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Sample records for cftr anion channel

  1. CFTR anion channel modulates expression of human transmembrane mucin MUC3 through the PDZ protein GOPC.

    Science.gov (United States)

    Pelaseyed, Thaher; Hansson, Gunnar C

    2011-09-15

    The transmembrane mucins in the enterocyte are type 1 transmembrane proteins with long and rigid mucin domains, rich in proline, threonine and serine residues that carry numerous O-glycans. Three of these mucins, MUC3, MUC12 and MUC17 are unique in harboring C-terminal class I PDZ motifs, making them suitable ligands for PDZ proteins. A screening of 123 different human PDZ domains for binding to MUC3 identified a strong interaction with the PDZ protein GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). This interaction was mediated by the C-terminal PDZ motif of MUC3, binding to the single GOPC PDZ domain. GOPC is also a binding partner for cystic fibrosis transmembrane conductance regulator (CFTR) that directs CFTR for degