; Cid, L Pablo

    2009-12-01

    ClC-2 chloride channel is present in the brain and some transporting epithelia where its function is poorly understood. We have now demonstrated that the surface channels are rapidly internalised and approximately the 70% of the surface membrane protein recycles after 4- to 8-min internalisation. Endocytosis of ClC-2 was dependent upon tyrosine 179 located within an endocytic motif. Rapid recycling accompanied by an even faster internalisation could account for the abundant presence of ClC-2 in intracellular membranous structures. At least a proportion of ClC-2 resides in lipid rafts. Use of beta-cyclodextrin led to an increase in cell surface channel, but, surprisingly, a decrease in functionally active channels. We suggest that ClC-2 requires residing in beta-cyclodextrin sensitive clusters with other molecules in order to remain active. Regulation of ClC-2 trafficking to and within the membrane could be a means of modulating its activity. PMID:19711355

  4. The chloride-channel blocker 9-anthracenecarboxylic acid reduces the nonlinear capacitance of prestin-associated charge movement.

    PubMed

    Harasztosi, Csaba; Gummer, Anthony W

    2016-04-01

    The basis of the extraordinary sensitivity and frequency selectivity of the cochlea is a chloride-sensitive protein called prestin which can produce an electromechanical response and which resides in the basolateral plasma membrane of outer hair cells (OHCs). The compound 9-anthracenecarboxylic acid (9-AC), an inhibitor of chloride channels, has been found to reduce the electromechanical response of the cochlea and the OHC mechanical impedance. To elucidate these 9-AC effects, the functional electromechanical status of prestin was assayed by measuring the nonlinear capacitance of OHCs from the guinea-pig cochlea and of prestin-transfected human embryonic kidney 293 (HEK 293) cells. Extracellular application of 9-AC caused reversible, dose-dependent and chloride-sensitive reduction in OHC nonlinear charge transfer, Qmax . Prestin-transfected cells also showed reversible reduction in Qmax . For OHCs, intracellular 9-AC application as well as reduced intracellular pH had no detectable effect on the reduction in Qmax by extracellularly applied 9-AC. In the prestin-transfected cells, cytosolic application of 9-AC approximately halved the blocking efficacy of extracellularly applied 9-AC. OHC inside-out patches presented the whole-cell blocking characteristics. Disruption of the cytoskeleton by preventing actin polymerization with latrunculin A or by decoupling of spectrin from actin with diamide did not affect the 9-AC-evoked reduction in Qmax . We conclude that 9-AC acts on the electromechanical transducer principally by interaction with prestin rather than acting via the cytoskeleton, chloride channels or pH. The 9-AC block presents characteristics in common with salicylate, but is almost an order of magnitude faster. 9-AC provides a new tool for elucidating the molecular dynamics of prestin function. PMID:26869218

  5. 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

  6. 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. PMID:27241308

  7. Characterization of the putative chloride channel xClC-5 expressed in Xenopus laevis oocytes and comparison with endogenous chloride currents.

    PubMed

    Schmieder, S; Lindenthal, S; Banderali, U; Ehrenfeld, J

    1998-09-01

    1. We recently cloned a putative chloride channel (xClC-5) from the renal cell line A6, which induced the appearance of a Cl- conductance not found in control oocytes after homologous expression in Xenopus oocytes. With the aim of increasing the Xenopus oocyte xClC-5 expression, we constructed a new plasmid in which the native 5' and 3' non-coding regions of xClC-5 were replaced by the non-coding regions of the Xenopus beta-globin sequence and in which a Kozak consensus site was introduced before the initiator ATG. 2. We then compared the induced currents Inative (induced by injection of cRNA presenting the native non-coding regions of xClC-5) and Ibeta-globin (induced by injection of cRNA presenting the non-coding regions of the Xenopus beta-globin sequence) investigating anion selectivity and anion blocker sensitivity. Several differences were found: (1) expression yield and oocyte surviving rate were largely increased by injecting (beta) xClC-5 cRNA, (2) the Ibeta-globin outward rectification score was 2.6 times that of Inative, (3) the anion conductivity sequence was nitrate > bromide > chloride > iodide > gluconate for Ibeta-globin and iodide > bromide > nitrate > chloride > gluconate for Inative, (4) 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), anthracene-9-carboxylic acid (9-AC), DIDS, lanthanum ions, cAMP and ionomycin-induced [Ca2+]i increase inhibited Inative but had no effect on Ibeta-globin, and (5) Inative showed considerable similarity to the previously reported endogenous current appearing after ClC-6 or pICln cRNA injection. 3. Comparison of Inative with the endogenous chloride current ICl,swell which develops under hyposmotic conditions demonstrated several similarities in their electrophysiological and pharmacological characteristics but were nevertheless distinguishable. 4. In vitro translation assays demonstrated that protein synthesis was much greater using the (beta) xClC-5 construct than that of xClC-5. Furthermore, immunoreactivity

  8. Characterization of volume-activated chloride currents in regulatory volume decrease of human cholangiocyte.

    PubMed

    Chen, Biyi; Jefferson, Douglas M; Cho, Won Kyoo

    2010-05-01

    Volume-activated chloride channel (VACC) plays vital roles in many physiological functions. In bile duct epithelium, VACC actively participates in biliary secretion and cell volume regulation, and it mediates regulatory volume decrease (RVD). Recently, we have shown that mouse cholangiocytes have an intact RVD via VACC and K(+) conductance. However, such cell volume regulation was not studied in the normal human cholangiocyte. Volume measurement by Coulter counter and whole-cell patch clamp technique were used to characterize the RVD and VACC in human cholangiocyte cell line (HBDC). When exposed to hypotonic solution, HBDC exhibited an intact RVD, which was inhibited by 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), NPPB (5-nitro-2'- (3-phenylpropylamino)-benzoate), DIDS (4,4'-diisothiocyanatostilbene-2-disulfonic acid), and tamoxifen, but was not affected by the removal of extracellular calcium. During RVD, HBDC exhibited large, outwardly rectifying currents and time-dependent inactivation at positive potential. The amplitude of the outward current was approximately 3 times of that of the inward current, and this volume-activated current returned to the baseline when switched to isotonic solution. The amplitude and reversal potential of the volume-activated current was dependent on Cl(-) concentration, and the VACC was significantly inhibited by replacing chloride with gluconate, glutamate, sucrose, and acetate in the hypotonic solution. In addition, classical VACC inhibitors, such as NPPB or tamoxifen, inhibited the VACC. These inhibitory effects were reversible with washing out the inhibitors from the bath solution. The present study is the first to characterize and show that HBDC has an intact RVD, mediated by VACC, which has similar electrophysiological characteristics as that in mouse cholangiocytes. PMID:20411247

  9. AB095. Increased expression of TMEM16A/Ano1 chloride channel associated with diabetic erectile dysfunction

    PubMed Central

    Ruan, Yajun; Chen, Yingwei; Li, Mingchao; Wang, Tao; Yang, Jun; Rao, Ke; Wang, Shaogang; Yang, Weimin; Liu, Jihong; Ye, Zhangqun

    2016-01-01

    Objective To investigate the presence, location and functional role of TMEM16A/anotamin-1 (Ano1) calcium-activated chloride channel (CaCC) in the penile of rats with diabetic erectile dysfunction. Methods Eight-week-old male Sprague-Dawley (SD) rats were administrated streptozotocin (diabetic) or citrate buffer (control) randomly. Erectile function was measured by cavernous nerve electrostimulation at 12th week after diabetes was induced. The effect of Ano1 specific inhibitor—T16Ainh-A01 on intracavernous pressure (ICP) was evaluated. Then the penile tissues were harvested for molecular exploration. Real-time PCR and Western Blotting were used to assess the expression of Ano1 in penile tissues. Immunofluorescent labelling of penile tissue allowed localization of Ano1. Cavernous smooth muscle cell (CSMC) was cultured in high glucose medium. The change of Ano1 was measured using Western Blotting. The proliferation of CSMC was evaluated by cell counting kit-8 (CCK-8). Results Erectile function was impaired in diabetic rats. The expression of Ano1 was increased in rats with diabetic erectile dysfunction at mRNA and protein levels. Immunofluorescent labelling revealed the presence of Ano1 mainly in cavernous smooth muscle cells. The inhibition of Ano1 increased the ICP of DED rats. High glucose in vitro enhanced the proliferation of CSMC and the expression level of Ano1. Conclusions Ano1 is expressed in rat penile tissue and is increased with diabetes mellitus. The inhibition of Ano1 increased the ICP of DED rats. The alerted Ano1 may be associated with diabetic erectile dysfunction. It is a potential therapy target for ED in the future.

  10. 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.

  11. Active channel for Fanno Creek, Oregon

    USGS Publications Warehouse

    Sobieszczyk, Steven

    2011-01-01

    Fanno Creek is a tributary to the Tualatin River and flows though parts of the southwest Portland metropolitan area. The stream is heavily influenced by urban runoff and shows characteristic flashy streamflow and poor water quality commonly associated with urban streams. This data set represents the active, wetted channel as derived from light detection and ranging (LiDAR) data and aerial photographic imagery. The wetted channel boundary is equivalent to the extent of water observed during a 2-yr high flow event.

  12. 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

  13. 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. PMID:26048979

  14. The anti-MMP activity of benzalkonium chloride

    PubMed Central

    Tezvergil-Mutluay, Arzu; Mutluay, M. Murat; Gu, Li-sha; Zhang, Kai; Agee, Kelli A.; Carvalho, Ricardo M.; Manso, Adriana; Carrilho, Marcela; Tay, Franklin R.; Breschi, Lorenzo; Suh, Byoung-In; Pashley, David H.

    2013-01-01

    SUMMARY Objective This study evaluated the ability of benzalkonium chloride (BAC) to bind to dentine and to inhibit soluble recombinant MMPs and bound dentine matrix metalloproteinases (MMPs). Methods Dentine powder was prepared from extracted human molars. Half was left mineralized; the other half was completely demineralized. The binding of BAC to dentine powder was followed by measuring changes in the supernatant concentration using UV spectrometry. The inhibitory effects of BAC on rhMMP-2, -8 and -9 were followed using a commercially available in vitro proteolytic assay. Matrix-bound endogenous MMP-activity was evaluated in completely demineralized beams. Each beam was either dipped into BAC and then dropped into 1 mL of a complete medium (CM) or they were placed in 1 mL of CM containing BAC for 30 d. After 30 d, changes in the dry mass of the beams or in the hydroxyproline (HYP) content of hydrolyzates of the media were quantitated as indirect measures of matrix collagen hydrolysis by MMPs. Results Demineralized dentine powder took up 10-times more BAC than did mineralized powder. Water rinsing removed about 50% of the bound BAC, while rinsing with 0.5 M NaCl removed more than 90% of the bound BAC. BAC concentrations 0.5 wt% produced 100% inhibition of soluble recombinant MMP-2, -8 or -9, and inhibited matrix-bound MMPs between 55-66% when measured as mass loss or 76-81% when measured as solubilization of collagen peptide fragments. Conclusions BAC is effective at inhibiting both soluble recombinant MMPs and matrix-bound dentine MMPs in the absence of resins. PMID:20951183

  15. 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

  16. Mouse cystic fibrosis transmembrane conductance regulator forms cAMP-PKA-regulated apical chloride channels in cortical collecting duct.

    PubMed

    Lu, Ming; Dong, Ke; Egan, Marie E; Giebisch, Gerhard H; Boulpaep, Emile L; Hebert, Steven C

    2010-03-30

    The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in many segments of the mammalian nephron, where it may interact with and modulate the activity of a variety of apical membrane proteins, including the renal outer medullary potassium (ROMK) K(+) channel. However, the expression of CFTR in apical cell membranes or its function as a Cl(-) channel in native renal epithelia has not been demonstrated. Here, we establish that CFTR forms protein kinase A (PKA)-activated Cl(-) channels in the apical membrane of principal cells from the cortical collecting duct obtained from mice. These Cl(-) channels were observed in cell-attached apical patches of principal cells after stimulation by forskolin/3-isobutyl-1-methylxanthine. Quiescent Cl(-) channels were present in patches excised from untreated tubules because they could be activated after exposure to Mg-ATP and the catalytic subunit of PKA. The single-channel conductance, kinetics, and anion selectivity of these Cl(-) channels were the same as those of recombinant mouse CFTR channels expressed in Xenopus laevis oocytes. The CFTR-specific closed-channel blocker CFTR(inh)-172 abolished apical Cl(-) channel activity in excised patches. Moreover, apical Cl(-) channel activity was completely absent in principal cells from transgenic mice expressing the DeltaF508 CFTR mutation but was present and unaltered in ROMK-null mice. We discuss the physiologic implications of open CFTR Cl(-) channels on salt handling by the collecting duct and on the functional CFTR-ROMK interactions in modulating the metabolic ATP-sensing of ROMK. PMID:20231442

  17. Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore

    PubMed Central

    Wang, Wuyang; El Hiani, Yassine

    2011-01-01

    Different transmembrane (TM) α helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl− channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84–T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the

  18. Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore.

    PubMed

    Wang, Wuyang; El Hiani, Yassine; Linsdell, Paul

    2011-08-01

    Different transmembrane (TM) α helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl(-) channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84-T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore

  19. Halide permeation in wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels.

    PubMed

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

    1997-10-01

    Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22 degrees C or 8.7 pS at 37 degrees C. The current-voltage relationship became linear when patches were excised into symmetrical, -tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22 degrees C to 10.9 pS at 37 degrees C. The conductance at 22 degrees C was approximately 1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl activity was hyperbolic and well fitted by a Michaelis-Menten-type function having a of approximately 38 mM and maximum conductance of 10 pS at 22 degrees C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P/P = 0.003-0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P(1.8) > P(1. 3) > P(1.0) > P(0.17), consistent with a "weak field strength" selectivity site. The same sequence was obtained for external halides, although inward F flow was not observed. Iodide currents were protocol dependent and became blocked after 1-2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I/Cl permeability ratio (P/P< 0.4). The switch to low I permeability was enhanced at potentials that favored Cl entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl and I ions may influence I permeation and be

  20. Novel regulation of cystic fibrosis transmembrane conductance regulator (CFTR) channel gating by external chloride.

    PubMed

    Wright, Angela M; Gong, Xiandi; Verdon, Burns; Linsdell, Paul; Mehta, Anil; Riordan, John R; Argent, Barry E; Gray, Mike A

    2004-10-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is vital for Cl(-) and HCO(3)(-) transport in many epithelia. As the HCO(3)(-) concentration in epithelial secretions varies and can reach as high as 140 mm, the lumen-facing domains of CFTR are exposed to large reciprocal variations in Cl(-) and HCO(3)(-) levels. We have investigated whether changes in the extracellular anionic environment affects the activity of CFTR using the patch clamp technique. In fast whole cell current recordings, the replacement of 100 mm external Cl(-) ((Cl(o)(-))) with HCO(3)(-), Br(-), NO(3)(-), or aspartate(-) inhibited inward CFTR current (Cl(-) efflux) by approximately 50% in a reversible manner. Lowering Cl(o)(-) alone by iso-osmotic replacement with mannitol also reduced Cl(-) efflux to a similar extent. The maximal inhibition of CFTR current was approximately 70%. Raising cytosolic calcium shifted the Cl(-) dose-inhibition curve to the left but did not alter the maximal current inhibition observed. In contrast, a reduction in the internal [Cl(-)] neither inhibited CFTR nor altered the block caused by reduced Cl(o)(-). Single channel recordings from outside-out patches showed that lowering Cl(o)(-) markedly reduced channel open probability with little effect on unitary conductance. Together, these results indicate that alterations in Cl(o)(-) alone and not the Cl(-)/HCO(3)(-) ratio regulate the gating of CFTR. Physiologically, our data have implications for current models of epithelial HCO(3)(-) secretion and for the control of pH at epithelial cell surfaces. PMID:15286085

  1. Chloride intracellular channel protein CLIC4 (p64H1) binds directly to brain dynamin I in a complex containing actin, tubulin and 14-3-3 isoforms.

    PubMed Central

    Suginta, W; Karoulias, N; Aitken, A; Ashley, R H

    2001-01-01

    Mammalian chloride intracellular channel (CLIC) (p64-related) proteins are widely expressed, with an unusual dual localization as both soluble and integral membrane proteins. The molecular basis for their cellular localization and ion channel activity remains unclear. To help in addressing these problems, we identified novel rat brain CLIC4 (p64H1) binding partners by affinity chromatography, mass spectrometric analysis and microsequencing. Brain CLIC4 binds dynamin I, alpha-tubulin, beta-actin, creatine kinase and two 14-3-3 isoforms; the interactions are confirmed in vivo by immunoprecipitation. Gel overlay and reverse pull-down assays indicate that the binding of CLIC4 to dynamin I and 14-3-3zeta is direct. In HEK-293 cells, biochemical and immunofluorescence analyses show partial co-localization of recombinant CLIC4 with caveolin and with functional caveolae, which is consistent with a dynamin-associated role for CLIC4 in caveolar endocytosis. We speculate that brain CLIC4 might be involved in the dynamics of neuronal plasma membrane microdomains (micropatches) containing caveolin-like proteins and might also have other cellular roles related to membrane trafficking. Our results provide the basis for new hypotheses concerning novel ways in which CLIC proteins might be associated with cell membrane remodelling, the control of cell shape, and anion channel activity. PMID:11563969

  2. Lubiprostone activates non-CFTR-dependent respiratory epithelial chloride secretion in cystic fibrosis mice

    PubMed Central

    MacDonald, Kelvin D.; McKenzie, Karen R.; Henderson, Mark J.; Hawkins, Charles E.; Vij, Neeraj; Zeitlin, Pamela L.

    2008-01-01

    Periciliary fluid balance is maintained by the coordination of sodium and chloride channels in the apical membranes of the airways. In the absence of the cystic fibrosis transmembrane regulator (CFTR), chloride secretion is diminished and sodium reabsorption exaggerated. ClC-2, a pH- and voltage-dependent chloride channel, is present on the apical membranes of airway epithelial cells. We hypothesized that ClC-2 agonists would provide a parallel pathway for chloride secretion. Using nasal potential difference (NPD) measurements, we quantified lubiprostone-mediated Cl− transport in sedated cystic fibrosis null (gut-corrected), C57Bl/6, and A/J mice during nasal perfusion of lubiprostone (a putative ClC-2 agonist). Baseline, amiloride-inhibited, chloride-free gluconate-substituted Ringer with amiloride and low-chloride Ringer plus lubiprostone (at increasing concentrations of lubiprostone) were perfused, and the NPD was continuously recorded. A clear dose-response relationship was detected in all murine strains. The magnitude of the NPD response to 20 μM lubiprostone was −5.8 ± 2.1 mV (CF, n = 12), −8.1 ± 2.6 mV (C57Bl/6 wild-type, n = 12), and −5.3 ± 1.2 mV (AJ wild-type, n = 8). A cohort of ClC-2 knockout mice did not respond to 20 μM lubiprostone (n = 6, P = 0.27). In C57Bl/6 mice, inhibition of CFTR with topical application of CFTR inhibitor-172 did not abolish the lubiprostone response, thus confirming the response seen is independent of CFTR regulation. RT-PCR confirmed expression of ClC-2 mRNA in murine lung homogenate. The direct application of lubiprostone in the CF murine nasal airway restores nearly normal levels of chloride secretion in nasal epithelia. PMID:18805957

  3. 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

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

    PubMed

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

    2000-06-01

    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

  5. Mechanism of voltage-dependent gating in skeletal muscle chloride channels.

    PubMed Central

    Fahlke, C; Rosenbohm, A; Mitrovic, N; George, A L; Rüdel, R

    1996-01-01

    Voltage-dependent gating was investigated in a recombinant human skeletal muscle Cl- channel, hCIC-1, heterologously expressed in human embryonic kidney (HEK-293) cells. Gating was found to be mediated by two qualitatively distinct processes. One gating step operates on a microsecond time scale and involves the rapid rearrangement of two identical intramembranous voltage sensors, each consisting of a single titratable residue. The second process occurs on a millisecond time scale and is due to a blocking-unblocking reaction mediated by a cytoplasmic gate that interacts with the ion pore of the channel. These results illustrate a rather simple structural basis for voltage sensing that has evolved in skeletal muscle Cl- channels and provides evidence for the existence of a cytoplasmic gating mechanism in an anion channel analogous to the "ball and chain" mechanism of voltage-gated cation channels. Images FIGURE 3 PMID:8842208

  6. Iron-Catalyzed Activation of Chloride from Saline Media

    NASA Astrophysics Data System (ADS)

    Wittmer, Julian; Bleicher, Sergej; Ofner, Johannes; Zetzsch, Cornelius

    2015-04-01

    Iron (Fe) occurs in highly saline media (e.g. sea salt aerosol, salt brines etc.). High salinity, low pH, and organic constituents promote the dissolution of iron, forming photosensitive complexes that are responsible for a gaseous Cl production when irradiated by sunlight [1]. We studied the production of atomic Cl, Br and OH radicals from modeled salt pans [2] and artificial sea-salt aerosols containing Fe(III) ions or pyrogenic Fe2O3particles (Sicotrans Orange, BASF) at various compositions in a Teflon smog-chamber. The samples were either spread on a Teflon sheet or they were nebulized from dilute brines (most abundant particle diameter: ~0.4 μm, initial surface area: up to 3-10-2 cm2cm-3) and exposed to simulated sunlight at 60-90% relative humidity. The photochemical formation of Cl, OH and Br (if possible) in the gas phase was quantified by the radical clock method [3] resulting in time profiles of the radical-production rates and total productions. Simultaneous monitoring of the aerosol surface area enabled us to determine the initial Cl production rate per cm2 aerosol surface. Whereas no significant Cl production was detected employing a molar Cl-/Fe(III) ratio of 13, it increased to ~2.8-1017 atoms cm-2s-1 (at a ratio of 101), ~4.1-1017 atoms cm-2s-1(at a ratio of 53) and ~1.9-1018 atoms cm-2s-1(at a ratio of 13). The presence of NO2 (~20 ppb) or O3 (630 ppb) in the gas phase additionally increased the Fe(III)-induced chloride activation to ~7-1018 atoms cm-2s-1 and ~9-1018 atoms cm-2s-1(at a Cl-/Fe(III) ratio of 13), respectively. SO2 slightly restrained the Cl formation. Artificial sea salt aerosols doped with Fe2O3 (Cl-/Fetot ~13) did not result in detectable Cl concentrations. However, decreasing the pH below 2 favored the dissolution of Fe and led to Cl production rates comparable to the Fe(III) experiments. These observations are in accord with the speciation of the photolabile, aqueous Fe(III) complexes obtained from an equilibrium model (PHREEQC

  7. Arachidonic acid activation of a new family of K+ channels in cultured rat neuronal cells.

    PubMed Central

    Kim, D; Sladek, C D; Aguado-Velasco, C; Mathiasen, J R

    1995-01-01

    1. The presence and properties of K+ channels activated by arachidonic acid were studied in neuronal cells cultured from the mesencephalic and hypothalamic areas of rat brain. 2. Arachidonic acid produced a concentration-dependent (5-50 microM) and reversible activation of whole-cell currents. 3. In excised membrane patches, arachidonic acid applied to the cytoplasmic or extracellular side of the membrane caused opening of three types of channels whose current-voltage relationships were slightly outwardly rectifying, inwardly rectifying and linear, and whose single channel slope conductances at +60 mV were 143, 45 and 52 pS, respectively. 4. All three currents were K+ selective and blocked by 2 mM Ba2+ but not by other K+ channel blockers such as tetraethylammonium chloride, 4-aminopyridine and quinidine. The outwardly and inwardly rectifying currents were slightly voltage dependent with higher channel activity at more depolarized potentials. 5. Arachidonic acid activated the K+ channels in cells treated with cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and nordihydroguaiaretic acid), indicating that arachidonic acid itself can directly activate the channels. Alcohol and methyl ester derivatives of arachidonic acid failed to activate the K+ channels, indicating that the charged carboxyl group is important for activation. 6. Certain unsaturated fatty acids (linoleic, linolenic and docosahexaenoic acids), but not saturated fatty acids (myristic, palmitic, stearic acids), also reversibly activated all three types of K+ channel. 7. All three K+ channels were activated by pressure applied to the membrane (i.e. channels were stretch sensitive) with a half-maximal pressure of approximately 18 mmHg. The K+ channels were not blocked by 100 microM GdCl3. 8. A decrease in intracellular pH (over the range 5.6-7.2) caused a reversible, pH-dependent increase in channel activity whether the channel was initially activated by arachidonic acid or stretch. 9. Glutamate

  8. Evidence that extracellular anions interact with a site outside the CFTR chloride channel pore to modify channel properties.

    PubMed

    Zhou, Jing-Jun; Linsdell, Paul

    2009-05-01

    Extracellular anions enter into the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, interacting with binding sites on the pore walls and with other anions inside the pore. There is increasing evidence that extracellular anions may also interact with sites away from the channel pore to influence channel properties. We have used site-directed mutagenesis and patch-clamp recording to identify residues that influence interactions with external anions. Anion interactions were assessed by the ability of extracellular Pt(NO2)42- ions to weaken the pore-blocking effect of intracellular Pt(NO2)42- ions, a long-range ion-ion interaction that does not appear to reflect ion interactions inside the pore. We found that mutations that remove positive charges in the 4th extracellular loop of CFTR (K892Q and R899Q) significantly alter the interaction between extracellular and intracellular Pt(NO2)42- ions. These mutations do not affect unitary Cl- conductance or block of single-channel currents by extracellular Pt(NO2)42- ions, however, suggesting that the mutated residues are not in the channel pore region. These results suggest that extracellular anions can regulate CFTR pore properties by binding to a site outside the pore region, probably by a long-range conformational change. Our findings also point to a novel function of the long 4th extracellular loop of the CFTR protein in sensing and (or) responding to anions in the extracellular solution. PMID:19448737

  9. Mosquitocidal properties of IgG targeting the glutamate-gated chloride channel in three mosquito disease vectors (Diptera: Culicidae)

    PubMed Central

    Meyers, Jacob I.; Gray, Meg; Foy, Brian D.

    2015-01-01

    ABSTRACT 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

  10. Calcium-activated chloride currents in primary cultures of rabbit distal convoluted tubule.

    PubMed

    Bidet, M; Tauc, M; Rubera, I; de Renzis, G; Poujeol, C; Bohn, M T; Poujeol, P

    1996-10-01

    Chloride (Cl-) conductances were studied in primary cultures of rabbit distal convoluted tubule (very early distal "bright" convoluted tubule, DCTb) by the whole cell patch-clamp technique. We identified a Cl- current activated by 2 microM extracellular ionomycin. The kinetics of the macroscopic current were time dependent for depolarizing potentials with a slow developing component. The steady state current presented outward rectification, and the ion selectivity sequence was I- > Br- > > Cl > glutamate. The current was inhibited by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and 1 mM diphenylamine-2-carboxylate. To identify the location of the Cl- conductance, 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence experiments were carried out in confluent cultures developed on collagen-coated permeable filters. Cl- removal from the apical solution induced a Cl- efflux that was stimulated by 10 microM forskolin. Forskolin had no effect on the basolateral Cl- permeability Cl- substitution in the basolateral solution induced an efflux stimulated by 2 microM ionomycin or 50 microM extracellular ATP Ionomycin had no effect on the apical Cl- fluxes. Thus cultured DCTb cells exhibit Ca(2+)-activated Cl- channels located in the basolateral membrane. This Cl- permeability was active at a resting membrane potential and could participate in the Cl- reabsorption across the DCTb in control conditions. PMID:8898026

  11. Activation of chloride current by P2-purinoceptors in rat ventricular myocytes.

    PubMed Central

    Kaneda, M.; Fukui, K.; Doi, K.

    1994-01-01

    1. Rat ventricular myocytes were dissociated and their responses to extracellularly applied ATP were recorded using patch pipettes under the whole cell configuration. 2. ATP initially induced an inward current followed by an outward current at -50 mV. With a Cs-rich pipette solution the late outward current was blocked, leaving a sustained inward current (IATPs) suggesting that a K+ conductance underlies the late response. 3. When the extracellular Cl- concentration was changed, the reversal potential of IATPs corresponded well to the shift of the Cl- equilibrium potential. IATPs was reversibly blocked by the chloride channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). 4. The concentration-response curve of IATPs had a Hill coefficient of 0.98 and an EC50 value of 5.2 x 10(-6) M. 5. ATP was more potent than ADP, while AMP and adenosine were ineffective, suggesting that P2-purinoceptor activation induced IATPs. 6. The activation of IATPs was depressed by depleting the extracellular Mg2+ and increased by adding Mg2+. 7. Our results strongly suggest that P2-purinoceptor activation by ATP induces both a Cl(-)-conductance (IATPs) and a K(+)-conductance in rat ventricular myocytes. PMID:8032621

  12. Probing Oxygen Activation Sites in Two Flavoprotein Oxidases Using Chloride as an Oxygen Surrogate

    SciTech Connect

    Kommoju, Phaneeswara-Rao; Chen, Zhi-wei; Bruckner, Robert C.; Mathews, F. Scott; Jorns, Marilyn Schuman

    2011-08-16

    A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a preorganized binding site for superoxide anion, an obligatory intermediate in the two-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. First, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX-chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX-chloride complex and a ternary MSOX-chloride-MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is an effective probe of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.

  13. Halide Permeation in Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

    PubMed Central

    Tabcharani, Joseph A.; Linsdell, Paul; Hanrahan, John W.

    1997-01-01

    Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl− pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22°C or 8.7 pS at 37°C. The current–voltage relationship became linear when patches were excised into symmetrical, N-tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22°C to 10.9 pS at 37°C. The conductance at 22°C was ∼1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl− activity was hyperbolic and well fitted by a Michaelis-Menten–type function having a Km of ∼38 mM and maximum conductance of 10 pS at 22°C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (PNa/PCl = 0.003–0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated PI (1.8) > PBr (1.3) > PCl (1.0) > PF (0.17), consistent with a “weak field strength” selectivity site. The same sequence was obtained for external halides, although inward F− flow was not observed. Iodide currents were protocol dependent and became blocked after 1–2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I−/Cl− permeability ratio (PI/PCl < 0.4). The switch to low I− permeability was enhanced at potentials that favored Cl− entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl− and I− ions may influence I− permeation and be

  14. [Preparation and optimum process of walnut peel activated carbon by zinc chloride as activating agent].

    PubMed

    Liu, Xiao-hong; Wang, Xing-wei; Zhao, Bo; Lü, Jun-fang; Kang, Ni-na; Zhang, Yao-jun

    2014-12-01

    Walnut peel as raw material, zinc chloride was used as activating agent for preparation walnut peel activated carbon in the muffle furnace in this experiment, using orthogonal design. Yield, the specific surface area and iodine number of walnut peel activated carbon were determined at all designed experimental conditions and the optimum technological condition of preparation was obtained. By analysis of aperture, infrared spectra and the content of acidic group in surface with Boehm, walnut peel activated carbon of prepared at the optimum condition was characterized. The results showed the optimum technological parameters of preparation: activation temperature (600 °C), activation time (1 h), the concentration of zinc chloride (50%), the particle size (60 mesh). The specific surface area of walnut peel activated carbon obtained at optimum condition was mounting to 1258.05 m2 · g(-1), the ratio of medium porous 32.18%. Therefore, walnut peel can be used in the preparation of the high-quality activated carbon of large surface area. Agricultural wastes, as walnut peel, not only were implemented recycle, but also didn't make any pollution. Meanwhile, a cheap adsorbent was provided and it was of great significance to open a new source of activated carbon. PMID:25881437

  15. A family of acetylcholine-gated chloride channel subunits in Caenorhabditis elegans.

    PubMed

    Putrenko, Igor; Zakikhani, Mahvash; Dent, Joseph A

    2005-02-25

    The genome of the nematode Caenorhabditis elegans encodes a surprisingly large and diverse superfamily of genes encoding Cys loop ligand-gated ion channels. Here we report the first cloning, expression, and pharmacological characterization of members of a family of anion-selective acetylcholine receptor subunits. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. These channels are blocked by d-tubocurarine but not by alpha-bungarotoxin. We provide evidence that two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors. PMID:15579462

  16. 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. PMID:19381710

  17. Relationship between anion binding and anion permeability revealed by mutagenesis within the cystic fibrosis transmembrane conductance regulator chloride channel pore

    PubMed Central

    Linsdell, Paul

    2001-01-01

    Anion binding within the pores of wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels, expressed in two different mammalian cell lines, was assayed using patch clamp recording. Specifically, experiments measured both the conductance of different anions and the ability of other permeant anions to block Cl− permeation through the pore. Under symmetrical ionic conditions, wild-type CFTR channels showed the conductance sequence Cl− >NO3− >Br−≥formate >F− >SCN−≈ ClO4−. High SCN− conductance was not observed, nor was there an anomalous mole fraction effect of SCN− on conductance under the conditions used. Iodide currents could not be measured under symmetrical ionic conditions, but under bi-ionic conditions I− conductance appeared low. Chloride currents through CFTR channels were blocked by low concentrations (10 mM) of SCN−, I− and ClO4−, implying relatively tight binding of these anions within the pore. Two mutations in CFTR which alter the anion permeability sequence, F337S and T338A, also altered the anion conductance sequence. Furthermore, block by SCN−, I− and ClO4− were weakened in both mutants. Both these effects are consistent with altered anion binding within the pore. The effects of mutations on anion permeability and relative anion conductance suggested that, for most anions, increased permeability was associated with increased conductance. This indicates that the CFTR channel pore does not achieve its anion selectivity by selective anion binding within the mutated region. Instead, it is suggested that entry of anions into the region around F337 and T338 facilitates their passage through the pore. In wild-type CFTR channels, anion entry into this crucial pore region is probably dominated by anion hydration energies. PMID:11179391

  18. Relationship between anion binding and anion permeability revealed by mutagenesis within the cystic fibrosis transmembrane conductance regulator chloride channel pore.

    PubMed

    Linsdell, P

    2001-02-15

    1. Anion binding within the pores of wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, expressed in two different mammalian cell lines, was assayed using patch clamp recording. Specifically, experiments measured both the conductance of different anions and the ability of other permeant anions to block Cl- permeation through the pore. 2. Under symmetrical ionic conditions, wild-type CFTR channels showed the conductance sequence Cl- > NO3- > Br- > or = formate > F- > SCN- congruent to ClO4-. 3. High SCN- conductance was not observed, nor was there an anomalous mole fraction effect of SCN- on conductance under the conditions used. Iodide currents could not be measured under symmetrical ionic conditions, but under bi-ionic conditions I- conductance appeared low. 4. Chloride currents through CFTR channels were blocked by low concentrations (10 mM) of SCN-, I- and ClO4-, implying relatively tight binding of these anions within the pore. 5. Two mutations in CFTR which alter the anion permeability sequence, F337S and T338A, also altered the anion conductance sequence. Furthermore, block by SCN-, I- and ClO4- were weakened in both mutants. Both these effects are consistent with altered anion binding within the pore. 6. The effects of mutations on anion permeability and relative anion conductance suggested that, for most anions, increased permeability was associated with increased conductance. This indicates that the CFTR channel pore does not achieve its anion selectivity by selective anion binding within the mutated region. Instead, it is suggested that entry of anions into the region around F337 and T338 facilitates their passage through the pore. In wild-type CFTR channels, anion entry into this crucial pore region is probably dominated by anion hydration energies. PMID:11179391

  19. The role of ClC-3 in volume-activated chloride currents and volume regulation in bovine epithelial cells demonstrated by antisense inhibition

    PubMed Central

    Wang, Liwei; Chen, Lixin; Jacob, Tim J C

    2000-01-01

    A chloride current with mild outward rectification was induced in the native bovine non-pigmented ciliary epithelial (NPCE) cells by a 23 % hypotonic solution. The current showed no or little inactivation at depolarized steps. ATP blocked 88 and 61 % of the outward and inward components of the volume-activated chloride current (ICl,vol) with an IC50 of 5.3 and 9.6 mm, respectively. The volume-activated chloride current was decreased and the activation of the current was delayed by inhibiting endogenous ClC-3 expression using a ClC-3 antisense oligonucleotide. The inhibition of the current as a function of antisense concentration was asymptotic with a maximum about 60 %. The remaining current was probably not derived from ClC-3 and was inhibited by ATP. ClC-3 expression in the bovine NPCE cells was verified by immunofluorescence studies. ClC-3 immunofluorescence was distributed throughout the cells but with the predominant location within the nucleus. The expression of ClC-3 protein was diminished by the ClC-3 antisense oligonucleotide with the greatest diminution occurring in the nuclear region. The size of the volume-activated chloride current was positively correlated with the ClC-3 immunofluorescence level. Regulatory volume decrease of the NPCE cells was reduced by ClC-3 antisense oligonucleotide. We conclude that endogenous ClC-3 is associated with the volume-activated chloride current and is involved in cell volume regulation, but that it can only contribute towards a proportion of the current in NPCE cells. The nuclear predominance of ClC-3 immunofluorescence in NPCE cells, the absence of basal activity of chloride current and the marked pharmacological differences between IClC-3 and ICl,vol argue against ClC-3 being the only, or even the main, volume-activated chloride channel in NPCE cells. PMID:10747184

  20. An improved chloride-conducting channelrhodopsin for light-induced inhibition of neuronal activity in vivo

    PubMed Central

    Wietek, Jonas; Beltramo, Riccardo; Scanziani, Massimo; Hegemann, Peter; Oertner, Thomas G.; Simon Wiegert, J.

    2015-01-01

    Channelrhodopsins are light-gated cation channels that have been widely used for optogenetic stimulation of electrically excitable cells. Replacement of a glutamic acid in the central gate with a positively charged amino acid residue reverses the ion selectivity and produces chloride-conducting ChRs (ChloCs). Expressed in neurons, published ChloCs produced a strong shunting effect but also a small, yet significant depolarization from the resting potential. Depending on the state of the neuron, the net result of illumination might therefore be inhibitory or excitatory with respect to action potential generation. Here we report two additional amino acid substitutions that significantly shift the reversal potential of improved ChloC (iChloC) to the reversal potential of endogenous GABAA receptors. As a result, light-evoked membrane depolarization was strongly reduced and spike initiation after current injection or synaptic stimulation was reliably inhibited in iChloC-transfected neurons in vitro. In the primary visual cortex of anesthetized mice, activation of iChloC suppressed spiking activity evoked by visual stimulation. Due to its high operational light sensitivity, iChloC makes it possible to inhibit neurons in a large volume of brain tissue from a small, point-like light source. PMID:26443033

  1. Substrates of multidrug resistance-associated proteins block the cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed

    Linsdell, P; Hanrahan, J W

    1999-03-01

    1. The effects of physiological substrates of multidrug resistance-associated proteins (MRPs) on cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel currents were examined using patch clamp recording from CFTR-transfected mammalian cell lines. 2. Two MRP substrates, taurolithocholate-3-sulphate (TLCS) and beta-estradiol 17-(beta-D-glucuronide) (E217betaG) caused a voltage-dependent block of macroscopic CFTR Cl- currents when applied to the intracellular face of excised membrane patches, with mean apparent dissociation constants (KDs) of 96+/-10 and 563+/-103 microM (at 0 mV) respectively. The unconjugated bile salts taurocholate and cholate were also effective CFTR channel blockers under these conditions, with KDs of 453+/-44 and 3760+/-710 microM (at 0 mV) respectively. 3. Reducing the extracellular Cl- concentration from 154 to 20 mM decreased the KD for block intracellular TLCS to 54+/-1 microM, and also significantly reduced the voltage dependence of block, by suggesting that TLCS blocks Cl- permeation through CFTR by binding within the channel pore. 4. Intracellular TLCS reduced the apparent amplitude of CFTR single channel currents, suggesting that the duration of block is very fast compared to the gating of the channel. 5. The apparent affinity of block by TLCs is comparable to that of other well-known CFTR channel blockers, suggesting that MRP substrates may comprise a novel class of probes of the CFTR channel pore. 6. These results also suggest that the related proteins CFTR and MRP may share a structurally similar anion binding site at the cytoplasmic face of the membrane. PMID:10217542

  2. Substrates of multidrug resistance-associated proteins block the cystic fibrosis transmembrane conductance regulator chloride channel

    PubMed Central

    Linsdell, Paul; Hanrahan, John W

    1999-01-01

    The effects of physiological substrates of multidrug resistance-associated proteins (MRPs) on cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel currents were examined using patch clamp recording from CFTR-transfected mammalian cell lines. Two MRP substrates, taurolithocholate-3-sulphate (TLCS) and β-estradiol 17-(β-D-glucuronide) (E217βG) caused a voltage-dependent block of macroscopic CFTR Cl− currents when applied to the intracellular face of excised membrane patches, with mean apparent dissociation constants (KDs) of 96±10 and 563±103 μM (at 0 mV) respectively. The unconjugated bile salts taurocholate and cholate were also effective CFTR channel blockers under these conditions, with KDs of 453±44 and 3760±710 μM (at 0 mV) respectively. Reducing the extracellular Cl− concentration from 154 to 20 mM decreased the KD for block intracellular TLCS to 54±1 μM, and also significantly reduced the voltage dependence of block, by suggesting that TLCS blocks Cl− permeation through CFTR by binding within the channel pore. Intracellular TLCS reduced the apparent amplitude of CFTR single channel currents, suggesting that the duration of block is very fast compared to the gating of the channel. The apparent affinity of block by TLCs is comparable to that of other well-known CFTR channel blockers, suggesting that MRP substrates may comprise a novel class of probes of the CFTR channel pore. These results also suggest that the related proteins CFTR and MRP may share a structurally similar anion binding site at the cytoplasmic face of the membrane. PMID:10217542

  3. Decreased chloride channel expression in the dorsolateral prefrontal cortex in schizophrenia.

    PubMed

    Sullivan, Courtney R; Funk, Adam J; Shan, Dan; Haroutunian, Vahram; McCullumsmith, Robert E

    2015-01-01

    Alterations in GABAergic neurotransmission are implicated in several psychiatric illnesses, including schizophrenia. The Na-K-Cl and K-Cl cotransporters regulate intracellular chloride levels. Abnormalities in cotransporter expression levels could shift the chloride electrochemical gradient and impair GABAergic transmission. In this study, we performed Western blot analysis to investigate whether the Na-K-Cl and K-Cl cotransporter protein is abnormally expressed in the dorsal lateral prefrontal cortex and the anterior cingulate cortex in patients with schizophrenia versus a control group. We found decreased K-Cl cotransporter protein expression in the dorsal lateral prefrontal cortex, but not the anterior cingulate cortex, in subjects with schizophrenia, supporting the hypothesis of region level abnormal GABAergic function in the pathophysiology of schizophrenia. Subjects with schizophrenia off antipsychotic medication at the time of death had decreased K-Cl cotransporter protein expression compared to both normal controls and subjects with schizophrenia on antipsychotics. Our results provide evidence for KCC2 protein abnormalities in schizophrenia and suggest that antipsychotic medications might reverse deficits of this protein in the illness. PMID:25826365

  4. Decreased Chloride Channel Expression in the Dorsolateral Prefrontal Cortex in Schizophrenia

    PubMed Central

    Sullivan, Courtney R.; Funk, Adam J.; Shan, Dan; Haroutunian, Vahram; McCullumsmith, Robert E.

    2015-01-01

    Alterations in GABAergic neurotransmission are implicated in several psychiatric illnesses, including schizophrenia. The Na-K-Cl and K-Cl cotransporters regulate intracellular chloride levels. Abnormalities in cotransporter expression levels could shift the chloride electrochemical gradient and impair GABAergic transmission. In this study, we performed Western blot analysis to investigate whether the Na-K-Cl and K-Cl cotransporter protein is abnormally expressed in the dorsal lateral prefrontal cortex and the anterior cingulate cortex in patients with schizophrenia versus a control group. We found decreased K-Cl cotransporter protein expression in the dorsal lateral prefrontal cortex, but not the anterior cingulate cortex, in subjects with schizophrenia, supporting the hypothesis of region level abnormal GABAergic function in the pathophysiology of schizophrenia. Subjects with schizophrenia off antipsychotic medication at the time of death had decreased K-Cl cotransporter protein expression compared to both normal controls and subjects with schizophrenia on antipsychotics. Our results provide evidence for KCC2 protein abnormalities in schizophrenia and suggest that antipsychotic medications might reverse deficits of this protein in the illness. PMID:25826365

  5. Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo electroplax.

    PubMed Central

    Tank, D W; Miller, C; Webb, W W

    1982-01-01

    Small unilamellar vesicles formed from purified phospholids by detergent/dialysis methods may be enlarged to 30-microns diameter by freezing and thawing. Very-high-resistance seals were formed by applying a glass micropipette to the surface of these large liposomes, and single bilayer "patches" of membrane were isolated from the liposome surface while remaining sealed to the micropipette. The exogenous channel-forming peptides gramicidin and alamethicin induced characteristic single-channel fluctuation behavior in these excised patches held under voltage-clamp conditions. Large liposomes were formed from the small unilamellar vesicles made from cholate extracts of Torpedo electroplax plasma membrane vesicles. Isolated patches formed from these reconstituted membranes displayed current fluctuations due to single voltage-gated Cl- channels from non-innervated-face membranes; the properties of these Cl- channels are identical to those observed in planar bilayer membranes after direct insertion from native membranes. This liposome-patch method combines the advantages of membrane protein incorporation into liposomes with high-resolution electrical recording methods and may provide a generally applicable approach to the study of integral membrane channel proteins after solubilization and reconstitution. Images PMID:6296849

  6. Location of a permeant anion binding site in the cystic fibrosis transmembrane conductance regulator chloride channel pore.

    PubMed

    Rubaiy, Hussein N; Linsdell, Paul

    2015-05-01

    In the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, lyotropic anions with high permeability also bind relatively tightly within the pore. However, the location of permeant anion binding sites, as well as their relationship to anion permeability, is not known. We have identified lysine residue K95 as a key determinant of permeant anion binding in the CFTR pore. Lyotropic anion binding affinity is related to the number of positively charged amino acids located in the inner vestibule of the pore. However, mutations that change the number of positive charges in this pore region have minimal effects on anion permeability. In contrast, a mutation at the narrow pore region alters permeability with minimal effects on anion binding. Our results suggest that a localized permeant anion binding site exists in the pore; however, anion binding to this site has little influence over anion permeability. Implications of this work for the mechanisms of anion recognition and permeability in CFTR are discussed. PMID:25673337

  7. 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.

  8. CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.

    PubMed

    Schwiebert, E M; Egan, M E; Hwang, T H; Fulmer, S B; Allen, S S; Cutting, G R; Guggino, W B

    1995-06-30

    The cystic fibrosis transmembrane conductance regulator (CFTR) functions to regulate both Cl- and Na+ conductive pathways; however, the cellular mechanisms whereby CFTR acts as a conductance regulator are unknown. CFTR and outwardly rectifying Cl- channels (ORCCs) are distinct channels but are linked functionally via an unknown regulatory mechanism. We present results from whole-cell and single-channel patch-clamp recordings, short-circuit current recordings, and [gamma-32P]ATP release assays of normal, CF, and wild-type or mutant CFTR-transfected CF airway cultured epithelial cells wherein CFTR regulates ORCCs by triggering the transport of the potent agonist, ATP, out of the cell. Once released, ATP stimulates ORCCs through a P2U purinergic receptor-dependent signaling mechanism. Our results suggest that CFTR functions to regulate other Cl- secretory pathways in addition to itself conducting Cl-. PMID:7541313

  9. Relative contribution of different transmembrane segments to the CFTR chloride channel pore.

    PubMed

    Wang, Wuyang; El Hiani, Yassine; Rubaiy, Hussein N; Linsdell, Paul

    2014-03-01

    The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) α-helices, arranged in 2 symmetrical groups of 6. However, those TMs that line the channel pore are not completely defined. We used patch clamp recording to compare the accessibility of cysteine-reactive reagents to cysteines introduced into different TMs. Several residues in TM11 were accessible to extracellular and/or intracellular cysteine reactive reagents; however, no reactive cysteines were identified in TMs 5 or 11. Two accessible residues in TM11 (T1115C and S1118C) were found to be more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels, as previously reported for T338C in TM6. However, the effects of mutagenesis at S1118 (TM11) on a range of pore functional properties were relatively minor compared to the large effects of mutagenesis at T338 (TM6). Our results suggest that the CFTR pore is lined by TM11 but not by TM5 or TM7. Comparison with previous works therefore suggests that the pore is lined by TMs 1, 6, 11, and 12, suggesting that the structure of the open channel pore is asymmetric in terms of the contributions of different TMs. Although TMs 6 and 11 appear to undergo similar conformational changes during channel opening and closing, the influence of these two TMs on the functional properties of the narrowest region of the pore is clearly unequal. PMID:23955087

  10. 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. PMID:25892339

  11. Gating of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels by Adenosine Triphosphate Hydrolysis

    PubMed Central

    Zeltwanger, Shawn; Wang, Fei; Wang, Guo-Tang; Gillis, Kevin D.; Hwang, Tzyh-Chang

    1999-01-01

    Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) involves a coordinated action of ATP on two nucleotide binding domains (NBD1 and NBD2). Previous studies using nonhydrolyzable ATP analogues and NBD mutant CFTR have suggested that nucleotide hydrolysis at NBD1 is required for opening of the channel, while hydrolysis of nucleotides at NBD2 controls channel closing. We studied ATP-dependent gating of CFTR in excised inside-out patches from stably transfected NIH3T3 cells. Single channel kinetics of CFTR gating at different [ATP] were analyzed. The closed time constant (τc) decreased with increasing [ATP] to a minimum value of ∼0.43 s at [ATP] >1.00 mM. The open time constant (τo) increased with increasing [ATP] with a minimal τo of ∼260 ms. Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBD2, reveals the presence of two open states. A short open state with a time constant of ∼250 ms is dominant at low ATP concentrations (10 μM) and a much longer open state with a time constant of ∼3 min is present at millimolar ATP. These data suggest that nucleotide binding and hydrolysis at NBD1 is coupled to channel opening and that the channel can close without nucleotide interaction with NBD2. A quantitative cyclic gating scheme with microscopic irreversibility was constructed based on the kinetic parameters derived from single-channel analysis. The estimated values of the kinetic parameters suggest that NBD1 and NBD2 are neither functionally nor biochemically equivalent. PMID:10102935

  12. Functional evaluation of human ClC-2 chloride channel mutations associated with idiopathic generalized epilepsies.

    PubMed

    Niemeyer, María Isabel; Yusef, Yamil R; Cornejo, Isabel; Flores, Carlos A; Sepúlveda, Francisco V; Cid, L Pablo

    2004-09-16

    The ClC-2 Cl- channel has been postulated to play a role in the inhibitory GABA response in neurons or to participate in astrocyte-dependent extracellular electrolyte homeostasis. Three different mutations in the CLCN2 gene, encoding the voltage-dependent homodimeric ClC-2 channel, have been associated with idiopathic generalized epilepsy (IGE). We study their function in vitro by patch clamp and confocal microscopy in transiently transfected HEK-293 cells. A first mutation predicts a premature stop codon (M200fsX231). An altered splicing, due to an 11-bp deletion in intron 2 (IVS2-14del11), predicts exon 3 skipping (Delta74-117). A third is a missense mutation (G715E). M200fsX231 and Delta74-117 are nonfunctional and do not affect the function of the normal (wild type, WT) channel. Neither M200fsX231 nor Delta74-117 reach the plasma membrane. Concerning the IVS2-14del11 mutation, we find no difference in the proportion of exon-skipped to normally spliced mRNA using a minigene approach and, on this basis, predict no alteration in channel expression in affected individuals. G715E has voltage dependence and intracellular Cl- dependence indistinguishable from WT channels. ClC-2 channels are shown to be sensitive to intracellular replacement of ATP by AMP, which accelerates the opening and closing kinetics. This effect is diminished in the G715E mutant and not significant in WT+G715E coexpression. We do not know whether, in a situation of cellular ATP depletion, this might become pathological in individuals carrying the mutation. We postulate that loss of function mutation M200fsX231 of ClC-2 might contribute to the IGE phenotype through a haploinsufficiency mechanism. PMID:15252188

  13. Volume-sensitive outwardly rectifying chloride channels are involved in oxidative stress-induced apoptosis of mesangial cells

    SciTech Connect

    Jiao Jundong; Xu Chaoqian; Yue Peng; Dong Deli; Li Zhe; Du Zhimin; Yang Baofeng . E-mail: yangbf@ems.hrbmu.edu.cn

    2006-02-03

    Volume-sensitive outwardly rectifying (VSOR) Cl{sup -} channels have been electrophysiologically identified in human and mouse mesangial cells, but the functional role of VSOR Cl{sup -} channels in mesangial cell apoptosis is not clear. The aim of the present study was to demonstrate the role of VSOR Cl{sup -} channels in oxidative stress-induced mesangial cell apoptosis. H{sub 2}O{sub 2}-induced Cl{sup -} currents showed phenotypic properties of VSOR Cl{sup -} channels, including outward rectification, voltage-dependent inactivation at more positive potentials, sensitivity to hyperosmolarity, and inhibition by VSOR Cl{sup -} channel blockers. Moreover, blockage of VSOR Cl{sup -} channels by DIDS (100 {mu}M), NPPB (10 {mu}M) or niflumic acid (10 {mu}M) rescued mesangial cell apoptosis induced by H{sub 2}O{sub 2}. Treatment with 150 {mu}M H{sub 2}O{sub 2} for 2 h resulted in significant reduction of cell volume, in contrast, nuclear condensation and/or fragmentation were not observed and the caspase-3 activity was also not increased. The early-phase alterations in cell volume were markedly abolished by pretreatment with VSOR Cl{sup -} channel blockers. We conclude that VSOR Cl{sup -}channels are involved in H{sub 2}O{sub 2}-induced apoptosis in cultured mesangial cells and its mechanism is associated with apoptotic volume decrease processes.

  14. Activity coefficients of aqueous sodium chloride from 15° to 50°C measured with a glass electrode

    USGS Publications Warehouse

    Truesdell, A.H.

    1968-01-01

    Values of the mean activity coefficient of sodium chloride at 15°, 25°, 38° and 50°C were determined for aqueous NaCl solutions of 0.01 to 1.0 molal from electromotive force measurements on the cell: (sodium-sensitive glass electrode, aqueous sodium chloride, silver chloride-silver).

  15. Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore

    PubMed Central

    Zhou, Jing-Jun; Li, Man-Song; Qi, Jiansong

    2010-01-01

    Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be “transplanted” to a residue in a different transmembrane (TM) region (S1141). Thus, the mutant channel K95S/S1141K showed Cl− conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby “rescuing” the effects of the charge-neutralizing K95S mutation. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). The S1141K mutant had similar Cl− conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO2)42− in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl− channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl− transport through a

  16. Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore.

    PubMed

    Zhou, Jing-Jun; Li, Man-Song; Qi, Jiansong; Linsdell, Paul

    2010-03-01

    Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be "transplanted" to a residue in a different transmembrane (TM) region (S1141). Thus, the mutant channel K95S/S1141K showed Cl(-) conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby "rescuing" the effects of the charge-neutralizing K95S mutation. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). The S1141K mutant had similar Cl(-) conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO(2))(4)(2-) in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl(-) channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl(-) transport through a

  17. Susceptibility of channel catfish (Ictalurus punctatus) fed dietary sodium chloride to nitrite toxicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Juvenile channel catfish (Ictalurus punctatus) were fed nutritionally complete, practical basal diets supplemented with NaCl at 0, 1, 2, or 4 % of diet to apparent satiation twice daily for 10 weeks. Catfish were exposed to nitrite after six (7.70 mg/l nitrite-N) and ten (7.18 mg/l nitrite-N) weeks ...

  18. The tyrosine kinase p60c-src regulates the fast gate of the cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed Central

    Fischer, H; Machen, T E

    1996-01-01

    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. PMID:8968578

  19. 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.

  20. 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

    may attenuate D-[3H]aspartate release by regulation of mitochondrial Ca2+ sequestration and by activation of a chloride channel, but not that governed by GABA(A) or strychnine-sensitive glycine receptors. PMID:17173871

  1. 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

    2016-01-01

    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. PMID:27294913

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

    PubMed Central

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

    2016-01-01

    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. PMID:27294913

  3. Thiocyanate as a probe of the cystic fibrosis transmembrane conductance regulator chloride channel pore.

    PubMed

    Linsdell, P

    2001-07-01

    Immediately following exposure to thiocyanate (SCN-)-containing solutions, the cystic fibrosis conductance regulator Cl- channel exhibits high unitary SCN conductance and anomalous mole fraction behaviour, suggesting the presence of multiple anion binding sites within the channel pore. However, under steady-state conditions SCN-conductance is very low. Here I show, using patch clamp recording from CFTR-transfected mammalian cell lines, that under steady-state conditions neither SCN- conductance nor SCN- permeability show anomalous mole fraction behaviour. Instead, SCN conductance, permeability, and block of Cl- permeation can all be reproduced by a rate theory model that assumes only a single intrapore anion binding site. These results suggest that under steady-state conditions the interaction between SCN- and the CFTR channel pore can be understood by a simple model whereby SCN- ions enter the pore more easily than Cl-, and bind within the pore more tightly than Cl-. The implications of these findings for investigating and understanding the mechanism of anion permeation are discussed. PMID:11478590

  4. Tuning of CFTR Chloride Channel Function by Location of Positive Charges within the Pore

    PubMed Central

    El Hiani, Yassine; Linsdell, Paul

    2012-01-01

    High unitary Cl− conductance in the cystic fibrosis transmembrane conductance regulator Cl− channel requires a functionally unique, positively charged lysine residue (K95) in the inner vestibule of the channel pore. Here we used a mutagenic approach to investigate the ability of other sites in the pore to host this important positive charge. The loss of conductance observed in the K95Q mutation was >50% rescued by substituting a lysine for each of five different pore-lining amino acids, suggesting that the exact location of the fixed positive charge is not crucial to support high conductance. Moving the positive charge also restored open-channel blocker interactions that are lost in K95Q. Introducing a second positive charge in addition to that at K95 did not increase conductance at any site, but did result in a striking increase in the strength of block by divalent Pt(NO2)42− ions. Based on the site dependence of these effects, we propose that although the exact location of the positive charge is not crucial for normal pore properties, transplanting this charge to other sites results in a diminution of its effectiveness that appears to depend on its location along the axis of the pore. PMID:23083715

  5. Tuning of CFTR chloride channel function by location of positive charges within the pore.

    PubMed

    El Hiani, Yassine; Linsdell, Paul

    2012-10-17

    High unitary Cl(-) conductance in the cystic fibrosis transmembrane conductance regulator Cl(-) channel requires a functionally unique, positively charged lysine residue (K95) in the inner vestibule of the channel pore. Here we used a mutagenic approach to investigate the ability of other sites in the pore to host this important positive charge. The loss of conductance observed in the K95Q mutation was >50% rescued by substituting a lysine for each of five different pore-lining amino acids, suggesting that the exact location of the fixed positive charge is not crucial to support high conductance. Moving the positive charge also restored open-channel blocker interactions that are lost in K95Q. Introducing a second positive charge in addition to that at K95 did not increase conductance at any site, but did result in a striking increase in the strength of block by divalent Pt(NO(2))(4)(2-) ions. Based on the site dependence of these effects, we propose that although the exact location of the positive charge is not crucial for normal pore properties, transplanting this charge to other sites results in a diminution of its effectiveness that appears to depend on its location along the axis of the pore. PMID:23083715

  6. Permeability of Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels to Polyatomic Anions

    PubMed Central

    Linsdell, Paul; Tabcharani, Joseph A.; Rommens, Johanna M.; Hou, Yue-Xian; Chang, Xiu-Bao; Tsui, Lap-Chee; Riordan, John R.; Hanrahan, John W.

    1997-01-01

    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 (PX/PCl) sequence NO3− > Cl− > HCO3− > 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 (PX/PCl < 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 ∼5.3 Å. 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. PMID:9379168

  7. 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. PMID:9379168

  8. Determination of the functional unit of the cystic fibrosis transmembrane conductance regulator chloride channel. One polypeptide forms one pore.

    PubMed

    Zhang, Zhi-Ren; Cui, Guiying; Liu, Xuehong; Song, Binlin; Dawson, David C; McCarty, Nael A

    2005-01-01

    The magnitudes and distributions of subconductance states were studied in chloride channels formed by the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and in CFTRs bearing amino acid substitutions in transmembrane segment 6. Within an open burst, it was possible to distinguish three distinct conductance states referred to as the full conductance, subconductance 1, and subconductance 2 states. Amino acid substitutions in transmembrane segment 6 altered the duration and probability of occurrence of these subconductance states but did not greatly alter their relative amplitudes. Results from real time measurements indicated that covalent modification of single R334C-CFTR channels by [2-(trimethylammonium)ethyl]methanethiosulfonate resulted in the simultaneous modification of all three conductance levels in what appeared to be a single step, without changing the proportion of time spent in each state. This behavior suggests that at least a portion of the conduction path is common to all three conducting states. The time course for the modification of R334C-CFTR, measured in outside-out macropatches using a rapid perfusion system, was also consistent with a single modification step as if each pore contained only a single copy of the cysteine at position 334. These results are consistent with a model for the CFTR conduction pathway in which a single anion-conducting pore is formed by a single CFTR polypeptide. PMID:15504728

  9. Epithelial sodium channel modulates platelet collagen activation.

    PubMed

    Cerecedo, Doris; Martínez-Vieyra, Ivette; Alonso-Rangel, Lea; Benítez-Cardoza, Claudia; Ortega, Arturo

    2014-03-01

    Activated platelets adhere to the exposed subendothelial extracellular matrix and undergo a rapid cytoskeletal rearrangement resulting in shape change and release of their intracellular dense and alpha granule contents to avoid hemorrhage. A central step in this process is the elevation of the intracellular Ca(2+) concentration through its release from intracellular stores and on throughout its influx from the extracellular space. The Epithelial sodium channel (ENaC) is a highly selective Na(+) channel involved in mechanosensation, nociception, fluid volume homeostasis, and control of arterial blood pressure. The present study describes the expression, distribution, and participation of ENaC in platelet migration and granule secretion using pharmacological inhibition with amiloride. Our biochemical and confocal analysis in suspended and adhered platelets suggests that ENaC is associated with Intermediate filaments (IF) and with Dystrophin-associated proteins (DAP) via α-syntrophin and β-dystroglycan. Migration assays, quantification of soluble P-selectin, and serotonin release suggest that ENaC is dispensable for migration and alpha and dense granule secretion, whereas Na(+) influx through this channel is fundamental for platelet collagen activation. PMID:24679405

  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. Coupled movement of permeant and blocking ions in the CFTR chloride channel pore

    PubMed Central

    Gong, Xiandi; Linsdell, Paul

    2003-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel pore is blocked in a voltage-dependent manner by a broad range of anionic substances added to the cytoplasmic side of the membrane. Here we investigate the origin of the voltage dependence of block by intracellular Au(CN)2−, a highly permeant lyotropic anion which also acts as a high-affinity blocker of Cl− permeation. Not only the affinity, but also the voltage dependence of block by intracellular Au(CN)2− ions is strongly dependent on extracellular Cl− concentration; following replacement of most extracellular Cl− by glucose or by impermeant anions, block by Au(CN)2− shows greatly weakened voltage dependence. This suggests that coupled movement of Au(CN)2− and Cl− ions within the pore contributes to the voltage dependence of block. This explanation requires that interactions between different anions take place within the pore, implying simultaneous binding of multiple anions to intrapore sites. Other anions are able to substitute for extracellular Cl− and interact with intracellular Au(CN)2− ions. Analysis of the effects of different extracellular anions on the apparent affinity and voltage dependence of block by intracellular Au(CN)2− ions suggests that extracellular anions do not need to permeate through the channel in order to destabilize Au(CN)2− binding within the pore, implying that this destabilizing effect results from binding to an externally accessible site in the permeation pathway. We propose that multiple anions can bind simultaneously within the CFTR channel pore, and that repulsive interactions between bound anions speeds anion exit from the pore. PMID:12679371

  12. Coupled movement of permeant and blocking ions in the CFTR chloride channel pore.

    PubMed

    Gong, Xiandi; Linsdell, Paul

    2003-06-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel pore is blocked in a voltage-dependent manner by a broad range of anionic substances added to the cytoplasmic side of the membrane. Here we investigate the origin of the voltage dependence of block by intracellular Au(CN)2-, a highly permeant lyotropic anion which also acts as a high-affinity blocker of Cl- permeation. Not only the affinity, but also the voltage dependence of block by intracellular Au(CN)2- ions is strongly dependent on extracellular Cl- concentration; following replacement of most extracellular Cl- by glucose or by impermeant anions, block by Au(CN)2- shows greatly weakened voltage dependence. This suggests that coupled movement of Au(CN)2- and Cl- ions within the pore contributes to the voltage dependence of block. This explanation requires that interactions between different anions take place within the pore, implying simultaneous binding of multiple anions to intrapore sites. Other anions are able to substitute for extracellular Cl- and interact with intracellular Au(CN)2- ions. Analysis of the effects of different extracellular anions on the apparent affinity and voltage dependence of block by intracellular Au(CN)2- ions suggests that extracellular anions do not need to permeate through the channel in order to destabilize Au(CN)2- binding within the pore, implying that this destabilizing effect results from binding to an externally accessible site in the permeation pathway. We propose that multiple anions can bind simultaneously within the CFTR channel pore, and that repulsive interactions between bound anions speeds anion exit from the pore. PMID:12679371

  13. Molecular determinants and role of an anion binding site in the external mouth of the CFTR chloride channel pore

    PubMed Central

    Gong, Xiandi; Linsdell, Paul

    2003-01-01

    Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is blocked by highly lyotropic permeant anions which bind tightly within the pore. Here we show that several different substitutions of a positively charged amino acid residue, arginine R334, in the putative outer mouth of the CFTR pore, greatly reduce the block caused by lyotropic Au(CN)2− ions applied to the intracellular side of the channel. Fixed positive charge at this site appears to play a role in Au(CN)2− binding, as judged by multiple substitutions of differently charged amino acid side chains and also by the pH dependence of block conferred by the R334H mutant. However, non-charge-dependent effects also appear to contribute to Au(CN)2− binding. Mutation of R334 also disrupts the apparent electrostatic interaction between intracellular Au(CN)2− ions and extracellular permeant anions, an interaction which normally acts to relieve channel block. All six mutations studied at R334 significantly weakened this interaction, suggesting that arginine possesses a unique ability to coordinate ion-ion interactions at this site in the pore. Our results suggest that lyotropic anions bind tightly to a site in the outer mouth of the CFTR pore that involves interaction with a fixed positive charge. Binding to this site is also involved in coordination of multiple permeant anions within the pore, suggesting that anion binding in the outer mouth of the pore is an important aspect in the normal anion permeation mechanism. PMID:12679372

  14. Molecular determinants and role of an anion binding site in the external mouth of the CFTR chloride channel pore.

    PubMed

    Gong, Xiandi; Linsdell, Paul

    2003-06-01

    Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is blocked by highly lyotropic permeant anions which bind tightly within the pore. Here we show that several different substitutions of a positively charged amino acid residue, arginine R334, in the putative outer mouth of the CFTR pore, greatly reduce the block caused by lyotropic Au(CN)2- ions applied to the intracellular side of the channel. Fixed positive charge at this site appears to play a role in Au(CN)2- binding, as judged by multiple substitutions of differently charged amino acid side chains and also by the pH dependence of block conferred by the R334H mutant. However, non-charge-dependent effects also appear to contribute to Au(CN)2- binding. Mutation of R334 also disrupts the apparent electrostatic interaction between intracellular Au(CN)2- ions and extracellular permeant anions, an interaction which normally acts to relieve channel block. All six mutations studied at R334 significantly weakened this interaction, suggesting that arginine possesses a unique ability to coordinate ion-ion interactions at this site in the pore. Our results suggest that lyotropic anions bind tightly to a site in the outer mouth of the CFTR pore that involves interaction with a fixed positive charge. Binding to this site is also involved in coordination of multiple permeant anions within the pore, suggesting that anion binding in the outer mouth of the pore is an important aspect in the normal anion permeation mechanism. PMID:12679372

  15. Paradoxical Contribution of SK3 and GIRK Channels to the Activation of Mouse Vomeronasal Organ

    PubMed Central

    Kim, SangSeong; Ma, Limei; Jensen, Kristi L.; Kim, Michelle M.; Bond, Chris T.; Adelman, John P.; Yu, C. Ron

    2012-01-01

    The vomeronasal organ (VNO) plays an essential role in intraspecies communication for terrestrial vertebrates. The ionic mechanisms of VNO activation remain unclear. We find that the calcium–activated potassium channel SK3 and G–protein activated potassium channel GIRK are part of an independent pathway for VNO activation. In slice preparations, the potassium channels attenuate inward currents carried by TRPC2 and calcium–activated chloride channels (CACCs). In intact tissue preparations, paradoxically, the potassium channels enhance urine–evoked inward currents. This discrepancy results from the loss of a high concentration of lumenal potassium, which enables the influx of potassium ions to depolarize the VNO neurons in vivo. SK3−/− and GIRK1−/− mice show deficits in both mating and aggressive behaviors and deficiency in SK3−/− is exacerbated by TRPC2 knockout. Our results suggest a model of VNO activation that is mediated by TRPC2, CACCs and two potassium channels, all contributing to the in vivo depolarization of VNO neurons. PMID:22842147

  16. Paradoxical contribution of SK3 and GIRK channels to the activation of mouse vomeronasal organ.

    PubMed

    Kim, SangSeong; Ma, Limei; Jensen, Kristi L; Kim, Michelle M; Bond, Chris T; Adelman, John P; Yu, C Ron

    2012-09-01

    The vomeronasal organ (VNO) is essential for intraspecies communication in many terrestrial vertebrates. The ionic mechanisms of VNO activation remain unclear. We found that the calcium-activated potassium channel SK3 and the G protein-activated potassium channel GIRK are part of an independent pathway for VNO activation. In slice preparations, the potassium channels attenuated inward currents carried by TRPC2 and calcium-activated chloride channels (CACCs). In intact tissue preparations, paradoxically, the potassium channels enhanced urine-evoked inward currents. This discrepancy resulted from the loss of a high concentration of lumenal potassium, which enabled the influx of potassium ions to depolarize the VNO neurons in vivo. Both Sk3 (also known as Kcnn3) and Girk1 (also known as Kcnj3) homozygous null mice showed deficits in mating and aggressive behaviors, and the deficiencies in Sk3(-/-) mice were exacerbated by Trpc2 knockout. Our results suggest that VNO activation is mediated by TRPC2, CACCs and two potassium channels, all of which contributed to the in vivo depolarization of VNO neurons. PMID:22842147

  17. 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. PMID:18449561

  18. 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. PMID:26659082

  19. Calcium-Activated Potassium Channels: Potential Target for Cardiovascular Diseases.

    PubMed

    Dong, De-Li; Bai, Yun-Long; Cai, Ben-Zhi

    2016-01-01

    Ca(2+)-activated K(+) channels (KCa) are classified into three subtypes: big conductance (BKCa), intermediate conductance (IKCa), and small conductance (SKCa) KCa channels. The three types of KCa channels have distinct physiological or pathological functions in cardiovascular system. BKCa channels are mainly expressed in vascular smooth muscle cells (VSMCs) and inner mitochondrial membrane of cardiomyocytes, activation of BKCa channels in these locations results in vasodilation and cardioprotection against cardiac ischemia. IKCa channels are expressed in VSMCs, endothelial cells, and cardiac fibroblasts and involved in vascular smooth muscle proliferation, migration, vessel dilation, and cardiac fibrosis. SKCa channels are widely expressed in nervous and cardiovascular system, and activation of SKCa channels mainly contributes membrane hyperpolarization. In this chapter, we summarize the physiological and pathological roles of the three types of KCa channels in cardiovascular system and put forward the possibility of KCa channels as potential target for cardiovascular diseases. PMID:27038376

  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. PMID:25587118

  1. 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.

  2. 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.

  3. Meroterpenoid Chrodrimanins Are Selective and Potent Blockers of Insect GABA-Gated Chloride Channels

    PubMed Central

    Ihara, Makoto; Ling, Yun; Yang, Xinling; Kai, Kenji; Hayashi, Hideo; Matsuda, Kazuhiko

    2015-01-01

    Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs. PMID:25902139

  4. Meroterpenoid Chrodrimanins Are Selective and Potent Blockers of Insect GABA-Gated Chloride Channels.

    PubMed

    Xu, Yan; Furutani, Shogo; Ihara, Makoto; Ling, Yun; Yang, Xinling; Kai, Kenji; Hayashi, Hideo; Matsuda, Kazuhiko

    2015-01-01

    Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to 10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs. PMID:25902139

  5. 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

  6. Cysteine-independent inhibition of the CFTR chloride channel by the cysteine-reactive reagent sodium (2-sulphonatoethyl) methanethiosulphonate

    PubMed Central

    Li, M-S; Demsey, AFA; Qi, J; Linsdell, P

    2009-01-01

    Background and purpose: Methanethiosulphonate (MTS) reagents are used extensively to modify covalently cysteine side chains in ion channel structure-function studies. We have investigated the interaction between a widely used negatively charged MTS reagent, (2-sulphonatoethyl) methanethiosulphonate (MTSES), and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Experimental approach: Patch clamp recordings were used to study a ‘cys-less’ variant of human CFTR, in which all 18 endogenous cysteine residues have been removed by mutagenesis, expressed in mammalian cell lines. Use of excised inside–out membrane patches allowed MTS reagents to be applied to the cytoplasmic face of active channels. Key results: Intracellular application of MTSES, but not the positively charged MTSET, inhibited the function of cys-less CFTR. Inhibition was voltage dependent, with a Kd of 1.97 mmol·L−1 at −80 mV increasing to 36 mmol·L−1 at +80 mV. Inhibition was completely reversed on washout of MTSES, inconsistent with covalent modification of the channel protein. At the single channel level, MTSES caused a concentration-dependent reduction in unitary current amplitude. This inhibition was strengthened when extracellular Cl− concentration was decreased. Conclusions and implications: Our results indicate that MTSES inhibits the function of CFTR in a manner that is independent of its ability to modify cysteine residues covalently. Instead, we suggest that MTSES functions as an open channel blocker that enters the CFTR channel pore from its cytoplasmic end to physically occlude Cl− permeation. Given the very widespread use of MTS reagents in functional studies, our findings offer a broadly applicable caveat to the interpretation of results obtained from such studies. PMID:19466983

  7. Epilepsy-Related Slack Channel Mutants Lead to Channel Over-Activity by Two Different Mechanisms.

    PubMed

    Tang, Qiong-Yao; Zhang, Fei-Fei; Xu, Jie; Wang, Ran; Chen, Jian; Logothetis, Diomedes E; Zhang, Zhe

    2016-01-01

    Twelve sodium-activated potassium channel (KCNT1, Slack) genetic mutants have been identified from severe early-onset epilepsy patients. The changes in biophysical properties of these mutants and the underlying mechanisms causing disease remain elusive. Here, we report that seven of the 12 mutations increase, whereas one mutation decreases, the channel's sodium sensitivity. Two of the mutants exhibit channel over-activity only when the intracellular Na(+) ([Na(+)]i) concentration is ∼80 mM. In contrast, single-channel data reveal that all 12 mutants increase the maximal open probability (Po). We conclude that these mutant channels lead to channel over-activity predominantly by increasing the ability of sodium binding to activate the channel, which is indicated by its maximal Po. The sodium sensitivity of these epilepsy causing mutants probably determines the [Na(+)]i concentration at which these mutants exert their pathological effects. PMID:26725113

  8. AUGMENTATION OF MURINE NATURAL KILLER CELL ACTIVITY BY MANGANESE CHLORIDE

    EPA Science Inventory

    Natural Killer (NK) cell activity of spleen cells from male CBA/J mice was augmented by a single parenteral injection of MnCl2 administered 1 day prior to testing by in vitro and in vivo isotope release assays. Increased cytotoxic activity was observed in vitro against both NK-se...

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

    NASA Astrophysics Data System (ADS)

    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.

    2014-03-01

    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.

  10. 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. PMID:25944907

  11. 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

  12. Dendritic NMDA receptors activate axonal calcium channels

    PubMed Central

    Christie, Jason M.; Jahr, Craig E.

    2008-01-01

    Summary NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca2+ and monovalent cations, they could alter release directly by increasing presynaptic Ca2+ or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca2+ channels (VSCCs). Using two-photon microscopy to measure Ca2+ excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca2+ transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca2+ entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca2+-dependent forms of presynaptic plasticity and release. PMID:18957221

  13. ALLYL CHLORIDE: THE MUTAGENIC ACTIVITY OF ITS PHOTOOXIDATION PRODUCTS

    EPA Science Inventory

    Irridations of C3H5Cl/NOx, C3H5Cl/C2H6/NOx, and C2/NOx mixtures were conducted in a 22.7-m3 Teflon smog chamber, operated in a static mode. he irridated mixtures were tested for mutagenic activity by periodically exposing Salmonella typhimurium strain TA100 to the smog chamber ef...

  14. Preparation of activated carbons from bituminous coals with zinc chloride activation

    SciTech Connect

    Teng, H.; Yeh, T.S.

    1998-01-01

    Activated carbons were prepared by chemical activation from two Australian bituminous coals in this study. The preparation process consisted of zinc chloride impregnation followed by carbonization in nitrogen. The carbonization temperature ranges from 400 to 700 C. Experimental results reveal that an acid-washing process following the carbonization with ZnCl{sub 2} is necessary for preparing high-porosity carbons. Surface area, pore volume, and average pore diameter of the resulting carbons increase with the carbonization temperature to a maximum at 500 C and then begin to decrease. The maximum values of surface area and pore volume are larger for the carbon prepared from the coal with a lower O/C atomic ratio, while earlier findings from physical activation with CO{sub 2} have shown an opposite trend. An increase in particle size of the coal precursor leads to a reduction in porosity of the resulting carbons. The duration of the carbonization period affects the porosity of the resulting carbons, and the influence varies with the activation temperature.

  15. A point mutation in a glutamate-gated chloride channel confers abamectin resistance in the two-spotted spider mite, Tetranychus urticae Koch.

    PubMed

    Kwon, D H; Yoon, K S; Clark, J M; Lee, S H

    2010-08-01

    The molecular mechanisms and genetics of abamectin resistance mediated by target site insensitivity in the two-spotted spider mite, Tetranychus urticae, were investigated by comparing two isogenic abamectin-susceptible (AbaS) and abamectin-resistant (AbaR) strains. Cloning and sequencing of full-length cDNA fragments of gamma-amino butyric acid (GABA)-gated chloride channel genes revealed no polymorphisms between the two strains. However, sequence comparison of the full-length cDNA fragment of a T. urticae glutamate-gated chloride channel gene (TuGluCl) identified a G323D point mutation as being tentatively related with abamectin resistance. In individual F(2) progenies obtained by backcrossing, the G323D genotype was confirmed to correlate with abamectin resistance. Bioassays using progeny from reciprocal crossings revealed that the abamectin resistance trait resulting from TuGluCl insensitivity is incompletely recessive. PMID:20522121

  16. [Heme oxygenase activity in rat organs during cadmium chloride administration].

    PubMed

    Strel'chenko, E V; Nikitchenko, I V; Kaliman, P A

    2002-01-01

    Heme oxygenase activity, the level of spontaneous and ascorbat-induced LPO in the liver, kidney and spleen homogenates of rats and blood serum absorption spectrum in the Soret region in different periods both after CdCl2 and prior alpha-tocopherol administration were studied. The increase in the hemolysis products content in the serum was observed in 15 min after CdCl2 injection and remained during 24 h. Heme oxygenase activity in the liver and kidney increased after 6 h and stayed at the same level 24 h after CdCl2 administration. The level of spontaneous LPO in the spleen increased after 6 h, and in the liver and kidney the level of spontaneous and ascorbat-induced LPO increased in 24 h after CdCl2 injection. The preliminary alpha-tocopherol administration did not prevent the accumulation of hemolysis products in the serum and the increase of heme oxygenase activity in the liver and kidney caused by CdCl2 administration. However, the increase in the ascorbat-induced LPO in these organs was completely blocked. The role of heme and LPO in the heme oxygenase induction by CdCl2 are discussed. PMID:12916165

  17. Ion channels activated by light in Limulus ventral photoreceptors

    PubMed Central

    1986-01-01

    The light-activated conductance of Limulus ventral photoreceptors was studied using the patch-clamp technique. Channels (40 pS) were observed whose probability of opening was greatly increased by light. In some cells the latency of channel activation was nearly the same as that of the macroscopic response, while in other cells the channel latency was much greater. Like the macroscopic conductance, channel activity was reduced by light adaptation but enhanced by the intracellular injection of the calcium chelator EGTA. The latter observation indicates that channel activation was not a secondary result of the light-induced rise in intracellular calcium. A two-microelectrode voltage-clamp method was used to measure the voltage dependence of the light-activated macroscopic conductance. It was found that this conductance is constant over a wide voltage range more negative than zero, but it increases markedly at positive voltages. The single channel currents measured over this same voltage range show that the single channel conductance is independent of voltage, but that channel gating properties are dependent on voltage. Both the mean channel open time and the opening rate increase at positive voltages. These properties change in a manner consistent with the voltage dependence of the macroscopic conductance. The broad range of similarities between the macroscopic and single channel currents supports the conclusion that the 40-pS channel that we have observed is the principal channel underlying the response to light in these photoreceptors. PMID:2419481

  18. Surface-Bonded Antimicrobial Activity of an Organosilicon Quaternary Ammonium Chloride

    PubMed Central

    Isquith, A. J.; Abbott, E. A.; Walters, P. A.

    1972-01-01

    The hydrolysis product of 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride exhibited antimicrobial activity against a broad range of microorganisms while chemically bonded to a variety of surfaces. The chemical was not removed from surfaces by repeated washing with water, and its antimicrobial activity could not be attributed to a slow release of the chemical, but rather to the surface-bonded chemical. Images PMID:4650597

  19. Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity

    PubMed Central

    Londino, James D.; Lazrak, Ahmed; Jurkuvenaite, Asta; Collawn, James F.; Noah, James W.

    2013-01-01

    The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl−) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H+) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o−) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H+, did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection. PMID:23457187

  20. 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. PMID:26592158

  1. Changes of Chloride Channels in the Lacrimal Glands of a Rabbit Model of Sjögren’s syndrome

    PubMed Central

    Nandoskar, Prachi; Wang, Yanru; Wei, Ruihua; Liu, Ying; Zhao, Ping; Lu, Michael; Huang, Jianyan; Thomas, Padmaja; Trousdale, Melvin D.; Ding, Chuanqing

    2011-01-01

    Purpose To test the hypothesis that expression of Na+-K+-2Cl− co-transporter-1 (NKCC1), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride channel 2 γ subunit (ClC2γ) in the lacrimal glands (LG) of rabbits with induced autoimmune dacryoadenitis (IAD) are changed. Methods LGs were obtained from adult female rabbits with IAD, and age-matched female control rabbits. LGs were processed for laser capture microdissection, real time RT-PCR, western blot, and immunofluorescence. Results In rabbits with IAD, mRNA abundances and protein expressions for NKCC1 and CFTR from whole LGs were significantly lower than in controls. mRNA abundances of NKCC1, CFTR, and ClC2γ from rabbits with IAD were significantly different from acini and ductal cells from controls. NKCC1 was localized to the basolateral membranes of all acinar and ductal cells, with weaker staining intensity in ductal cells, and the staining pattern from rabbits with IAD appeared similar to that from controls. CFTR was found as punctate aggregates in the apical cytoplasm of all acinar and ductal cells, with the intensity in ductal cells much stronger, and no significant difference between controls and rabbits with IAD. ClC2γ was also localized to the apical cytoplasm as punctate aggregates of all acinar cells, but not in ductal cells, and similar staining pattern was observed in rabbits with IAD to control rabbits. Conclusions Our data demonstrated significant changes of mRNA and protein expressions of NKCC1, CFTR, and ClC2γ in rabbits with IAD, suggesting that these changes may contribute to the altered lacrimal secretion, particularly Cl− transport, in rabbits with IAD. PMID:22157573

  2. Preferential Phosphorylation of R-domain Serine 768 Dampens Activation of CFTR Channels by PKA

    PubMed Central

    Csanády, László; Seto-Young, Donna; Chan, Kim W.; Cenciarelli, Cristina; Angel, Benjamin B.; Qin, Jun; McLachlin, Derek T.; Krutchinsky, Andrew N.; Chait, Brian T.; Nairn, Angus C.; Gadsby, David C.

    2005-01-01

    CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (Po) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted Po-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The

  3. Synthesis and structure-activity relationship of aminoarylthiazole derivatives as correctors of the chloride transport defect in cystic fibrosis.

    PubMed

    Pesce, Emanuela; Bellotti, Marta; Liessi, Nara; Guariento, Sara; Damonte, Gianluca; Cichero, Elena; Galatini, Andrea; Salis, Annalisa; Gianotti, Ambra; Pedemonte, Nicoletta; Zegarra-Moran, Olga; Fossa, Paola; Galietta, Luis J V; Millo, Enrico

    2015-06-24

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel present in the membrane of epithelial cells. Mutations affecting the CFTR gene cause cystic fibrosis (CF), a multi-organ severe disease. The most common CF mutation, F508del, impairs the processing and activity (gating) of CFTR protein. Other mutations, like G551D, only cause a gating defect. Processing and gating defects can be targeted by small molecules called generically correctors and potentiators, respectively. Aminoarylthiazoles (AATs) represent an interesting class of compounds that includes molecules with dual activity, as correctors and potentiators. With the aim to improve the activity profile of AATs, we have now designed and synthesized a library of novel compounds in order to establish an initial SAR that may provide indications about the chemical groups that are beneficial or detrimental for rescue activity. The new compounds were tested as correctors and potentiators in CFBE41o-expressing F508del-CFTR using a functional assay. A dual active compound, AAT-4a, characterized by improved efficacy and marked synergy when combined with the corrector VX-809 has been identified. Moreover, by computational methods, a possible binding site for AATs in nucleotide binding domain NBD1 has been detected. These results will direct the synthesis of new analogues with possibly improved activity. PMID:26041577

  4. Structure of Thermally Activated TRP Channels

    PubMed Central

    Cohen, Matthew R.; Moiseenkova-Bell, Vera Y.

    2015-01-01

    Temperature sensation is important for adaptation and survival of organisms. While temperature has the potential to affect all biological macromolecules, organisms have evolved specific thermosensitive molecular detectors that are able to transduce temperature changes into physiologically relevant signals. Among these thermosensors are ion channels from the transient receptor potential (TRP) family. Prime candidates include TRPV1–4, TRPA1, and TRPM8 (the so-called “thermoTRP” channels), which are expressed in sensory neurons and gated at specific temperatures. Electrophysiological and thermodynamic approaches have been employed to determine the nature by which thermoTRPs detect temperature and couple temperature changes to channel gating. To further understand how thermoTRPs sense temperature, high-resolution structures of full-length thermoTRPs channels will be required. Here, we will discuss current progress in unraveling the structures of thermoTRP channels. PMID:25366237

  5. Calcium influx through stretch-activated channels mediates microfilament reorganization in osteoblasts under simulated weightlessness

    NASA Astrophysics Data System (ADS)

    Luo, Mingzhi; Yang, Zhouqi; Li, Jingbao; Xu, Huiyun; Li, Shengsheng; Zhang, Wei; Qian, Airong; Shang, Peng

    2013-06-01

    We have explored the role of Ca2+ signaling in microfilament reorganization of osteoblasts induced by simulated weightlessness using a random positioning machine (RPM). The RPM-induced alterations of cell morphology, microfilament distribution, cell proliferation, cell migration, cytosol free calcium concentration ([Ca2+]i), and protein expression in MG63 osteoblasts were investigated. Simulated weightlessness reduced cell size, disrupted microfilament, inhibited cellular proliferation and migration, and induced an increase in [Ca2+]i in MG63 human osteosarcoma cells. Gadolinium chloride (Gd), an inhibitor for stretch-activated channels, attenuated the increase in [Ca2+]i and microfilament disruption. Further, the expression of calmodulin was significantly increased by simulated weightlessness, and an inhibitor of calmodulin, W-7, aggravated microfilament disruption. Our findings demonstrate that simulated weightlessness induces Ca2+ influx through stretch-activated channels, then results in microfilament disruption.

  6. Cumulative Activation of Voltage-Dependent KVS-1 Potassium Channels

    PubMed Central

    Rojas, Patricio; Garst-Orozco, Jonathan; Baban, Beravan; de Santiago-Castillo, Jose Antonio; Covarrubias, Manuel; Salkoff, Lawrence

    2008-01-01

    In this study, we reveal the existence of a novel use-dependent phenomenon in potassium channels, which we refer to as cumulative activation (CA). CA consists of an increase in current amplitude in response to repetitive depolarizing step pulses to the same potential. CA persists for up to 20 s and is similar to a phenomenon called “voltage-dependent facilitation” observed in some calcium channels. The KVS-1 K+ channel, which exhibits CA, is a rapidly activating and inactivating voltage-dependent potassium channel expressed in chemosensory and other neurons of Caenorhabditis elegans. It is unusual in being most closely related to the Shab (Kv2) family of potassium channels, which typically behave like delayed rectifier K+ channels in other species. The magnitude of CA depends on the frequency, voltage, and duration of the depolarizing step pulse. CA also radically changes the activation and inactivation kinetics of the channel, suggesting that the channel may undergo a physical modification in a use-dependent manner; thus, a model that closely simulates the behavior of the channel postulates the existence of two populations of channels, unmodified and modified. Use-dependent changes in the behavior of potassium channels, such as CA observed in KVS-1, could be involved in functional mechanisms of cellular plasticity such as synaptic depression that represent the cellular basis of learning and memory. PMID:18199775

  7. Cyclic AMP-and beta-agonist-activated chloride conductance of a toad skin epithelium.

    PubMed

    Willumsen, N J; Vestergaard, L; Larsen, E H

    1992-04-01

    1. The control by intracellular cyclic AMP and beta-adrenergic stimulation of chloride conductance was studied in toad skin epithelium mounted in a chamber on the stage of an upright microscope. Impalement of identified principal cells from the serosal side with single-barrelled conventional or double-barrelled Cl(-)-sensitive microelectrodes was performed at x500 magnification. For blocking the active sodium current 50 microM-amiloride was present in the mucosal bath. 2. When clamped at transepithelial potential difference V = 0 mV, the preparations generated clamping currents of 0.9 +/- 1 microA/cm2 (mean +/- S.E.M.; number of observations n = 55). The intracellular potential of principal cells (Vb) was -96 +/- 2 mV with a fractional resistance of the basolateral membrane (fRb) of 0.016 +/- 0.003 (n = 54), and an intracellular Cl- activity of 40 +/- 2 mM (n = 24). 3. At V = 0 mV, serosal application of a cyclic AMP analogue, dibutyryl cyclic AMP (500 microM) or a beta-adrenergic agonist, isoprenaline (5 microM) resulted in a sixfold increase in transepithelial Cl- conductance identified by standard 36Cl- tracer technique. 4. The clamping current at V = 0 mV was unaffected by cyclic AMP (short-circuit current Isc = 0.1 +/- 0.3 microA/cm2, n = 16) indicating that subepidermal Cl(-)-secreting glands are not functioning in our preparations obtained by collagenase treatment. 5. Cyclic AMP- or isoprenaline-induced chloride conductance (Gcl) activation (V = 0 mV) was not reflected in membrane potential and intracellular Cl- activity in principal cells. Intracellular chloride activity was constant at approximately 40 mM at membrane potentials between -90 and -100 mV. Therefore, it can be concluded that the principal cells are not contributing to activated Cl- currents. 6. At V = -100 mV where the voltage-dependent chloride conductance of mitochondria-rich (MR) cells was already fully activated, GCl was unaffected by cyclic AMP or isoprenaline. The major effect of these

  8. The PDZ-binding chloride channel ClC-3B localizes to the Golgi and associates with cystic fibrosis transmembrane conductance regulator-interacting PDZ proteins.

    PubMed

    Gentzsch, Martina; Cui, Liying; Mengos, April; Chang, Xiu-Bao; Chen, Jey-Hsin; Riordan, John R

    2003-02-21

    ClC chloride channels are widely distributed in organisms across the evolutionary spectrum, and members of the mammalian family play crucial roles in cellular function and are mutated in several human diseases (Jentsch, T. J., Stein, V., Weinreich, F., and Zdebik, A. A. (2002) Physiol. Rev. 82, 503-568). Within the ClC-3, -4, -5 branch of the family that are intracellular channels, two alternatively spliced ClC-3 isoforms were recognized recently (Ogura, T., Furukawa, T., Toyozaki, T., Yamada, K., Zheng, Y. J., Katayama, Y., Nakaya, H., and Inagaki, N. (2002) FASEB J. 16, 863-865). ClC-3A resides in late endosomes where it serves as an anion shunt during acidification. We show here that the ClC-3B PDZ-binding isoform resides in the Golgi where it co-localizes with a small amount of the other known PDZ-binding chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator). Both channel proteins bind the Golgi PDZ protein, GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). Interestingly, however, when overexpressed, GOPC, which is thought to influence traffic in the endocytic/secretory pathway, causes a large reduction in the amounts of both channels, probably by leading them to the degradative end of this pathway. ClC-3B as well as CFTR also binds EBP50 (ERM-binding phosphoprotein 50) and PDZK1, which are concentrated at the plasma membrane. However, only PDZK1 was found to promote interaction between the two channels, perhaps because they were able to bind to two different PDZ domains in PDZK1. Thus while small portions of the populations of ClC-3B and CFTR may associate and co-localize, the bulk of the two populations reside in different organelles of cells where they are expressed heterologously or endogenously, and therefore their cellular functions are likely to be distinct and not primarily related. PMID:12471024

  9. A voltage-gated chloride channel in ascidian embryos modulated by both the cell cycle clock and cell volume.

    PubMed Central

    Villaz, M; Cinniger, J C; Moody, W J

    1995-01-01

    1. Eggs of the ascidian Boltenia villosa have an inwardly rectifying Cl- current whose amplitude varies by more than 10-fold during each cell cycle, the largest amplitude being at exit from M-phase. We examined whether this current was also sensitive to changes in cell volume. 2. Cell swelling, produced by direct inflation through a whole-cell recording pipette, greatly increased the amplitude of the Cl- current at all stages of the cell cycle in activated eggs. Swelling was much less effective in unfertilized eggs. 3. The increase in Cl- current amplitude continued for 10-20 min after an increase in diameter that was complete in 10 s, suggesting the involvement of a second messenger system in the response. 4. Treatment of unfertilized eggs with 6-dimethylaminopurine (DMAP), an inhibitor of cell cycle-dependent protein kinases, increased the amplitude of the Cl- current and its sensitivity to swelling to levels characteristic of fertilized eggs. 5. Osmotically produced swelling also increased Cl- current amplitude in unfertilized eggs. 6. We propose that dephosphorylation renders the Cl- channel functional, and that swelling or activation of the egg increases the sensitivity of the channel to dephosphorylation, perhaps by disrupting its links to the cytoskeleton. PMID:8576858

  10. Allosterism and Structure in Thermally Activated Transient Receptor Potential Channels.

    PubMed

    Diaz-Franulic, Ignacio; Poblete, Horacio; Miño-Galaz, Germán; González, Carlos; Latorre, Ramón

    2016-07-01

    The molecular sensors that mediate temperature changes in living organisms are a large family of proteins known as thermosensitive transient receptor potential (TRP) ion channels. These membrane proteins are polymodal receptors that can be activated by cold or hot temperatures, depending on the channel subtype, voltage, and ligands. The stimuli sensors are allosterically coupled to a pore domain, increasing the probability of finding the channel in its ion conductive conformation. In this review we first discuss the allosteric coupling between the temperature and voltage sensor modules and the pore domain, and then discuss the thermodynamic foundations of thermo-TRP channel activation. We provide a structural overview of the molecular determinants of temperature sensing. We also posit an anisotropic thermal diffusion model that may explain the large temperature sensitivity of TRP channels. Additionally, we examine the effect of several ligands on TRP channel function and the evidence regarding their mechanisms of action. PMID:27297398

  11. Adsorption properties of CFC and CFC replacements on activated carbon containing introduced ionic fluoride and chloride

    SciTech Connect

    Tanada, Seiki; Kawasaki, Naohito; Nakamura, Takeo; Abe, Ikuo

    1996-10-15

    Plasma technology has been available for the chlorofluorocarbon (CFC) decomposition or etching of silicone. The adsorption properties of CFC (CFC113) and CFC replacements (HCFC141b, HCFC225cb, and 5FP) on several kinds of plasma-treated activated carbons (P-ACs) prepared under different treatment gases were investigated using the adsorption isotherms, the limiting pore volume and the affinity coefficient and energy of adsorption calculated by the Dubinin-Radushkevich plot, and the quality and kinds of introduced fluoride and chloride. The dissolved fluoride and chloride atoms were introduced to the surface of activated carbon by CFC113, HCFC141b, and HCFC225cb, while the dissolved fluoride atoms were those from 5FP and tetrafluoromethane. The adsorbed amount of CFC and CFC replacements, except for 5FP, on P-ACs was larger than that on U-AC. The specific adsorption site on plasma-treated activated carbon of the CFC and CFC replacements was the fluoride atoms which were introduced by plasma treatment. It is concluded that the plasma-treated activated carbon was suitable for the recovery of CFC and CFC replacements, because the adsorbed amount of CFC and CFC replacements was larger than that on untreated activated carbon, and the adsorbed CFC and CFC replacements on activated carbon were decomposed by the plasma treatment.

  12. Synthesis and in vitro antimicrobial activity of 1-methyl-3-sulfonylthio-4-aminoquinolinium chlorides.

    PubMed

    Zieba, Andrzej; Wojtyczka, Robert D; Idzik, Danuta; Kepa, Małgorzata

    2013-01-01

    Synthesis and in vitro antimicrobial activity (MIC) of novel 1-methyl-3-sulfonylthio-4-aminoquinolinium chlorides 2 are described. Compounds 2 were obtained in the reaction of 1-methyl-4-aminoquinolinium 3-thiolates 1 with sulfonyl chlorides. Antimicrobial activity of compounds 2 was investigated using Gram-positive Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus epidermidis, Micrococcus luteus, Enterococcus faecalis, Bacillus subtilis, Corynebacterium pseudodiphtericum and Gram-negative Escherichia coil, Klebsiella pneumoniae, Proteus vulgaris, Serratia marcescens, Citrobacter freundii, Pseudomonas aeruginosa strains as well as Candida albicans. The investigated compounds exhibited growth-inhibitory activity towards Gram-positive bacteria in the 4-64 microg/mL range whereas that towards Gram-negative bacteria covered the 128-1024 microg/mL range. The highest activity was shown by compound 2c, featuring a phenylsulfonylthio substituent in the 3-quinoline position and a 4-chlorophenylamine group in the position 4. All of the investigated compounds 2 showed antifungal activity in the 32-128 microg/mL range. Correlations between antimicrobial activity and chemical structure of the tested compounds were observed. PMID:23610972

  13. Friction induced surface activity of some simple organic chlorides and hydrocarbons with iron.

    NASA Technical Reports Server (NTRS)

    Buckley, D. H.

    1973-01-01

    Sliding friction studies were conducted on an iron surface with exposure of that surface to various hydrocarbons and organic chlorides. The hydrocarbons included ethane, ethylene, ethyl chloride, methyl chloride and vinyl chloride. Auger cylindrical-mirror analysis was used to follow interactions of the hydrocarbon and organic chlorides with the iron surface. Results with vinyl chloride indicate friction-induced surface reactivity, adsorption to surface oxides, friction sensitivity to concentration and polymerization. Variation in the loads employed influence adsorption and, accordingly, friction. Unlike results with ethyl and vinyl chloride, friction-induced surface reactivity was not observed with ethane and ethylene.

  14. Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel.

    PubMed

    Heusser, Stephanie A; Yoluk, Özge; Klement, Göran; Riederer, Erika A; Lindahl, Erik; Howard, Rebecca J

    2016-07-01

    The superfamily of pentameric ligand-gated ion channels includes neurotransmitter receptors that mediate fast synaptic transmission in vertebrates, and are targets for drugs including alcohols, anesthetics, benzodiazepines, and anticonvulsants. However, the mechanisms of ion channel opening, gating, and modulation in these receptors leave many open questions, despite their pharmacological importance. Subtle conformational changes in both the extracellular and transmembrane domains are likely to influence channel opening, but have been difficult to characterize given the limited structural data available for human membrane proteins. Recent crystal structures of a modified Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in multiple states offer an appealing model system for structure-function studies. However, the pharmacology of the crystallographic GluCl construct is not well established. To establish the functional relevance of this system, we used two-electrode voltage-clamp electrophysiology in Xenopus oocytes to characterize activation of crystallographic and native-like GluCl constructs by L-glutamate and ivermectin. We also tested modulation by ethanol and other anesthetic agents, and used site-directed mutagenesis to explore the role of a region of Loop F which was implicated in ligand gating by molecular dynamics simulations. Our findings indicate that the crystallographic construct functionally models concentration-dependent agonism and allosteric modulation of pharmacologically relevant receptors. Specific substitutions at residue Leu174 in loop F altered direct L-glutamate activation, consistent with computational evidence for this region's role in ligand binding. These insights demonstrate conservation of activation and modulation properties in this receptor family, and establish a framework for GluCl as a model system, including new possibilities for drug discovery. In this study, we elucidate the validity of a modified glutamate

  15. Iron(III)-induced activation of chloride and bromide from modeled salt pans.

    PubMed

    Wittmer, Julian; Bleicher, Sergej; Zetzsch, Cornelius

    2015-05-14

    The photochemistry of halides in sea spray aerosol, on salt pans, and on other salty surfaces leads to a formation of reactive halogen species. We investigated the photochemical formation of atomic chlorine (Cl) and bromine (Br) in the gas phase in the presence of laboratory-modeled salt pans consisting of sodium chloride doped with iron(III) chloride hexahydrate (0.5 and 2 wt %). The samples were spread on a Teflon sheet and exposed to simulated sunlight in a Teflon smog chamber in purified, humidified air in the presence of a test mixture of hydrocarbons at the ppb level to determine Cl, Br, and OH formation by the radical clock method. Driven by the photolytic reduction of Fe(III) to Fe(II), the production rates of the Fe(III)-doped NaCl salt samples (up to10(7) atoms cm(-3) s(-1)) exceeded the release of Cl above a pure NaCl sample by more than an order of magnitude in an initially O3-free environment at low NOX. In bromide-doped samples (0.5 wt % NaBr), a part of the Cl release was replaced by Br when Fe(III) was present. Additions of sodium sulfate, sodium oxalate, oxalic acid, and catechol to NaCl/FeCl3 samples were found to restrain the activation of chloride. PMID:25243918

  16. The Ca2+-activated Cl- channel ANO1/TMEM16A regulates primary ciliogenesis.

    PubMed

    Ruppersburg, Chelsey Chandler; Hartzell, H Criss

    2014-06-01

    Many cells possess a single, nonmotile, primary cilium highly enriched in receptors and sensory transduction machinery that plays crucial roles in cellular morphogenesis. Although sensory transduction requires ion channels, relatively little is known about ion channels in the primary cilium (with the exception of TRPP2). Here we show that the Ca(2+)-activated Cl ((-)) channel anoctamin-1 (ANO1/TMEM16A) is located in the primary cilium and that blocking its channel function pharmacologically or knocking it down with short hairpin RNA interferes with ciliogenesis. Before ciliogenesis, the channel becomes organized into a torus-shaped structure ("the nimbus") enriched in proteins required for ciliogenesis, including the small GTPases Cdc42 and Arl13b and the exocyst complex component Sec6. The nimbus excludes F-actin and coincides with a ring of acetylated microtubules. The nimbus appears to form before, or independent of, apical docking of the mother centriole. Our data support a model in which the nimbus provides a scaffold for staging of ciliary components for assembly very early in ciliogenesis and chloride transport by ANO1/TMEM16A is required for the genesis or maintenance of primary cilia. PMID:24694595

  17. Down-Regulation of ClC-3 Expression Reduces Epidermal Stem Cell Migration by Inhibiting Volume-Activated Chloride Currents.

    PubMed

    Guo, Rui; Pan, Fuqiang; Tian, Yanping; Li, Hongli; Li, Shirong; Cao, Chuan

    2016-06-01

    ClC-3, a member of the ClC chloride (Cl(-)) channel family, has recently been proposed as the primary Cl(-) channel involved in cell volume regulation. Changes in cell volume influence excitability, contraction, migration, pathogen-host interactions, cell proliferation, and cell death processes. In this study, expression and function of ClC-3 channels were investigated during epidermal stem cell (ESC) migration. We observed differential expression of CLC-3 regulates migration of ESCs. Further, whole-cell patch-clamp recordings and image analysis demonstrated ClC-3 expression affected volume-activated Cl(-) current (I Cl,Vol) within ESCs. Live cell imaging systems, designed to observe cellular responses to overexpression and suppression of ClC-3 in real time, indicated ClC-3 may regulate ESC migratory dynamics. We employed IMARIS software to analyze the velocity and distance of ESC migration in vitro to demonstrate the function of ClC-3 channel in ESCs. As our data suggest volume-activated Cl(-) channels play a vital role in migration of ESCs, which contribute to skin repair by migrating from neighboring unwounded epidermis infundibulum, hair follicle or sebaceous glands, ClC-3 may represent a new and valuable target for stem cell therapies. PMID:26769712

  18. Determination of optical birefringence of S-benzylisothiouronium chloride and S-benzylisothiouronium nitrate using Cauchy’s two term model by the channelled spectrum method

    NASA Astrophysics Data System (ADS)

    Hemalatha, P.; Veeravazhuthi, V.; Shashidharan Nair, C. K.

    2009-07-01

    The channelled spectrum method was used for measuring the birefringence of anisotropic organic single crystals S-benzylisothiouronium chloride (SBTC) and S-benzylisothiouronium nitrate (SBTN). Crystals of uniform thickness were placed between crossed polarizers and illuminated with a tungsten source. The resulting pattern was analyzed to deduce the birefringence and its dispersion across the visible spectrum. Cauchy's two term formula was applied to fit the experimental data. The Cauchy's coefficients A and B were obtained by solving the linear equations using MATLAB ®. The wavelength dispersion of birefringence was calculated.

  19. Role of the Juxtamembrane Region of Cytoplasmic Loop 3 in the Gating and Conductance of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

    PubMed Central

    2012-01-01

    Opening and closing of the cystic fibrosis transmembrane conductance regulator chloride channel are controlled by interactions of ATP with its cytoplasmic nucleotide binding domains (NBDs). The NBDs are connected to the transmembrane pore via four cytoplasmic loops. These loops have been suggested to play roles both in channel gating and in forming a cytoplasmic extension of the channel pore. To investigate the structure and function of one of these cytoplasmic loops, we have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced into loop 3. We find that methanethiosulfonate (MTS) reagents modify cysteines introduced at 14 of 16 sites studied in the juxtamembrane region of loop 3, in all cases leading to inhibition of channel function. In most cases, both the functional effects of modification and the rate of modification were similar for negatively and positively charged MTS reagents. Single-channel recordings indicated that, at all sites, inhibition was the result of an MTS reagent-induced decrease in channel open probability; in no case was the Cl– conductance of open channels altered by modification. These results indicate that loop 3 is readily accessible to the cytoplasm and support the involvement of this region in the control of channel gating. However, our results do not support the hypothesis that this region is close enough to the Cl– permeation pathway to exert any influence on permeating Cl– ions. We propose that either the cytoplasmic pore is very wide or cytoplasmic Cl– ions use other routes to access the transmembrane pore. PMID:22545782

  20. Role of the juxtamembrane region of cytoplasmic loop 3 in the gating and conductance of the cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed

    El Hiani, Yassine; Linsdell, Paul

    2012-05-15

    Opening and closing of the cystic fibrosis transmembrane conductance regulator chloride channel are controlled by interactions of ATP with its cytoplasmic nucleotide binding domains (NBDs). The NBDs are connected to the transmembrane pore via four cytoplasmic loops. These loops have been suggested to play roles both in channel gating and in forming a cytoplasmic extension of the channel pore. To investigate the structure and function of one of these cytoplasmic loops, we have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced into loop 3. We find that methanethiosulfonate (MTS) reagents modify cysteines introduced at 14 of 16 sites studied in the juxtamembrane region of loop 3, in all cases leading to inhibition of channel function. In most cases, both the functional effects of modification and the rate of modification were similar for negatively and positively charged MTS reagents. Single-channel recordings indicated that, at all sites, inhibition was the result of an MTS reagent-induced decrease in channel open probability; in no case was the Cl(-) conductance of open channels altered by modification. These results indicate that loop 3 is readily accessible to the cytoplasm and support the involvement of this region in the control of channel gating. However, our results do not support the hypothesis that this region is close enough to the Cl(-) permeation pathway to exert any influence on permeating Cl(-) ions. We propose that either the cytoplasmic pore is very wide or cytoplasmic Cl(-) ions use other routes to access the transmembrane pore. PMID:22545782

  1. Involvements of chloride ion in decolorization of Acid Orange 7 by activated peroxydisulfate or peroxymonosulfate oxidation.

    PubMed

    Wang, Ping; Yang, Shiying; Shan, Liang; Niu, Rui; Shao, Xueting

    2011-01-01

    The effects of chloride anion (Cl-) (up to 1.0 mol/L) on the decolorization of a model compound, azo dye Acid Orange 7 (AO7), by sulfate radical (SO4-*) based-peroxydisulfate (PS) or peroxymonosulfate (PMS) oxidation under various activated conditions (UV254 nm/PS, Thermal (70 degrees C/PS, UV254 nm/PMS, Co2+/PMS) were investigated. Methanol and NH4+ were used as quenching reagents to determine the contributions of active chlorine species (dichloride radical (Cl2-*) and hypochlorous acid (HClO)). The results indicated that the effects of Cl- on the reaction mechanism were different under various activated conditions. For UV/PS and Thermal/PS, the inhibition tendency became more clear as the Cl- concentration increased, probably due to the reaction between Cl- and SO4-* and the generation of Cl2-* or HCIO. For UV/PMS, Cl- did not exhibit inhibition when the concentration was below 0.1 mol/L. As Cl- concentration reached to 1.0 mol/L, the decolorization rate of AO7 was, however, accelerated, possibly because PMS directly reacts with Cl- to form HClO. For Co2+/PMS, Cl- exhibited a significant inhibiting effect even at low concentration (< or = 0.01 mol/L). When Cl- concentration exceeded 0.1 mol/L, the activation of PMS by Co2+ was almost completely inhibited. Under this condition, HClO maybe played a major role in decolorization of AO7. The results implicated that chloride ion is an important factor in SO4(-*) -based degradation of organic contamination in chloride-containing water. PMID:22432303

  2. Slack, Slick, and Sodium-Activated Potassium Channels

    PubMed Central

    Kaczmarek, Leonard K.

    2013-01-01

    The Slack and Slick genes encode potassium channels that are very widely expressed in the central nervous system. These channels are activated by elevations in intracellular sodium, such as those that occur during trains of one or more action potentials, or following activation of nonselective cationic neurotransmitter receptors such as AMPA receptors. This review covers the cellular and molecular properties of Slack and Slick channels and compares them with findings on the properties of sodium-activated potassium currents (termed KNa currents) in native neurons. Human mutations in Slack channels produce extremely severe defects in learning and development, suggesting that KNa channels play a central role in neuronal plasticity and intellectual function. PMID:24319675

  3. Ca2+ current and Ca(2+)-activated chloride current in isolated smooth muscle cells of the sheep urethra.

    PubMed Central

    Cotton, K D; Hollywood, M A; McHale, N G; Thornbury, K D

    1997-01-01

    1. Isolated sheep urethral cells were studied using the perforated patch clamp technique (T = 37 degrees C). Depolarizing steps ranging from -40 to -10 mV evoked an inward current that peaked within 10 ms and a slower inward current. Stepping back to the holding potential of -80 mV evoked large inward tail currents. All three currents were abolished by nifedipine (1 microM). Substitution of external Ca2+ with Ba2+ resulted in potentiation of the fast inward current and blockade of the slow current and tails. 2. Changing the chloride equilibrium potential (ECl) from 0 to +27 mV shifted the reversal potential of the tail currents from 1 +/- 1 to 27 +/- 1 mV (number of cells, n = 5). Chloride channel blockers, niflumic acid (10 microM) and anthracene-9-carboxylic acid (9AC, 1 mM), reduced the slow current and tails suggesting that these were Ca(2+)-activated Cl- currents, ICl(Ca). 4. Caffeine (10 mM) induced currents that reversed at ECl and were blocked by niflumic acid (10 microM). 5. In current clamp mode, some cells developed spontaneous transient depolarizations (STDs) and action potentials. Short exposure to nifedipine blocked the action potentials and unmasked STDs. In contrast, 9AC and niflumic acid reduced the amplitude of the STDs and blocked the action potentials. 6. In conclusion, these cells have both L-type ICa and ICl(Ca). The former appears to be responsible for the upstroke of the action potential, while the latter may act as a pacemaker current. PMID:9409476

  4. SLO2 Channels Are Inhibited by All Divalent Cations That Activate SLO1 K+ Channels.

    PubMed

    Budelli, Gonzalo; Sun, Qi; Ferreira, Juan; Butler, Alice; Santi, Celia M; Salkoff, Lawrence

    2016-04-01

    Two members of the family of high conductance K(+)channels SLO1 and SLO2 are both activated by intracellular cations. However, SLO1 is activated by Ca(2+)and other divalent cations, while SLO2 (Slack or SLO2.2 from rat) is activated by Na(+) Curiously though, we found that SLO2.2 is inhibited by all divalent cations that activate SLO1, with Zn(2+)being the most effective inhibitor with an IC50of ∼8 μmin contrast to Mg(2+), the least effective, with an IC50of ∼ 1.5 mm Our results suggest that divalent cations are not SLO2 pore blockers, but rather inhibit channel activity by an allosteric modification of channel gating. By site-directed mutagenesis we show that a histidine residue (His-347) downstream of S6 reduces inhibition by divalent cations. An analogous His residue present in some CNG channels is an inhibitory cation binding site. To investigate whether inhibition by divalent cations is conserved in an invertebrate SLO2 channel we cloned the SLO2 channel fromDrosophila(dSLO2) and compared its properties to those of rat SLO2.2. We found that, like rat SLO2.2, dSLO2 was also activated by Na(+)and inhibited by divalent cations. Inhibition of SLO2 channels in mammals andDrosophilaby divalent cations that have second messenger functions may reflect the physiological regulation of these channels by one or more of these ions. PMID:26823461

  5. Tonic PKA Activity Regulates SK Channel Nanoclustering and Somatodendritic Distribution.

    PubMed

    Abiraman, Krithika; Sah, Megha; Walikonis, Randall S; Lykotrafitis, George; Tzingounis, Anastasios V

    2016-06-01

    Small-conductance calcium-activated potassium (SK) channels mediate a potassium conductance in the brain and are involved in synaptic plasticity, learning, and memory. SK channels show a distinct subcellular localization that is crucial for their neuronal functions. However, the mechanisms that control this spatial distribution are unknown. We imaged SK channels labeled with fluorophore-tagged apamin and monitored SK channel nanoclustering at the single molecule level by combining atomic force microscopy and toxin (i.e., apamin) pharmacology. Using these two complementary approaches, we found that native SK channel distribution in pyramidal neurons, across the somatodendritic domain, depends on ongoing cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) levels, strongly limiting SK channel expression at the pyramidal neuron soma. Furthermore, tonic cAMP-PKA levels also controlled whether SK channels were expressed in nanodomains as single entities or as a group of multiple channels. Our study reveals a new level of regulation of SK channels by cAMP-PKA and suggests that ion channel topography and nanoclustering might be under the control of second messenger cascades. PMID:27107637

  6. Oxidative Stress and Maxi Calcium-Activated Potassium (BK) Channels

    PubMed Central

    Hermann, Anton; Sitdikova, Guzel F.; Weiger, Thomas M.

    2015-01-01

    All cells contain ion channels in their outer (plasma) and inner (organelle) membranes. Ion channels, similar to other proteins, are targets of oxidative impact, which modulates ion fluxes across membranes. Subsequently, these ion currents affect electrical excitability, such as action potential discharge (in neurons, muscle, and receptor cells), alteration of the membrane resting potential, synaptic transmission, hormone secretion, muscle contraction or coordination of the cell cycle. In this chapter we summarize effects of oxidative stress and redox mechanisms on some ion channels, in particular on maxi calcium-activated potassium (BK) channels which play an outstanding role in a plethora of physiological and pathophysiological functions in almost all cells and tissues. We first elaborate on some general features of ion channel structure and function and then summarize effects of oxidative alterations of ion channels and their functional consequences. PMID:26287261

  7. 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

  8. Biodegradation of didecyldimethylammonium chloride by Pseudomonas fluorescens TN4 isolated from activated sludge.

    PubMed

    Nishihara, T; Okamoto, T; Nishiyama, N

    2000-04-01

    Bacteria that degrade didecyldimethylammonium chloride (DDAC) were isolated from activated sludge from a municipal sewage treatment plant by enrichment culture with DDAC as a sole carbon source. One of the isolates, Pseudomonas fluorescens TN4, degraded DDAC to produce decyldimethylamine and subsequently, dimethylamine, as the intermediates. The TN4 strain also assimilated the other quaternary ammonium compounds (QACs), alkyltrimethyl- and alkylbenzyldimethyl-ammonium salts, but not alkylpyridinium salts. TN4 was highly resistant to these QACs and degraded them by an N-dealkylation process. These data mean that there are some QAC-resistant and QAC-degrading bacteria such as TN4 in the environment. PMID:10792522

  9. Voltage is a partial activator of rat thermosensitive TRP channels

    PubMed Central

    Matta, José A; Ahern, Gerard P

    2007-01-01

    TRPV1 and TRPM8 are sensory nerve ion channels activated by heating and cooling, respectively. A variety of physical and chemical stimuli activate these receptors in a synergistic manner but the underlying mechanisms are unclear. Both channels are voltage sensitive, and temperature and ligands modulate this voltage dependence. Thus, a voltage-sensing mechanism has become an attractive model to explain the generalized gating of these and other thermo-sensitive TRP channels. We show here using whole-cell and single channel measurements that voltage produces only a partial activation of TRPV1 and TRPM8. At room temperature (20–25°C) membrane depolarization evokes responses that saturate at ∼50–60% of the maximum open probability. Furthermore, high concentrations of capsaicin (10 μm), resiniferatoxin (5 μm) and menthol (6 mm) reveal voltage-independent gating. Similarly, other modes of TRPV1 regulation including heat, protein kinase C-dependent phosphorylation, and protons enhance both the efficacy and sensitivity of voltage activation. In contrast, the TRPV1 antagonist capsazepine produces the opposite effects. These data can be explained by an allosteric model in which voltage, temperature, agonists and inverse agonists are independently coupled, either positively or negatively, to channel gating. Thus, voltage acts separately but in concert with other stimuli to regulate channel activation, and, therefore, a voltage-sensitive mechanism is unlikely to represent a final, gating mechanism for these channels. PMID:17932142

  10. Oxidative Regulation of Large Conductance Calcium-Activated Potassium Channels

    PubMed Central

    Tang, Xiang D.; Daggett, Heather; Hanner, Markus; Garcia, Maria L.; McManus, Owen B.; Brot, Nathan; Weissbach, Herbert; Heinemann, Stefan H.; Hoshi, Toshinori

    2001-01-01

    Reactive oxygen/nitrogen species are readily generated in vivo, playing roles in many physiological and pathological conditions, such as Alzheimer's disease and Parkinson's disease, by oxidatively modifying various proteins. Previous studies indicate that large conductance Ca2+-activated K+ channels (BKCa or Slo) are subject to redox regulation. However, conflicting results exist whether oxidation increases or decreases the channel activity. We used chloramine-T, which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation in the cloned human Slo (hSlo) channel expressed in mammalian cells. In the virtual absence of Ca2+, the oxidant shifted the steady-state macroscopic conductance to a more negative direction and slowed deactivation. The results obtained suggest that oxidation enhances specific voltage-dependent opening transitions and slows the rate-limiting closing transition. Enhancement of the hSlo activity was partially reversed by the enzyme peptide methionine sulfoxide reductase, suggesting that the upregulation is mediated by methionine oxidation. In contrast, hydrogen peroxide and cysteine-specific reagents, DTNB, MTSEA, and PCMB, decreased the channel activity. Chloramine-T was much less effective when concurrently applied with the K+ channel blocker TEA, which is consistent with the possibility that the target methionine lies within the channel pore. Regulation of the Slo channel by methionine oxidation may represent an important link between cellular electrical excitability and metabolism. PMID:11222629

  11. Curcumin stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity.

    PubMed

    Berger, Allan L; Randak, Christoph O; Ostedgaard, Lynda S; Karp, Philip H; Vermeer, Daniel W; Welsh, Michael J

    2005-02-18

    Compounds that enhance either the function or biosynthetic processing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel may be of value in developing new treatments for cystic fibrosis (CF). Previous studies suggested that the herbal extract curcumin might affect the processing of a common CF mutant, CFTR-DeltaF508. Here, we tested the hypothesis that curcumin influences channel function. Curcumin increased CFTR channel activity in excised, inside-out membrane patches by reducing channel closed time and prolonging the time channels remained open. Stimulation was dose-dependent, reversible, and greater than that observed with genistein, another compound that stimulates CFTR. Curcumin-dependent stimulation required phosphorylated channels and the presence of ATP. We found that curcumin increased the activity of both wild-type and DeltaF508 channels. Adding curcumin also increased Cl(-) transport in differentiated non-CF airway epithelia but not in CF epithelia. These results suggest that curcumin may directly stimulate CFTR Cl(-) channels. PMID:15582996

  12. Antimicrobial activity of biogenic silver nanoparticles, and silver chloride nanoparticles: an overview and comments.

    PubMed

    Durán, Nelson; Nakazato, Gerson; Seabra, Amedea B

    2016-08-01

    The antimicrobial impact of biogenic-synthesized silver-based nanoparticles has been the focus of increasing interest. As the antimicrobial activity of nanoparticles is highly dependent on their size and surface, the complete and adequate characterization of the nanoparticle is important. This review discusses the characterization and antimicrobial activity of biogenic synthesized silver nanoparticles and silver chloride nanoparticles. By revising the literature, there is confusion in the characterization of these two silver-based nanoparticles, which consequently affects the conclusion regarding to their antimicrobial activities. This review critically analyzes recent publications on the synthesis of biogenic silver nanoparticles and silver chloride nanoparticles by attempting to correlate the characterization of the nanoparticles with their antimicrobial activity. It was difficult to correlate the size of biogenic nanoparticles with their antimicrobial activity, since different techniques are employed for the characterization. Biogenic synthesized silver-based nanoparticles are not completely characterized, particularly the nature of capped proteins covering the nanomaterials. Moreover, the antimicrobial activity of theses nanoparticles is assayed by using different protocols and strains, which difficult the comparison among the published papers. It is important to select some bacteria as standards, by following international foundations (Pharmaceutical Microbiology Manual) and use the minimal inhibitory concentration by broth microdilution assays from Clinical and Laboratory Standards Institute, which is the most common assay used in antibiotic ones. Therefore, we conclude that to have relevant results on antimicrobial effects of biogenic silver-based nanoparticles, it is necessary to have a complete and adequate characterization of these nanostructures, followed by standard methodology in microbiology protocols. PMID:27289481

  13. An anion channel in Arabidopsis hypocotyls activated by blue light.

    PubMed Central

    Cho, M H; Spalding, E P

    1996-01-01

    A rapid, transient depolarization of the plasma membrane in seedling stems is one of the earliest effects of blue light detected in plants. It appears to play a role in transducing blue light into inhibition of hypocotyl (stem) elongation, and perhaps other responses. The possibility that activation of a Cl- conductance is part of the depolarization mechanism was raised previously and addressed here. By patch clamping hypocotyl cells isolated from dark-grown (etiolated) Arabidopsis seedlings, blue light was found to activate an anion channel residing at the plasma membrane. An anion-channel blocker commonly known as NPPB 15-nitro-2-(3-phenylpropylamino)-benzoic acid] potently and reversibly blocked this anion channel. NPPB also blocked the blue-light-induced depolarization in vivo and decreased the inhibitory effect of blue light on hypocotyl elongation. These results indicate that activation of this anion channel plays a role in transducing blue light into growth inhibition. PMID:8755616

  14. An anion channel in Arabidopsis hypocotyls activated by blue light

    NASA Technical Reports Server (NTRS)

    Cho, M. H.; Spalding, E. P.; Evans, M. L. (Principal Investigator)

    1996-01-01

    A rapid, transient depolarization of the plasma membrane in seedling stems is one of the earliest effects of blue light detected in plants. It appears to play a role in transducing blue light into inhibition of hypocotyl (stem) elongation, and perhaps other responses. The possibility that activation of a Cl- conductance is part of the depolarization mechanism was raised previously and addressed here. By patch clamping hypocotyl cells isolated from dark-grown (etiolated) Arabidopsis seedlings, blue light was found to activate an anion channel residing at the plasma membrane. An anion-channel blocker commonly known as NPPB 15-nitro-2-(3-phenylpropylamino)-benzoic acid] potently and reversibly blocked this anion channel. NPPB also blocked the blue-light-induced depolarization in vivo and decreased the inhibitory effect of blue light on hypocotyl elongation. These results indicate that activation of this anion channel plays a role in transducing blue light into growth inhibition.

  15. Location of a common inhibitor binding site in the cytoplasmic vestibule of the cystic fibrosis transmembrane conductance regulator chloride channel pore.

    PubMed

    Linsdell, Paul

    2005-03-11

    Chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is inhibited by a broad range of organic anions that enter the channel pore from its cytoplasmic end, physically occluding the Cl- permeation pathway. These open channel blocker molecules are presumed to bind within a relatively wide pore inner vestibule that shows little discrimination between different large anions. The present study uses patch clamp recording to identify a pore-lining lysine residue, Lys-95, that acts to attract large blocker molecules into this inner vestibule. Mutations that remove the fixed positive charge associated with this amino acid residue dramatically weaken the blocking effects of five structurally unrelated open channel blockers (glibenclamide, 4,4'-dinitrostilbene-2,2'-disulfonic acid, lonidamine, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and taurolithocholate-3-sulfate) when applied to the cytoplasmic face of the membrane. Mutagenesis of Lys-95 also induced amino acid side chain charge-dependent rectification of the macroscopic current-voltage relationship, consistent with the fixed positive charge on this residue normally acting to attract Cl- ions from the intracellular solution into the pore. These results identify Lys-95 as playing an important role in attracting permeant anions into the channel pore inner vestibule, probably by an electrostatic mechanism. This same electrostatic attraction mechanism also acts to attract larger anionic molecules into the relatively wide inner vestibule, where these substances bind to block Cl- permeation. Thus, structurally diverse open channel blockers of CFTR appear to share a common molecular mechanism of action that involves interaction with a positively charged amino acid side chain located in the inner vestibule of the pore. PMID:15634668

  16. Basolateral K channel activated by carbachol in the epithelial cell line T84.

    PubMed

    Tabcharani, J A; Harris, R A; Boucher, A; Eng, J W; Hanrahan, J W

    1994-11-01

    Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (Isc), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T84 cells. Carbachol had little effect on Isc when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent Isc response to calcium ionophores. Carbachol (100 microM) transiently elevated intracellular free calcium ([Ca2+]i) by approximately 3-fold in confluent cells cultured on glass coverslips with a time course resembling the Isc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (-54 mV) with pipette solution containing 150 mM KCl (37 degrees C). This rectification persisted when patches were bathed in symmetrical 150 mM KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na > Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mM barium. It is referred to as the KBIC channel based on its most distinctive properties (Ba

  17. Ion permeation of AQP6 water channel protein. Single channel recordings after Hg2+ activation.

    PubMed

    Hazama, Akihiro; Kozono, David; Guggino, William B; Agre, Peter; Yasui, Masato

    2002-08-01

    Aquaporin-6 (AQP6) has recently been identified as an intracellular vesicle water channel with anion permeability that is activated by low pH or HgCl2. Here we present direct evidence of AQP6 channel gating using patch clamp techniques. Cell-attached patch recordings of AQP6 expressed in Xenopus laevis oocytes indicated that AQP6 is a gated channel with intermediate conductance (49 picosiemens in 100 mm NaCl) induced by 10 microm HgCl2. Current-voltage relationships were linear, and open probability was fairly constant at any given voltage, indicating that Hg2+-induced AQP6 conductance is voltage-independent. The excised outside-out patch recording revealed rapid activation of AQP6 channels immediately after application of 10 microm HgCl2. Reduction of both Na+ and Cl- concentrations from 100 to 30 mm did not shift the reversal potential of the Hg2+-induced AQP6 current, suggesting that Na+ is as permeable as Cl-. The Na+ permeability of Hg2+-induced AQP6 current was further demonstrated by 22Na+ influx measurements. Site-directed mutagenesis identified Cys-155 and Cys-190 residues as the sites of Hg2+ activation both for water permeability and ion conductance. The Hill coefficient from the concentration-response curve for Hg2+-induced conductance was 1.1 +/- 0.3. These data provide the first evidence of AQP6 channel gating at a single-channel level and suggest that each monomer contains the pore region for ions based on the number of Hg2+-binding sites and the kinetics of Hg2+-activation of the channel. PMID:12034750

  18. 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. PMID:27063443

  19. Synthesis and micellar properties of surface-active ionic liquids: 1-alkyl-3-methylimidazolium chlorides.

    PubMed

    El Seoud, Omar A; Pires, Paulo Augusto R; Abdel-Moghny, Thanaa; Bastos, Erick L

    2007-09-01

    A series of surface-active ionic liquids, RMeImCl, has been synthesized by the reaction of purified 1-methylimidazole and 1-chloroalkanes, RCl, R=C(10),C(12),C(14), and C(16), respectively. Adsorption and aggregation of these surfactants in water have been studied by surface tension measurement. Additionally, solution conductivity, electromotive force, fluorescence quenching of micelle-solubilized pyrene, and static light scattering have been employed to investigate micelle formation. The following changes resulted from an increase in the length of R: an increase of micelle aggregation number; a decrease of: minimum area/surfactant molecule at solution/air interface; critical micelle concentration, and degree of counter-ion dissociation. Theoretically-calculated aggregation numbers and those based on quenching of pyrene are in good agreement. Gibbs free energies of adsorption at solution/air interface, DeltaG(ads)(0), and micelle formation in water, DeltaG(mic)(0), were calculated, and compared to those of three surfactant series, alkylpyridinium chlorides, RPyCl, alkylbenzyldimethylammonium chlorides, RBzMe(2)Cl, and benzyl(3-acylaminoethyl)dimethylammonium chlorides, R(')AEtBzMe(2)Cl, respectively. Contributions to the above-mentioned Gibbs free energies from surfactant methylene groups (in the hydrophobic tail) and the head-group were calculated. For RMeImCl, the former energy is similar to that of other cationic surfactants. The corresponding free energy contribution of the head-group to DeltaG(mic)(0) showed the following order: RPyCl approximately RBzMe(2)Cl>RMeImCl>R(')AEtBzMe(2)Cl. The head-groups of the first two surfactant series are more hydrophobic than the imidazolium ring of RMeImCl, this should favor their aggregation. Micellization of RMeImCl, however, is driven by a relatively strong hydrogen-bonding between the chloride ion and the hydrogens in the imidazolium ring, in particular the relatively acidic H2. This interaction more than compensates for

  20. Maximization of the rate of chloride conduction in the CFTR channel pore by ion-ion interactions.

    PubMed

    Gong, Xiandi; Linsdell, Paul

    2004-06-01

    Multi-ion pore behaviour has been identified in many Cl(-) channel types but its biophysical significance is uncertain. Here, we show that mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel that disrupt anion-anion interactions within the pore are associated with drastically reduced single channel conductance. These results are consistent with models suggesting that rapid Cl(-) permeation in CFTR results from repulsive ion-ion interactions between Cl(-) ions bound concurrently inside the pore. Naturally occurring mutations that disrupt these interactions can result in cystic fibrosis. PMID:15130785

  1. Chlorine activation indoors and outdoors via surface-mediated reactions of nitrogen oxides with hydrogen chloride

    PubMed Central

    Raff, Jonathan D.; Njegic, Bosiljka; Chang, Wayne L.; Gordon, Mark S.; Dabdub, Donald; Gerber, R. Benny; Finlayson-Pitts, Barbara J.

    2009-01-01

    Gaseous HCl generated from a variety of sources is ubiquitous in both outdoor and indoor air. Oxides of nitrogen (NOy) are also globally distributed, because NO formed in combustion processes is oxidized to NO2, HNO3, N2O5 and a variety of other nitrogen oxides during transport. Deposition of HCl and NOy onto surfaces is commonly regarded as providing permanent removal mechanisms. However, we show here a new surface-mediated coupling of nitrogen oxide and halogen activation cycles in which uptake of gaseous NO2 or N2O5 on solid substrates generates adsorbed intermediates that react with HCl to generate gaseous nitrosyl chloride (ClNO) and nitryl chloride (ClNO2), respectively. These are potentially harmful gases that photolyze to form highly reactive chlorine atoms. The reactions are shown both experimentally and theoretically to be enhanced by water, a surprising result given the availability of competing hydrolysis reaction pathways. Airshed modeling incorporating HCl generated from sea salt shows that in coastal urban regions, this heterogeneous chemistry increases surface-level ozone, a criteria air pollutant, greenhouse gas and source of atmospheric oxidants. In addition, it may contribute to recently measured high levels of ClNO2 in the polluted coastal marine boundary layer. This work also suggests the potential for chlorine atom chemistry to occur indoors where significant concentrations of oxides of nitrogen and HCl coexist. PMID:19620710

  2. Exogenous Magnesium Chloride Reduces the Activated Partial Thromboplastin Times of Lupus Anticoagulant-Positive Patients

    PubMed Central

    Tokutake, Takayoshi; Baba, Hisami; Shimada, Yuji; Takeda, Wataru; Sato, Keijiro; Hiroshima, Yuki; Kirihara, Takehiko; Shimizu, Ikuo; Nakazawa, Hideyuki; Kobayashi, Hikaru; Ishida, Fumihiro

    2016-01-01

    The activated partial thromboplastin time (APTT) assay is a basic hemostatic assay based on the time it takes for clots to form in plasma samples after the addition of calcium chloride. It is used to screen for various coagulation disorders. Several previous reports have suggested that magnesium (Mg) might contribute to coagulation reactions by binding to specific coagulation proteins. We investigated the effects of Mg on the APTT. In healthy controls, the APTT was significantly prolonged in proportion to the increase in the concentration of magnesium chloride in the range from 2.1 to 16.7 mmol/L. Among eight samples from patients with various disorders that exhibited prolonged APTT, two samples demonstrated shorter APTT when Mg was added, both of which were from patients that were positive for lupus anticoagulant. When we examined 206 clinical APTT samples, we found that Mg shortened the APTT of two samples. These two samples were also from lupus anticoagulant-positive patients (p-value: <0.003). Our findings regarding the unique effects of exogenous Mg on the APTT of lupus anticoagulant-positive patients might shed light on the role of Mg in APTT assays and lead to the development of a novel screening method for lupus anticoagulant. PMID:27355205

  3. 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. PMID:27574292

  4. Multi-channel fiber photometry for population neuronal activity recording

    PubMed Central

    Guo, Qingchun; Zhou, Jingfeng; Feng, Qiru; Lin, Rui; Gong, Hui; Luo, Qingming; Zeng, Shaoqun; Luo, Minmin; Fu, Ling

    2015-01-01

    Fiber photometry has become increasingly popular among neuroscientists as a convenient tool for the recording of genetically defined neuronal population in behaving animals. Here, we report the development of the multi-channel fiber photometry system to simultaneously monitor neural activities in several brain areas of an animal or in different animals. In this system, a galvano-mirror modulates and cyclically couples the excitation light to individual multimode optical fiber bundles. A single photodetector collects excited light and the configuration of fiber bundle assembly and the scanner determines the total channel number. We demonstrated that the system exhibited negligible crosstalk between channels and optical signals could be sampled simultaneously with a sample rate of at least 100 Hz for each channel, which is sufficient for recording calcium signals. Using this system, we successfully recorded GCaMP6 fluorescent signals from the bilateral barrel cortices of a head-restrained mouse in a dual-channel mode, and the orbitofrontal cortices of multiple freely moving mice in a triple-channel mode. The multi-channel fiber photometry system would be a valuable tool for simultaneous recordings of population activities in different brain areas of a given animal and different interacting individuals. PMID:26504642

  5. Active Integrated Filters for RF-Photonic Channelizers

    PubMed Central

    Nagdi, Amr El; Liu, Ke; LaFave, Tim P.; Hunt, Louis R.; Ramakrishna, Viswanath; Dabkowski, Mieczyslaw; MacFarlane, Duncan L.; Christensen, Marc P.

    2011-01-01

    A theoretical study of RF-photonic channelizers using four architectures formed by active integrated filters with tunable gains is presented. The integrated filters are enabled by two- and four-port nano-photonic couplers (NPCs). Lossless and three individual manufacturing cases with high transmission, high reflection, and symmetric couplers are assumed in the work. NPCs behavior is dependent upon the phenomenon of frustrated total internal reflection. Experimentally, photonic channelizers are fabricated in one single semiconductor chip on multi-quantum well epitaxial InP wafers using conventional microelectronics processing techniques. A state space modeling approach is used to derive the transfer functions and analyze the stability of these filters. The ability of adapting using the gains is demonstrated. Our simulation results indicate that the characteristic bandpass and notch filter responses of each structure are the basis of channelizer architectures, and optical gain may be used to adjust filter parameters to obtain a desired frequency magnitude response, especially in the range of 1–5 GHz for the chip with a coupler separation of ∼9 mm. Preliminarily, the measurement of spectral response shows enhancement of quality factor by using higher optical gains. The present compact active filters on an InP-based integrated photonic circuit hold the potential for a variety of channelizer applications. Compared to a pure RF channelizer, photonic channelizers may perform both channelization and down-conversion in an optical domain. PMID:22319352

  6. Curcumin Cross-links Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Polypeptides and Potentiates CFTR Channel Activity by Distinct Mechanisms*

    PubMed Central

    Bernard, Karen; Wang, Wei; Narlawar, Rajeshwar; Schmidt, Boris; Kirk, Kevin L.

    2009-01-01

    Cystic fibrosis (CF) is caused by loss-of-function mutations in the CFTR chloride channel. Wild type and mutant CFTR channels can be activated by curcumin, a well tolerated dietary compound with some appeal as a prospective CF therapeutic. However, we show here that curcumin has the unexpected effect of cross-linking CFTR polypeptides into SDS-resistant oligomers. This effect occurred for CFTR channels in microsomes as well as in intact cells and at the same concentrations that are effective for promoting CFTR channel activity (5–50 μm). Both mature CFTR polypeptides at the cell surface and immature CFTR protein in the endoplasmic reticulum were cross-linked by curcumin, although the latter pool was more susceptible to this modification. Curcumin cross-linked two CF mutant channels (ΔF508 and G551D) as well as a variety of deletion constructs that lack the major cytoplasmic domains. In vitro cross-linking could be prevented by high concentrations of oxidant scavengers (i.e. reduced glutathione and sodium azide) indicating a possible oxidation reaction with the CFTR polypeptide. Importantly, cyclic derivatives of curcumin that lack the reactive β diketone moiety had no cross-linking activity. One of these cyclic derivatives stimulated the activities of wild type CFTR channels, Δ1198-CFTR channels, and G551D-CFTR channels in excised membrane patches. Like the parent compound, the cyclic derivative irreversibly activated CFTR channels in excised patches during prolonged exposure (>5 min). Our results raise a note of caution about secondary biochemical effects of reactive compounds like curcumin in the treatment of CF. Cyclic curcumin derivatives may have better therapeutic potential in this regard. PMID:19740743

  7. cAMP- and Ca2+-independent activation of cystic fibrosis transmembrane conductance regulator channels by phenylimidazothiazole drugs.

    PubMed

    Becq, F; Verrier, B; Chang, X B; Riordan, J R; Hanrahan, J W

    1996-07-01

    Patch-clamp, iodide efflux, and biochemical techniques were used to evaluate the ability of phenylimidazothiazoles to open normal and mutated cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels and to investigate the mechanism of activation. As reported previously for bromotetramisole, levamisole activated wild-type CFTR channels stably expressed in Chinese hamster ovary cells in the absence of other secretagogues and without elevating intracellular cAMP or calcium. The protein kinase A (PKA) inhibitor N - (2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesul-fonamid e abolished activation by forskolin but only partially inhibited stimulation by levamisole, suggesting the involvement of other kinases. CFTR channels bearing mutations at multiple phosphorylation sites, in the membrane domains, and in the first nucleotide binding domain (including the disease-causing mutations G551D and DeltaF508) all responded to phenylimidazothiazoles. Moreover, levamisole and bromotetramisole increased the activity of wild-type and mutant channels already exposed to PKA + MgATP, consistent with the inhibition of a constitutive, membrane-associated phosphatase activity. We conclude that phenylimidazothiazole drugs can open normal and mutated CFTR channels by stabilization of phosphoforms of CFTR that are produced by basal activity of PKA and alternative protein kinases. If similar stimulation is observed in humans in vivo, phenylimidazothiazoles may be useful in the development of pharmacological therapies for cystic fibrosis. PMID:8663098

  8. 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

  9. Asymmetric structure of the cystic fibrosis transmembrane conductance regulator chloride channel pore suggested by mutagenesis of the twelfth transmembrane region.

    PubMed

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

    2001-06-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel contains 12 membrane-spanning regions which are presumed to form the transmembrane pore. Although a number of findings have suggested that the sixth transmembrane region plays a key role in forming the pore and determining its functional properties, the role of other transmembrane regions is currently not well established. Here we assess the functional importance of the twelfth transmembrane region, which occupies a homologous position in the carboxy terminal half of the CFTR molecule to that of the sixth transmembrane region in the amino terminal half. Five residues in potentially important regions of the twelfth transmembrane region were mutated individually to alanines, and the function of the mutant channels was examined using patch clamp recording following expression in mammalian cell lines. Three of the five mutations significantly weakened block of unitary Cl(-) currents by SCN(-), implying a partial disruption of anion binding within the pore. Two of these mutations also caused a large reduction in the steady-state channel mean open probability, suggesting a role for the twelfth transmembrane region in channel gating. However, in direct contrast to analogous mutations in the sixth transmembrane region, all mutants studied here had negligible effects on the anion selectivity and unitary Cl(-) conductance of the channel. The relatively minor effects of these five mutations on channel permeation properties suggests that, despite their symmetrical positions within the CFTR protein, the sixth and twelfth transmembrane regions make highly asymmetric contributions to the functional properties of the pore. PMID:11380256

  10. 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. PMID:25143385

  11. Modulation of a recombinant invertebrate γ-aminobutyric acid receptor-chloride channel complex by isoflurane: effects of a point mutation in the M2 domain

    PubMed Central

    Edwards, Michelle D; Lees, George

    1997-01-01

    Inhalational anaesthetics modulate ligand-gated ion channels at clinical concentrations. In this paper we address submolecular mechanisms for γ-aminobutyric acid (GABA) receptor modulation by isoflurane. Wild-type Drosophila melanogaster homo-oligomeric GABA receptors were characterized and compared with an ion-channel mutant (alanine substituted to a serine in M2) by means of two-electrode voltage-clamp in membrane-invariant Xenopus oocytes. Both channel receptor isoforms generated outwardly rectifying, bicuculline-insensitive currents with reversal potentials characteristic of a chloride current. As previously shown, the point mutation in the M2 domain conferred a profound resistance to the blocking action of 10 μM picrotoxinin (PTX): circa 7 fold reduction at the GABA EC20. Isoflurane, 195–389 μM, enhanced GABA conductance in both receptor variants by significantly increasing the affinity of the agonist for its receptor without changing Hill slope or maximal response. Relative potencies were statistically indistinguishable. Isoflurane concentration-response curves (on circa GABA EC25) demonstrated that enhancement was effected at around 100–195 μM for both receptor subtypes, but a dramatic divergence was evident at concentrations above 400 μM: wild-type receptors exhibited concentration-dependent block, whilst mutant conductances continued to increase over the same concentration range, showing no tendency to saturate (up to 3330 μM). The above divergence was not attributable to differential desensitization: neither wild-type nor mutant conductance desensitized significantly (P>0.05) in the absence or presence of anaesthetic. This work demonstrates that modulatory sites for anaesthetic are present on a relatively primitive insect ion channel. The depression of GABA response at high isoflurane concentrations, in WT receptors, (typical of a variety of anaesthetic agents) may reflect low affinity channel block via the PTX site. The non

  12. Effects of salinity on chloride cells and Na+, K(+)-ATPase activity in the teleost Gillichthys mirabilis.

    PubMed

    Yoshikawa, J S; McCormick, S D; Young, G; Bern, H A

    1993-06-01

    1. Longjawed mudsuckers, Gillichthys mirabilis, in 30 ppt seawater (SW) were transferred to 1.5, 30 and 60 ppt SW. 2. In the first 1-3 days after transfer, plasma chloride level and plasma osmolarity rose in the 60 ppt SW fish, and decreased in the 1.5 ppt SW fish. 3. By day 21, however, plasma chloride and osmolarity were at or near the levels seen in the controls (30 ppt). 4. Branchial and jawskin Na+, K(+)-ATPase activities were high in all salinities, and did not differ significantly among treatments. 5. The vital fluorescent stains DASPEI and anthroylouabain were used to detect mitochondria and Na+, K(+)-ATPase, respectively, in chloride cells. 6. Both stains indicated that jawskin chloride cell density did not differ among treatment groups. 7. In contrast, chloride cell size increased significantly with increasing salinity. 8. The chloride cells of fish in 60 ppt SW were noticeably angular in outline, whereas those of both the 1.5 and 30 ppt SW fish were circular. 9. The results are discussed in relation to the ion transport requirements encountered in the intertidal habitat of the mudsucker. PMID:8101158

  13. Characterization of the hyperpolarization-activated chloride current in dissociated rat sympathetic neurons.

    PubMed

    Clark, S; Jordt, S E; Jentsch, T J; Mathie, A

    1998-02-01

    1. Dissociated rat superior cervical ganglion (SCG) neurons have been shown to possess a hyperpolarization-activated inwardly rectifying chloride current. The current was not altered by changes in external potassium concentration, replacing external cations with NMDG (N-methyl-D-glucamine) or by addition of 10 mM caesium or barium ions. 2. The reversal potential of the current was altered by changing external anions. The anion selectivity of the current was Cl- > Br- > I- > cyclamate. All substituted permeant anions also blocked the current. 3. The current was blocked by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid), 9AC (anthracene-9-carboxylic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) but was unaffected by SITS (4-acetamido-4'-isothiocyanatostilbene- 2,2'-disulphonic acid) and niflumic acid. The effective blockers were voltage dependent; DIDS and NPPB were more effective at depolarized potentials while 9AC was more effective at hyperpolarized potentials. 4. The current was enhanced by extracellular acidification and reduced by extracellular alkalinization. Reducing external osmolarity was without effect in conventional whole-cell recording but enhanced current amplitude in those perforated-patch recordings where little current was evident in control external solution. 5. The current in SCG neurons was blocked by external cadmium and zinc. ClC-2 chloride currents expressed in Xenopus oocytes were also sensitive to block by these divalent ions and by DIDS but the sensitivity of ClC-2 to block by cadmium ions was lower than that of the current in SCG neurons. 6. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed the presence of mRNA for ClC-2 in SCG neurons but not in rat cerebellar granule cells which do not possess a hyperpolarization-activated Cl- current. 7. The data suggest that ClC-2 may be functionally expressed in rat SCG neurons. This current may play a role in regulating the internal chloride

  14. 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

    2014-01-01

    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. PMID:24328042

  15. Hydrostatic and osmotic pressure activated channel in plant vacuole

    PubMed Central

    Alexandre, Joel; Lassalles, Jean-Paul

    1991-01-01

    The vacuolar membrane of red beet vacuoles contains a channel which was not gated by voltage or Ca2+ ions. Its unit conductance was 20 pS in 200 mM symmetrical KCl solutions. It was stretch activated: the conductance remained constant but the probability of opening was increased by suction or pressure applied to a membrane patch. A 1.5-kNm-2 suction applied to isolated patches or a 0.08-kNm-2 pressure applied to a 45-μm diameter vacuole induced an e-fold change in the mean current. A 75% inhibition of the channel current was obtained with 10 μM Gd3+ on the cytoplasmic side. The channel was more permeable for K+ than for Cl- (PK/PCl ∼ 3). A possible clustering for this channel was suggested by the recordings of the patch current. The channel properties were not significantly affected by a change in sorbitol osmolality in the solutions under isoosmotic conditions, between 0.6 and 1 mol/kg sorbitol. However, the channel was very sensitive to an osmotic gradient. A 0.2-mol/kg sorbitol gradient induced a two-fold increase in unit conductance and a thirty-fold increase in the mean patch current of the channel. A current was measured, when the osmotic gradient was the only driving force applied to the vacuolar membrane. The hydrostatic and osmotic pressure (HOP) activated channel described in this paper could be gated in vivo condition by a change in osmolality, without the need of a change in the turgor pressure in the cell. The HOP channel represents a possible example of an osmoreceptor for plant cells. PMID:19431814

  16. Anti-Diarrheal Mechanism of the Traditional Remedy Uzara via Reduction of Active Chloride Secretion

    PubMed Central

    Fromm, Anja; Günzel, Dorothee

    2011-01-01

    Background and Purpose The root extract of the African Uzara plant is used in traditional medicine as anti-diarrheal drug. It is known to act via inhibition of intestinal motility, but malabsorptive or antisecretory mechanisms are unknown yet. Experimental Approach HT-29/B6 cells and human colonic biopsies were studied in Ussing experiments in vitro. Uzara was tested on basal as well as on forskolin- or cholera toxin-induced Cl− secretion by measuring short-circuit current (ISC) and tracer fluxes of 22Na+ and 36Cl−. Para- and transcellular resistances were determined by two-path impedance spectroscopy. Enzymatic activity of the Na+/K+-ATPase and intracellular cAMP levels (ELISA) were measured. Key Results In HT-29/B6 cells, Uzara inhibited forskolin- as well as cholera toxin-induced ISC within 60 minutes indicating reduced active chloride secretion. Similar results were obtained in human colonic biopsies pre-stimulated with forskolin. In HT-29/B6, the effect of Uzara on the forskolin-induced ISC was time- and dose-dependent. Analyses of the cellular mechanisms of this Uzara effect revealed inhibition of the Na+/K+-ATPase, a decrease in forskolin-induced cAMP production and a decrease in paracellular resistance. Tracer flux experiments indicate that the dominant effect is the inhibition of the Na+/K+-ATPase. Conclusion and Implications Uzara exerts anti-diarrheal effects via inhibition of active chloride secretion. This inhibition is mainly due to an inhibition of the Na+/K+-ATPase and to a lesser extent to a decrease in intracellular cAMP responses and paracellular resistance. The results imply that Uzara is suitable for treating acute secretory diarrhea. PMID:21479205

  17. Immobilization of horseradish peroxidase on amidoximated acrylic polymer activated by cyanuric chloride.

    PubMed

    Mohamed, Saleh A; Al-Ghamdi, Saeed S; El-Shishtawy, Reda M

    2016-10-01

    Horseradish peroxidase (HRP) was immobilized on amidoximated acrylic fabric after being activated with cyanuric chloride. FT-IR spectroscopy and scanning electron microscopy were used to characterize fabrics. The maximum immobilization efficiency of HRP (70%) was detected at 4% cyanuric chloride and pH 7.0. The immobilized enzyme retained 45% of its initial activity after ten reuses. The immobilization of enzyme on the carrier is saturated after 6h of incubation time. The pH was shifted from 7.0 for soluble HRP to 7.5-8.0 for the immobilized enzyme. The soluble HRP and immobilized HRP had the same optimum activity at 40°C. The immobilized HRP is more thermal stable than soluble HRP. Substrate analogues were oxidized by immobilized HRP with higher efficiencies than those of soluble HRP. Km values of the soluble HRP and the immobilized HRP were 31 and 37mM for guiacol and 5.0 and 7.8mM for H2O2, respectively. The immobilized HRP had higher efficiency for removal of phenol than that of soluble HRP. The immobilized HRP had higher resistance toward heavy metal ions compared to the soluble enzyme. The immobilized HRP was more stable against high concentration of urea, Triton X-100 and isopropanol. The immobilized HRP exhibited high resistance to proteolysis by trypsin than soluble enzyme. In conclusion, the immobilized HRP could be used as a potential efficient catalyst for the removal of aromatic pollutants from wastewater. PMID:27264646

  18. Novel Activation of Voltage-gated K+ Channels by Sevoflurane*

    PubMed Central

    Barber, Annika F.; Liang, Qiansheng; Covarrubias, Manuel

    2012-01-01

    Voltage-gated ion channels are modulated by halogenated inhaled general anesthetics, but the underlying molecular mechanisms are not understood. Alkanols and halogenated inhaled anesthetics such as halothane and isoflurane inhibit the archetypical voltage-gated Kv3 channel homolog K-Shaw2 by stabilizing the resting/closed states. By contrast, sevoflurane, a more heavily fluorinated ether commonly used in general anesthesia, specifically activates K-Shaw2 currents at relevant concentrations (0.05–1 mm) in a rapid and reversible manner. The concentration dependence of this modulation is consistent with the presence of high and low affinity interactions (KD = 0.06 and 4 mm, respectively). Sevoflurane (<1 mm) induces a negative shift in the conductance-voltage relation and increases the maximum conductance. Furthermore, suggesting possible roles in general anesthesia, mammalian Kv1.2 and Kv1.5 channels display similar changes. Quantitative description of the observations by an economical allosteric model indicates that sevoflurane binding favors activation gating and eliminates an unstable inactivated state outside the activation pathway. This study casts light on the mechanism of the novel sevoflurane-dependent activation of Kv channels, which helps explain how closely related inhaled anesthetics achieve specific actions and suggests strategies to develop novel Kv channel activators. PMID:23038249

  19. Calcium-activated chloride current expression in axotomized sensory neurons: what for?

    PubMed Central

    Boudes, Mathieu; Scamps, Frédérique

    2012-01-01

    Calcium-activated chloride currents (CaCCs) are activated by an increase in intracellular calcium concentration. Peripheral nerve injury induces the expression of CaCCs in a subset of adult sensory neurons in primary culture including mechano- and proprioceptors, though not nociceptors. Functional screenings of potential candidate genes established that Best1 is a molecular determinant for CaCC expression among axotomized sensory neurons, while Tmem16a is acutely activated by inflammatory mediators in nociceptors. In nociceptors, such CaCCs are preferentially activated under receptor-induced calcium mobilization contributing to cell excitability and pain. In axotomized mechano- and proprioceptors, CaCC activation does not promote electrical activity and prevents firing, a finding consistent with electrical silencing for growth competence of adult sensory neurons. In favor of a role in the process of neurite growth, CaCC expression is temporally correlated to neurons displaying a regenerative mode of growth. This perspective focuses on the molecular identity and role of CaCC in axotomized sensory neurons and the future directions to decipher the cellular mechanisms regulating CaCC during neurite (re)growth. PMID:22461766

  20. Stretch-activated cation channel from larval bullfrog skin.

    PubMed

    Hillyard, Stanley D; Willumsen, Niels J; Marrero, Mario B

    2010-05-01

    Cell-attached patches from isolated epithelial cells from larval bullfrog skin revealed a cation channel that was activated by applying suction (-1 kPa to -4.5 kPa) to the pipette. Activation was characterized by an initial large current spike that rapidly attenuated to a stable value and showed a variable pattern of opening and closing with continuing suction. Current-voltage plots demonstrated linear or inward rectification and single channel conductances of 44-56 pS with NaCl or KCl Ringer's solution as the pipette solution, and a reversal potential (-V(p)) of 20-40 mV. The conductance was markedly reduced with N-methyl-D-glucamide (NMDG)-Cl Ringer's solution in the pipette. Neither amiloride nor ATP, which are known to stimulate an apical cation channel in Ussing chamber preparations of larval frog skin, produced channel activation nor did these compounds affect the response to suction. Stretch activation was not affected by varying the pipette concentrations of Ca(2+) between 0 mmol l(-1) and 4 mmol l(-1) or by varying pH between 6.8 and 8.0. However, conductance was reduced with 4 mmol l(-1) Ca(2+). Western blot analysis of membrane homogenates from larval bullfrog and larval toad skin identified proteins that were immunoreactive with mammalian TRPC1 and TRPC5 (TRPC, canonical transient receptor potential channel) antibodies while homogenates of skin from newly metamorphosed bullfrogs were positive for TRPC1 and TRPC3/6/7 antibodies. The electrophysiological response of larval bullfrog skin resembles that of a stretch-activated cation channel characterized in Xenopus oocytes and proposed to be TRPC1. These results indicate this channel persists in all life stages of anurans and that TRP isoforms may be important for sensory functions of their skin. PMID:20435829

  1. Point mutations in the pore region directly or indirectly affect glibenclamide block of the CFTR chloride channel.

    PubMed

    Gupta, Jyoti; Linsdell, Paul

    2002-03-01

    The sulfonylurea glibenclamide is a relatively potent inhibitor of the CFTR Cl(-) channel. This inhibition is thought to be via an open channel block mechanism. However, nothing is known about the physical nature of the glibenclamide-binding site on CFTR. Here we show that mutations in the pore-forming 6th and 12th transmembrane regions of CFTR affect block by intracellular glibenclamide, confirming previous suggestions that glibenclamide enters the pore in order to block the channel. Two mutations in the 6th transmembrane region, F337A and T338A, significantly weakened glibenclamide block, consistent with a direct interaction between glibenclamide and this region of the pore. Interestingly, two mutations in the 12th transmembrane region (N1138A and T1142A) significantly strengthened block. These two mutations also abolished the dependence of block on the extracellular Cl(-) concentration, which in wild-type CFTR suggests an interaction between Cl(-) and glibenclamide within the channel pore that limits block. We suggest that mutations in the 12th transmembrane region strengthen glibenclamide block not by directly altering interactions between glibenclamide and the pore walls, but indirectly by reducing interactions between Cl(-) ions and glibenclamide within the pore. This work demonstrates that glibenclamide binds within the CFTR channel pore and begins to define its intrapore binding site. PMID:11889571

  2. Smog Chamber Investigation on the Iron-Catalyzed Activation of Chloride from Modeled Saltpans

    NASA Astrophysics Data System (ADS)

    Wittmer, Julian; Bleicher, Sergej; Oeste <, Franz Dietrich; Zetzsch, Cornelius

    2014-05-01

    Halogen activation on sea spray aerosols and other halide surfaces and thus the formation of reactive halogen species (RHS), influencing trace and greenhouse gases, has become an important topic of research in recent years. In this context the chloride and bromide activation, in particular the formation of RHS by photochemically induced halogen release from (sea) salt surface and reactions with ozone (O3) and nitrogen oxides (NOx), came into focus [1,2]. Our studies concentrate on the quantification of atomic chlorine (Cl), bromine (Br) and hydroxyl (OH) radicals in the gas phase above lab-models of salt pans, enriched in iron(III) chloride (FeCl3), that are exposed to simulated sunlight in a smog chamber. The applied radical clock method [3] results in time profiles and source strengths for Cl, Br and OH, which are combined with the various compositions of humidified salts. In particular, the influence of bromine, sulfate, oxalate, and catechol on the FeCl3 enriched salt is investigated. Comparable investigations only exist for the aqueous phase chemistry of FeCl3 (e.g. [4]). Driven by the photolytic reduction from Fe(III) to Fe(II), an enormous amount of chlorine atoms (>107 cm-3) could be detected for sodium chloride (NaCl) salt pans with low addition of FeCl3 (0.5 - 2 wt%), even in an O3 and NOx free environment. The Cl2 source strength reaches a maximum of 8×1011 Cl2 molecules per cm3 within the first hour of the experiment, corresponding to a Cl2 mixing ratio of 30 ppbv at standard pressure. These concentrations exceeded the release above pure NaCl samples by a factor of 1000. A crucial factor for the Cl2 release is the pH and thus the formation of iron(III) complexes on the salt crystals that differ in their sensitivity for photolysis. Whereas the presence of sodium bromide normally strengthens the chlorine release, a suppression accompanied by strong bromine activation (>1010 cm-3) could be observed for iron enriched samples. Furthermore, the addition of

  3. Calcium-activated potassium channels and endothelial dysfunction: therapeutic options?

    PubMed Central

    Félétou, Michel

    2009-01-01

    The three subtypes of calcium-activated potassium channels (KCa) of large, intermediate and small conductance (BKCa, IKCa and SKCa) are present in the vascular wall. In healthy arteries, BKCa channels are preferentially expressed in vascular smooth muscle cells, while IKCa and SKCa are preferentially located in endothelial cells. The activation of endothelial IKCa and SKCa contributes to nitric oxide (NO) generation and is required to elicit endothelium-dependent hyperpolarizations. In the latter responses, the hyperpolarization of the smooth muscle cells is evoked either via electrical coupling through myo-endothelial gap junctions or by potassium ions, which by accumulating in the intercellular space activate the inwardly rectifying potassium channel Kir2.1 and/or the Na+/K+-ATPase. Additionally, endothelium-derived factors such as cytochrome P450-derived epoxyeicosatrienoic acids and under some circumstances NO, prostacyclin, lipoxygenase products and hydrogen peroxide (H2O2) hyperpolarize and relax the underlying smooth muscle cells by activating BKCa. In contrast, cytochrome P450-derived 20-hydroxyeicosatetraenoic acid and various endothelium-derived contracting factors inhibit BKCa. Aging and cardiovascular diseases are associated with endothelial dysfunctions that can involve a decrease in NO bioavailability, alterations of EDHF-mediated responses and/or enhanced production of endothelium-derived contracting factors. Because potassium channels are involved in these endothelium-dependent responses, activation of endothelial and/or smooth muscle KCa could prevent the occurrence of endothelial dysfunction. Therefore, direct activators of these potassium channels or compounds that regulate their activity or their expression may be of some therapeutic interest. Conversely, blockers of IKCa may prevent restenosis and that of BKCa channels sepsis-dependent hypotension. PMID:19187341

  4. 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.

  5. Rapid dye degradation with reactive oxidants generated by chloride-induced peroxymonosulfate activation.

    PubMed

    Lou, Xiao-Yi; Guo, Yao-Guang; Xiao, Dong-Xue; Wang, Zhao-Hui; Lu, Shu-Yu; Liu, Jian-She

    2013-09-01

    Transition-metal is known to catalyze peroxymonosulfate (PMS) decomposition to produce sulfate radicals. Here we report reactions between PMS and chloride, without a need of transition metals, also can be used to degrade organic dye pollutant (Rhodamine B, (RhB)). Some important operating parameters, such as dosages of PMS and Cl(-), pH of solution, temperature, ionic strength, and several common cations, were systematically investigated. Almost complete decoloration of RhB was achieved within 5 min ([PMS] = 0.5 mM, [Cl(-)] = 120 mM, and pH 3.0), and RhB bleaching rate increased with the increased dosages of both PMS and chloride ion, following the pseudo-first-order kinetic model. However, the total organic carbon (TOC) removal results demonstrated that the decoloration of RhB was due to the destruction of chromophore rather than complete degradation. RhB decoloration could be significantly accelerated due to the high ionic strength. Increasing of the reaction temperature from 273 K to 333 K was beneficial to the RhB degradation, and the activation energy was determined to be 32.996 kJ/mol. Bleaching rate of RhB with the examined cations increased with the order of NH4 (+) < Na(+) < K(+) < Al(3+) < Ca(2+) < Mg(2+). Some major degradation products of RhB were identified by GC-MS. The present study may have active technical implications for the treatment of dyestuff wastewater in practice. PMID:23589259

  6. Activation of peripheral KCNQ channels relieves gout pain

    PubMed Central

    Zheng, Yueming; Xu, Haiyan; Zhan, Li; Zhou, Xindi; Chen, Xueqin; Gao, Zhaobing

    2015-01-01

    Abstract Intense inflammatory pain caused by urate crystals in joints and other tissues is a major symptom of gout. Among therapy drugs that lower urate, benzbromarone (BBR), an inhibitor of urate transporters, is widely used because it is well tolerated and highly effective. We demonstrate that BBR is also an activator of voltage-gated KCNQ potassium channels. In cultured recombinant cells, BBR exhibited significant potentiation effects on KCNQ channels comparable to previously reported classical activators. In native dorsal root ganglion neurons, BBR effectively overcame the suppression of KCNQ currents, and the resultant neuronal hyperexcitability caused by inflammatory mediators, such as bradykinin (BK). Benzbromarone consistently attenuates BK-, formalin-, or monosodium urate–induced inflammatory pain in rat and mouse models. Notably, the analgesic effects of BBR are largely mediated through peripheral and not through central KCNQ channels, an observation supported both by pharmacokinetic studies and in vivo experiments. Moreover, multiple residues in the superficial part of the voltage sensing domain of KCNQ channels were identified critical for the potentiation activity of BBR by a molecular determinant investigation. Our data indicate that activation of peripheral KCNQ channels mediates the pain relief effects of BBR, potentially providing a new strategy for the development of more effective therapies for gout. PMID:25735002

  7. Positive Charges at the Intracellular Mouth of the Pore Regulate Anion Conduction in the CFTR Chloride Channel

    PubMed Central

    Aubin, Chantal N. St.; Linsdell, Paul

    2006-01-01

    Many different ion channel pores are thought to have charged amino acid residues clustered around their entrances. The so-called surface charges contributed by these residues can play important roles in attracting oppositely charged ions from the bulk solution on one side of the membrane, increasing effective local counterion concentration and favoring rapid ion movement through the channel. Here we use site-directed mutagenesis to identify arginine residues contributing important surface charges in the intracellular mouth of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel pore. While wild-type CFTR was associated with a linear current–voltage relationship with symmetrical solutions, strong outward rectification was observed after mutagenesis of two arginine residues (R303 and R352) located near the intracellular ends of the fifth and sixth transmembrane regions. Current rectification was dependent on the charge present at these positions, consistent with an electrostatic effect. Furthermore, mutagenesis-induced rectification was more pronounced at lower Cl− concentrations, suggesting that these mutants had a reduced ability to concentrate Cl− ions near the inner pore mouth. R303 and R352 mutants exhibited reduced single channel conductance, especially at negative membrane potentials, that was dependent on the charge of the amino acid residue present at these positions. However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl− concentration. Modification of an introduced cysteine residue at position 303 by charged methanethiosulfonate reagents reproduced charge-dependent effects on current rectification. Mutagenesis of arginine residues in the second and tenth transmembrane regions also altered channel permeation properties, however these effects were not consistent with changes in channel surface charges. These results suggest that positively charged arginine

  8. Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel.

    PubMed

    Aubin, Chantal N St; Linsdell, Paul

    2006-11-01

    Many different ion channel pores are thought to have charged amino acid residues clustered around their entrances. The so-called surface charges contributed by these residues can play important roles in attracting oppositely charged ions from the bulk solution on one side of the membrane, increasing effective local counterion concentration and favoring rapid ion movement through the channel. Here we use site-directed mutagenesis to identify arginine residues contributing important surface charges in the intracellular mouth of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. While wild-type CFTR was associated with a linear current-voltage relationship with symmetrical solutions, strong outward rectification was observed after mutagenesis of two arginine residues (R303 and R352) located near the intracellular ends of the fifth and sixth transmembrane regions. Current rectification was dependent on the charge present at these positions, consistent with an electrostatic effect. Furthermore, mutagenesis-induced rectification was more pronounced at lower Cl(-) concentrations, suggesting that these mutants had a reduced ability to concentrate Cl(-) ions near the inner pore mouth. R303 and R352 mutants exhibited reduced single channel conductance, especially at negative membrane potentials, that was dependent on the charge of the amino acid residue present at these positions. However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl(-) concentration. Modification of an introduced cysteine residue at position 303 by charged methanethiosulfonate reagents reproduced charge-dependent effects on current rectification. Mutagenesis of arginine residues in the second and tenth transmembrane regions also altered channel permeation properties, however these effects were not consistent with changes in channel surface charges. These results suggest that positively charged arginine residues

  9. Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. Quantitative analysis of a cyclic gating scheme.

    PubMed

    Zeltwanger, S; Wang, F; Wang, G T; Gillis, K D; Hwang, T C

    1999-04-01

    Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) involves a coordinated action of ATP on two nucleotide binding domains (NBD1 and NBD2). Previous studies using nonhydrolyzable ATP analogues and NBD mutant CFTR have suggested that nucleotide hydrolysis at NBD1 is required for opening of the channel, while hydrolysis of nucleotides at NBD2 controls channel closing. We studied ATP-dependent gating of CFTR in excised inside-out patches from stably transfected NIH3T3 cells. Single channel kinetics of CFTR gating at different [ATP] were analyzed. The closed time constant (tauc) decreased with increasing [ATP] to a minimum value of approximately 0.43 s at [ATP] >1.00 mM. The open time constant (tauo) increased with increasing [ATP] with a minimal tauo of approximately 260 ms. Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBD2, reveals the presence of two open states. A short open state with a time constant of approximately 250 ms is dominant at low ATP concentrations (10 microM) and a much longer open state with a time constant of approximately 3 min is present at millimolar ATP. These data suggest that nucleotide binding and hydrolysis at NBD1 is coupled to channel opening and that the channel can close without nucleotide interaction with NBD2. A quantitative cyclic gating scheme with microscopic irreversibility was constructed based on the kinetic parameters derived from single-channel analysis. The estimated values of the kinetic parameters suggest that NBD1 and NBD2 are neither functionally nor biochemically equivalent. PMID:10102935

  10. 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. PMID:24785711

  11. Na(+) -Activated K(+) Channels in Rat Supraoptic Neurones.

    PubMed

    Bansal, V; Fisher, T E

    2016-06-01

    The magnocellular neurosecretory cells (MNCs) of the hypothalamus secrete the neurohormones vasopressin and oxytocin. The systemic release of these hormones depends on the rate and pattern of MNC firing and it is therefore important to identify the ion channels that contribute to the electrical behaviour of MNCs. In the present study, we report evidence for the presence of Na(+) -activated K(+) (KN a ) channels in rat MNCs. KN a channels mediate outwardly rectifying K(+) currents activated by the increases in intracellular Na(+) that occur during electrical activity. Although the molecular identity of native KN a channels is unclear, their biophysical properties are consistent with those of expressed Slick (slo 2.1) and Slack (slo 2.2) proteins. Using immunocytochemistry and Western blot experiments, we found that both Slick and Slack proteins are expressed in rat MNCs. Using whole cell voltage clamp techniques on acutely isolated rat MNCs, we found that inhibiting Na(+) influx by the addition of the Na(+) channel blocker tetrodotoxin or the replacement of Na(+) in the external solution with Li(+) caused a significant decrease in sustained outward currents. Furthermore, the evoked outward current density was significantly higher in rat MNCs using patch pipettes containing 60 mm Na(+) than it was when patch pipettes containing 0 mm Na(+) were used. Our data show that functional KN a channels are expressed in rat MNCs. These channels could contribute to the activity-dependent afterhyperpolarisations that have been identified in the MNCs and thereby play a role in the regulation of their electrical behaviour. PMID:27091544

  12. Use of Small Fluorescent Molecules to Monitor Channel Activity

    NASA Astrophysics Data System (ADS)

    Jones, Sharon; Stringer, Sarah; Naik, Rajesh; Stone, Morley

    2001-03-01

    The Mechanosensitive channel of Large conductance (MscL) allows bacteria to rapidly adapt to changing environmental conditions such as osmolarity. The MscL channel opens in response to increases in membrane tension, which allows for the efflux of cytoplasmic constituents. Here we describe the cloning and expression of Salmonella typhimurium MscL (St-MscL). Using a fluorescence efflux assay, we demonstrate that efflux through the MscL channel during hypoosmotic shock can be monitored using endogenously produced fluorophores. In addition, we observe that thermal stimulation, i.e., heat shock, can also induce efflux through MscL. We present the first evidence of thermal activation of MscL efflux by heat shocking cells expressing the S. typhimurium protein variant. This finding has significant biosensor implications, especially for investigators exploring the use of channel proteins in biosensor applications. Thermal biosensors are relatively unexplored, but would have considerable commercial and military utility.

  13. Sinupret Activates CFTR and TMEM16A-Dependent Transepithelial Chloride Transport and Improves Indicators of Mucociliary Clearance

    PubMed Central

    Zhang, Shaoyan; Skinner, Daniel; Hicks, Stephen Bradley; Bevensee, Mark O.; Sorscher, Eric J.; Lazrak, Ahmed; Matalon, Sadis; McNicholas, Carmel M.; Woodworth, Bradford A.

    2014-01-01

    Introduction We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl−) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid. Methods Primary murine nasal septal epithelial (MNSE) [wild type (WT) and transgenic CFTR−/−], human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl− transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca2+]i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis. Results Sinupret-mediated Cl− secretion [ΔISC(µA/cm2)] was pronounced in WT MNSE (20.7+/−0.9 vs. 5.6+/−0.9(control), p<0.05), CFTR−/− MNSE (10.1+/−1.0 vs. 0.9+/−0.3(control), p<0.05) and HSNE (20.7+/−0.3 vs. 6.4+/−0.9(control), p<0.05). The formulation activated Ca2+ signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl− secretion. Sinupret also enhanced CBF and ASL depth. Conclusion Sinupret stimulates CBF, promotes transepithelial Cl− secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca2+-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl

  14. Neuronal modulation of calcium channel activity in cultured rat astrocytes

    SciTech Connect

    Corvalan, V.; Cole, R.; De Vellis, J.; Hagiwara, Susumu )

    1990-06-01

    The patch-clamp technique was used to study whether cocultivation of neurons and astrocytes modulates the expression of calcium channel activity in astrocytes. Whole-cell patch-clamp recordings from rat brain astrocytes cocultured with rat embryonic neurons revealed two types of voltage-dependent inward currents carried by Ca{sup 2+} and blocked by either Cd{sup 2+} or Co{sup 2+} that otherwise were not detected in purified astrocytes. This expression of calcium channel activity in astrocytes was neuron dependent and was not observed when astrocytes were cocultured with purified oligodendrocytes.

  15. Identification of a second blocker binding site at the cytoplasmic mouth of the cystic fibrosis transmembrane conductance regulator chloride channel pore.

    PubMed

    St Aubin, Chantal N; Zhou, Jing-Jun; Linsdell, Paul

    2007-05-01

    Chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is inhibited by a broad range of substances that bind within a wide inner vestibule in the pore and physically occlude Cl(-) permeation. Binding of many of these so-called open-channel blockers involves electrostatic interactions with a positively charged lysine residue (Lys95) located in the pore. Here, we use site-directed mutagenesis to identify a second blocker binding site located at the cytoplasmic mouth of the pore. Mutagenesis of a positively charged arginine at the cytoplasmic mouth of the pore, Arg303, leads to significant weakening of the blocking effects of suramin, a large negatively charged organic molecule. Apparent suramin affinity is correlated with the side chain charge at this position, consistent with an electrostatic interaction. In contrast, block by suramin is unaffected by mutagenesis of Lys95, suggesting that it does not approach close to this important pore-forming lysine residue. We propose that the CFTR pore inner vestibule contains two distinct blocker binding sites. Relatively small organic anions enter deeply into the pore to interact with Lys95, causing an open-channel block that is sensitive to both the membrane potential and the extracellular Cl(-) concentration. Larger anionic molecules can become lodged in the cytoplasmic mouth of the pore where they interact with Arg303, causing a distinct type of open-channel block that is insensitive to membrane potential or extracellular Cl(-) ions. The pore may narrow significantly between the locations of these two blocker binding sites. PMID:17293558

  16. Lithium chloride antileukemic activity in acute promyelocytic leukemia is GSK-3 and MEK/ERK dependent.

    PubMed

    Zassadowski, F; Pokorna, K; Ferre, N; Guidez, F; Llopis, L; Chourbagi, O; Chopin, M; Poupon, J; Fenaux, P; Ann Padua, R; Pla, M; Chomienne, C; Cassinat, B

    2015-12-01

    We recently identified that the MEK/ERK1/2 pathway synergized with retinoic acid (RA) to restore both transcriptional activity and RA-induced differentiation in RA-resistant acute promyelocytic leukemia (APL) cells. To target the MEK/ERK pathway, we identified glycogen synthase kinase-3β (GSK-3β) inhibitors including lithium chloride (LiCl) as activators of this pathway in APL cells. Using NB4 (RA-sensitive) and UF-1 (RA-resistant) APL cell lines, we observed that LiCl as well as synthetic GSK-3β inhibitors decreased proliferation, induced apoptosis and restored, in RA-resistant cells, the expression of RA target genes and the RA-induced differentiation. Inhibition of the MEK/ERK1/2 pathway abolished these effects. These results were corroborated in primary APL patient cells and translated in vivo using an APL preclinical mouse model in which LiCl given alone was as efficient as RA in increasing survival of leukemic mice compared with untreated mice. When LiCl was combined with RA, we observed a significant survival advantage compared with mice treated by RA alone. In this work, we demonstrate that LiCl, a well-tolerated agent in humans, has antileukemic activity in APL and that it has the potential to restore RA-induced transcriptional activation and differentiation in RA-resistant APL cells in an MEK/ERK-dependent manner. PMID:26108692

  17. 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. PMID:21796426

  18. Multi-Ion Mechanism for Ion Permeation and Block in the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

    PubMed Central

    Linsdell, Paul; Tabcharani, Joseph A.; Hanrahan, John W.

    1997-01-01

    The mechanism of Cl− ion permeation through single cystic fibrosis transmembrane conductance regulator (CFTR) channels was studied using the channel-blocking ion gluconate. High concentrations of intracellular gluconate ions cause a rapid, voltage-dependent block of CFTR Cl− channels by binding to a site ∼40% of the way through the transmembrane electric field. The affinity of gluconate block was influenced by both intracellular and extracellular Cl− concentration. Increasing extracellular Cl− concentration reduced intracellular gluconate affinity, suggesting that a repulsive interaction occurs between Cl− and gluconate ions within the channel pore, an effect that would require the pore to be capable of holding more than one ion simultaneously. This effect of extracellular Cl− is not shared by extracellular gluconate ions, suggesting that gluconate is unable to enter the pore from the outside. Increasing the intracellular Cl− concentration also reduced the affinity of intracellular gluconate block, consistent with competition between intracellular Cl− and gluconate ions for a common binding site in the pore. Based on this evidence that CFTR is a multi-ion pore, we have analyzed Cl− permeation and gluconate block using discrete-state models with multiple occupancy. Both two- and three-site models were able to reproduce all of the experimental data with similar accuracy, including the dependence of blocker affinity on external Cl− (but not gluconate) ions and the dependence of channel conductance on Cl− concentration. The three-site model was also able to predict block by internal and external thiocyanate (SCN−) ions and anomalous mole fraction behavior seen in Cl−/SCN− mixtures. PMID:9379169

  19. Multi-Ion mechanism for ion permeation and block in the cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed

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

    1997-10-01

    The mechanism of Cl ion permeation through single cystic fibrosis transmembrane conductance regulator (CFTR) channels was studied using the channel-blocking ion gluconate. High concentrations of intracellular gluconate ions cause a rapid, voltage-dependent block of CFTR Cl channels by binding to a site approximately 40% of the way through the transmembrane electric field. The affinity of gluconate block was influenced by both intracellular and extracellular Cl concentration. Increasing extracellular Cl concentration reduced intracellular gluconate affinity, suggesting that a repulsive interaction occurs between Cl and gluconate ions within the channel pore, an effect that would require the pore to be capable of holding more than one ion simultaneously. This effect of extracellular Cl is not shared by extracellular gluconate ions, suggesting that gluconate is unable to enter the pore from the outside. Increasing the intracellular Cl concentration also reduced the affinity of intracellular gluconate block, consistent with competition between intracellular Cl and gluconate ions for a common binding site in the pore. Based on this evidence that CFTR is a multi-ion pore, we have analyzed Cl permeation and gluconate block using discrete-state models with multiple occupancy. Both two- and three-site models were able to reproduce all of the experimental data with similar accuracy, including the dependence of blocker affinity on external Cl (but not gluconate) ions and the dependence of channel conductance on Cl concentration. The three-site model was also able to predict block by internal and external thiocyanate (SCN) ions and anomalous mole fraction behavior seen in Cl/SCN mixtures. PMID:9379169

  20. Sodium channel activation mechanisms. Insights from deuterium oxide substitution

    SciTech Connect

    Alicata, D.A.; Rayner, M.D.; Starkus, J.G. )

    1990-04-01

    Schauf and Bullock, using Myxicola giant axons, demonstrated that solvent substitution with deuterium oxide (D2O) significantly affects both sodium channel activation and inactivation kinetics without corresponding changes in gating current or tail current rates. They concluded that (a) no significant component of gating current derives from the final channel opening step, and (b) channels must deactivate (during tail currents) by a different pathway from that used in channel opening. By contrast, Oxford found in squid axons that when a depolarizing pulse is interrupted by a brief (approximately 100 microseconds) return to holding potential, subsequent reactivation (secondary activation) is very rapid and shows almost monoexponential kinetics. Increasing the interpulse interval resulted in secondary activation rate returning towards control, sigmoid (primary activation) kinetics. He concluded that channels open and close (deactivate) via the same pathway. We have repeated both sets of observations in crayfish axons, confirming the results obtained in both previous studies, despite the apparently contradictory conclusions reached by these authors. On the other hand, we find that secondary activation after a brief interpulse interval (50 microseconds) is insensitive to D2O, although reactivation after longer interpulse intervals (approximately 400 microseconds) returns towards a D2O sensitivity similar to that of primary activation. We conclude that D2O-sensitive primary activation and D2O-insensitive tail current deactivation involve separate pathways. However, D2O-insensitive secondary activation involves reversal of the D2O-insensitive deactivation step. These conclusions are consistent with parallel gate models, provided that one gating particle has a substantially reduced effective valence.

  1. Regulation of heme oxygenase activity in rat liver during oxidative stress induced by cobalt chloride and mercury chloride.

    PubMed

    Kaliman, P A; Nikitchenko, I V; Sokol, O A; Strel'chenko, E V

    2001-01-01

    Activities of heme oxygenase and tryptophan-2,3-dioxygenase and cytochrome P450 content in liver as well as absorption of the Soret band and optical density at 280 nm in serum were determined 2 and 24 h after administration of HgCl(2) and CoCl(2) and after co-administration of the metal salts with alpha-tocopherol. Administration of HgCl(2) and CoCl(2) increased the contents of hemolysis products in the serum, induced heme oxygenase, and decreased cytochrome P450 content in the liver. Injection of HgCl(2) increased the activity of tryptophan-2,3-dioxygenase holoenzyme and enzyme saturation with the heme, but administration of CoCl(2) decreased these parameters. Pretreatment with alpha-tocopherol completely blocked the changes induced by HgCl(2) after 24 h. Induction of heme oxygenase induced by CoCl(2) was not blocked by alpha-tocopherol, but this antioxidant normalized the increase in the level of hemolysis products in the serum and decrease in tryptophan-2,3-dioxygenase holoenzyme activity and cytochrome P450 content. Mechanisms of regulation of heme oxygenase by mercury and cobalt ions are discussed. PMID:11240397

  2. Phorbol ester activation of chloride current in guinea-pig ventricular myocytes.

    PubMed Central

    Shuba, L. M.; Asai, T.; McDonald, T. F.

    1996-01-01

    1. Although earlier studies with phorbol esters indicate that protein kinase C (PKC) may be an important regulator of Cl- current (Icl) in cardiac cells, there is a need for additional quantitative data and investigation of conflicting findings. Our objectives were to measure the magnitude, time course, and concentration-dependence of Icl activated in guinea-pig ventricular myocytes by phorbol 12-myristate 13-acetate (PMA), evaluate its PKC dependence, and examine its modification by external and internal ions. 2. The whole-cell patch clamp technique was used to apply short depolarizing and hyperpolarizing pulses to myocytes superfused with Na(+)-, K(+)-, Ca(2+)-free solution (36 degrees C) and dialysed with Cs+ solution. Stimulation of membrane currents by PMA (threshold < or = 1nM, EC50 approximately equal to 14 nM, maximal 40% increase with > or = 100 nM) plateaued within 6-10 min. 3. PMA-activated current was time-independent, and suppressed by l mM 9-anthracenecarboxylic acid (9-AC). Its reversal potential (Erev) was sensitive to changes in the Cl- gradient, and outward rectification of the current-voltage (I-V) relationship was more pronounced with 30 mM than 140 mM Cl- dialysate. 4. The relative permeability of PMA-activated channels estimated from Erev measurements was I- > Cl- > > aspartate. Channel activation was independent of external Na+. 5. PMA failed to activate Icl in myocytes pretreated with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or dialysed with pCa 10.5 solution. Lack of response to 4 alpha-phorbol 12, 13-didecanoate (alpha PDD) was a further indication of mediation by PKC. 6. Icl induced by 2 microM forskolin was far larger than that induced by PMA, suggesting that endogenous protein kinase A is a much stronger Cl- channel activator than endogenous PKC in these myocytes. 7. The macroscopic properties of PMA-induced Icl appear to be indistinguishable from those of PKA-activated Icl. We discount stimulation of PKA by PMA as an

  3. Detection of single ion channel activity with carbon nanotubes

    PubMed Central

    Zhou, Weiwei; Wang, Yung Yu; Lim, Tae-Sun; Pham, Ted; Jain, Dheeraj; Burke, Peter J.

    2015-01-01

    Many processes in life are based on ion currents and membrane voltages controlled by a sophisticated and diverse family of membrane proteins (ion channels), which are comparable in size to the most advanced nanoelectronic components currently under development. Here we demonstrate an electrical assay of individual ion channel activity by measuring the dynamic opening and closing of the ion channel nanopores using single-walled carbon nanotubes (SWNTs). Two canonical dynamic ion channels (gramicidin A (gA) and alamethicin) and one static biological nanopore (α-hemolysin (α-HL)) were successfully incorporated into supported lipid bilayers (SLBs, an artificial cell membrane), which in turn were interfaced to the carbon nanotubes through a variety of polymer-cushion surface functionalization schemes. The ion channel current directly charges the quantum capacitance of a single nanotube in a network of purified semiconducting nanotubes. This work forms the foundation for a scalable, massively parallel architecture of 1d nanoelectronic devices interrogating electrophysiology at the single ion channel level. PMID:25778101

  4. Detection of single ion channel activity with carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Zhou, Weiwei; Wang, Yung Yu; Lim, Tae-Sun; Pham, Ted; Jain, Dheeraj; Burke, Peter J.

    2015-03-01

    Many processes in life are based on ion currents and membrane voltages controlled by a sophisticated and diverse family of membrane proteins (ion channels), which are comparable in size to the most advanced nanoelectronic components currently under development. Here we demonstrate an electrical assay of individual ion channel activity by measuring the dynamic opening and closing of the ion channel nanopores using single-walled carbon nanotubes (SWNTs). Two canonical dynamic ion channels (gramicidin A (gA) and alamethicin) and one static biological nanopore (α-hemolysin (α-HL)) were successfully incorporated into supported lipid bilayers (SLBs, an artificial cell membrane), which in turn were interfaced to the carbon nanotubes through a variety of polymer-cushion surface functionalization schemes. The ion channel current directly charges the quantum capacitance of a single nanotube in a network of purified semiconducting nanotubes. This work forms the foundation for a scalable, massively parallel architecture of 1d nanoelectronic devices interrogating electrophysiology at the single ion channel level.

  5. Detection of single ion channel activity with carbon nanotubes.

    PubMed

    Zhou, Weiwei; Wang, Yung Yu; Lim, Tae-Sun; Pham, Ted; Jain, Dheeraj; Burke, Peter J

    2015-01-01

    Many processes in life are based on ion currents and membrane voltages controlled by a sophisticated and diverse family of membrane proteins (ion channels), which are comparable in size to the most advanced nanoelectronic components currently under development. Here we demonstrate an electrical assay of individual ion channel activity by measuring the dynamic opening and closing of the ion channel nanopores using single-walled carbon nanotubes (SWNTs). Two canonical dynamic ion channels (gramicidin A (gA) and alamethicin) and one static biological nanopore (α-hemolysin (α-HL)) were successfully incorporated into supported lipid bilayers (SLBs, an artificial cell membrane), which in turn were interfaced to the carbon nanotubes through a variety of polymer-cushion surface functionalization schemes. The ion channel current directly charges the quantum capacitance of a single nanotube in a network of purified semiconducting nanotubes. This work forms the foundation for a scalable, massively parallel architecture of 1d nanoelectronic devices interrogating electrophysiology at the single ion channel level. PMID:25778101

  6. Phosphatidylinositol-3-kinase regulates mast cell ion channel activity.

    PubMed

    Lam, Rebecca S; Shumilina, Ekaterina; Matzner, Nicole; Zemtsova, Irina M; Sobiesiak, Malgorzata; Lang, Camelia; Felder, Edward; Dietl, Paul; Huber, Stephan M; Lang, Florian

    2008-01-01

    Stimulation of the mast cell IgE-receptor (FcepsilonRI) by antigen leads to stimulation of Ca(2+) entry with subsequent mast cell degranulation and release of inflammatory mediators. Ca(2+) further activates Ca(2+)-activated K(+) channels, which in turn provide the electrical driving force for Ca(2+) entry. Since phosphatidylinositol (PI)-3-kinase has previously been shown to be required for mast cell activation and degranulation, we explored, whether mast cell Ca(2+) and Ca(2+)-activated K(+) channels may be sensitive to PI3-kinase activity. Whole-cell patch clamp experiments and Fura-2 fluorescence measurements for determination of cytosolic Ca(2+) concentration were performed in mouse bone marrow-derived mast cells either treated or untreated with the PI3-kinase inhibitors LY-294002 (10 muM) and wortmannin (100 nM). Antigen-stimulated Ca(2+) entry but not Ca(2+) release from the intracellular stores was dramatically reduced upon PI3-kinase inhibition. Ca(2+) entry was further inhibited by TRPV blocker ruthenium red (10 muM). Ca(2+) entry following readdition after Ca(+)-store depletion with thapsigargin was again decreased by LY-294002, pointing to inhibition of store-operated channels (SOCs). Moreover, inhibition of PI3-kinase abrogated IgE-stimulated, but not ionomycin-induced stimulation of Ca(2+)-activated K(+) channels. These observations disclose PI3-kinase-dependent regulation of Ca(2+) entry and Ca(2+)-activated K(+)-channels, which in turn participate in triggering mast cell degranulation. PMID:18769043

  7. Voltage-dependent and -independent titration of specific residues accounts for complex gating of a ClC chloride channel by extracellular protons.

    PubMed

    Niemeyer, María Isabel; Cid, L Pablo; Yusef, Yamil R; Briones, Rodolfo; Sepúlveda, Francisco V

    2009-04-01

    The ClC transport protein family comprises both Cl(-) ion channel and H(+)/Cl(-) and H(+)/NO(3)(-) 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 approximately 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 pK(a) 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

  8. 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

  9. TRPV3 channels mediate strontium-induced mouse egg activation

    PubMed Central

    Carvacho, Ingrid; Lee, Hoi Chang; Fissore, Rafael A.; Clapham, David E.

    2014-01-01

    SUMMARY In mammals, calcium influx is required for oocyte maturation and egg activation. The molecular identities of the calcium-permeant channels that underlie the initiation of embryonic development are not established. Here, we describe a Transient Receptor Potential (TRP) ion channel current activated by TRP agonists that is absent in TrpV3−/− eggs. TRPV3 current is differentially expressed during oocyte maturation, reaching a peak of maximum density and activity at metaphase of meiosis II (MII), the stage of fertilization. Selective activation of TRPV3 channels provokes egg activation by mediating massive calcium entry. Widely used to activate eggs, strontium application is known to yield normal offspring in combination with somatic cell nuclear transfer. We show that TRPV3 is required for strontium influx, as TrpV3−/− eggs failed to permeate Sr2+ or undergo strontium-induced activation. We propose that TRPV3 is the major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation. PMID:24316078

  10. Direct and indirect effects of mutations at the outer mouth of the cystic fibrosis transmembrane conductance regulator chloride channel pore.

    PubMed

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

    2007-04-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore is thought to contain multiple binding sites for permeant and impermeant anions. Here, we investigate the effects of mutation of different positively charged residues in the pore on current inhibition by impermeant Pt(NO(2)) (4) (2-) and suramin anions. We show that mutations that remove positive charges (K95, R303) influence interactions with intracellular, but not extracellular, Pt(NO(2))(4)(2-) ions, consistent with these residues being situated within the pore inner vestibule. In contrast, mutation of R334, supposedly located in the outer vestibule of the pore, affects block by both extracellular and intracellular Pt(NO(2))(4)(2-). Inhibition by extracellular Pt(NO(2))(4)(2-) requires a positive charge at position 334, consistent with a direct electrostatic interaction resulting in either open channel block or surface charge screening. In contrast, inhibition by intracellular Pt(NO(2))(4)(2-) is weakened in all R334-mutant forms of the channel studied, inconsistent with a direct interaction. Furthermore, mutation of R334 had similar effects on block by intracellular suramin, a large organic molecule that is apparently unable to enter deeply into the channel pore. Mutation of R334 altered interactions between intracellular Pt(NO(2))(4)(2-) and extracellular Cl(-) but not those between intracellular Pt(NO(2))(4)(2-) and extracellular Pt(NO(2))(4)(2-). We propose that while the positive charge of R334 interacts directly with extracellular anions, mutation of this residue also alters interactions with intracellular anions by an indirect mechanism, due to mutation-induced conformational changes in the protein that are propagated some distance from the site of the mutation in the outer mouth of the pore. PMID:17673962

  11. On the origin of asymmetric interactions between permeant anions and the cystic fibrosis transmembrane conductance regulator chloride channel pore.

    PubMed

    Fatehi, Mohammad; St Aubin, Chantal N; Linsdell, Paul

    2007-02-15

    Single channel and macroscopic current recording was used to investigate block of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore by the permeant anion Au(CN)2(-). Block was 1-2 orders of magnitude stronger when Au(CN)2(-) was added to the intracellular versus the extracellular solution, depending on membrane potential. A point mutation within the pore, T-338A, strongly decreased the asymmetry of block, by weakening block by intracellular Au(CN)2(-) and at the same time strengthening block by external Au(CN)2(-). Block of T-338A, but not wild-type, was strongest at the current reversal potential and weakened by either depolarization or hyperpolarization. In contrast to these effects, the T-338A mutation had no impact on block by the impermeant Pt(NO2)4(2-) ion. We suggest that the CFTR pore has at least two anion binding sites at which Au(CN)2(-) and Pt(NO2)4(2-) block Cl- permeation. The T-338A mutation decreases a barrier for Au(CN)2(-) movement between different sites, leading to significant changes in its blocking action. Our finding that apparent blocker binding affinity can be altered by mutagenesis of a residue which does not contribute to a blocker binding site has important implications for interpreting the effects of mutagenesis on channel blocker effects. PMID:17142267

  12. Physiological mechanisms for the modulation of pannexin 1 channel activity

    PubMed Central

    Sandilos, Joanna K; Bayliss, Douglas A

    2012-01-01

    It is widely recognized that ATP, along with other nucleotides, subserves important intercellular signalling processes. Among various nucleotide release mechanisms, the relatively recently identified pannexin 1 (Panx1) channel is gaining prominence by virtue of its ability to support nucleotide permeation and release in a variety of different tissues. Here, we review recent advances in our understanding of the factors that control Panx1 channel activity. By using electrophysiological and biochemical approaches, diverse mechanisms that dynamically regulate Panx1 channel function have been identified in various settings; these include, among others, activation by caspase-mediated channel cleavage in apoptotic immune cells, by G protein-coupled receptors in vascular smooth muscle, by low oxygen tension in erythrocytes and neurons, by high extracellular K+ in various cell types and by stretch/strain in airway epithelia. Delineating the distinct mechanisms of Panx1 modulation that prevail in different physiological contexts provides the possibility that these channels, and ATP release, could ultimately be targeted in a context-dependent manner. PMID:23070703

  13. Cloning and characterization of CLCN5, the human kidney chloride channel gene implicated in Dent disease (an X-linked hereditary nephrolithiasis).

    PubMed

    Fisher, S E; van Bakel, I; Lloyd, S E; Pearce, S H; Thakker, R V; Craig, I W

    1995-10-10

    Dent disease, an X-linked familial renal tubular disorder, is a form of Fanconi syndrome associated with proteinuria, hypercalciuria, nephrocalcinosis, kidney stones, and eventual renal failure. We have previously used positional cloning to identify the 3' part of a novel kidney-specific gene (initially termed hClC-K2, but now referred to as CLCN5), which is deleted in patients from one pedigree segregating Dent disease. Mutations that disrupt this gene have been identified in other patients with this disorder. Here we describe the isolation and characterization of the complete open reading frame of the human CLCN5 gene, which is predicted to encode a protein of 746 amino acids, with significant homology to all known members of the ClC family of voltage-gated chloride channels. CLCN5 belongs to a distinct branch of this family, which also includes the recently identified genes CLCN3 and CLCN4. We have shown that the coding region of CLCN5 is organized into 12 exons, spanning 25-30 kb of genomic DNA, and have determined the sequence of each exon-intron boundary. The elucidation of the coding sequence and exon-intron organization of CLCN5 will both expedite the evaluation of structure/function relationships of these ion channels and facilitate the screening of other patients with renal tubular dysfunction for mutations at this locus. PMID:8575751

  14. The Effects of the KCNQ Openers Retigabine and Flupirtine on Myotonia in Mammalian Skeletal Muscle Induced by a Chloride Channel Blocker.

    PubMed

    Su, Tzu-Rong; Zei, Wen-Shan; Su, Ching-Chyuan; Hsiao, George; Lin, Min-Jon

    2012-01-01

    The purpose of this study was to investigate the effect of KCNQ (potassium channel, voltage-gated, KQT-like subfamily) openers in preventing myotonia caused by anthracene-9-carboxylic acid (9-AC, a chloride channel blocker). An animal model of myotonia can be elicited in murine skeletal muscle by 9-AC treatment. KCNQ openers, such as retigabine and flupirtine, can inhibit the increased twitch amplitude (0.1 Hz stimulation) and reduce the tetanic fade (20 Hz stimulations) observed in the presence of 9-AC. Furthermore, the prolonged twitch duration of skeletal muscle was also inhibited by retigabine or flupirtine. Lamotrigine (an anticonvulsant drug) has a lesser effect on the muscle twitch amplitude, tetanic fade, and prolonged twitch duration as compared with KCNQ openers. In experiments using intracellular recordings, retigabine and flupirtine clearly reduced the firing frequencies of repetitive action potentials induced by 9-AC. These data suggested that KCNQ openers prevent the myotonia induced by 9-AC, at least partly through enhancing potassium conductance in skeletal muscle. Taken together, these results indicate that KCNQ openers are potential alternative therapeutic agents for the treatment of myotonia. PMID:22536291

  15. Cloning and characterization of CLCN5, the human kidney chloride channel gene implicated in Den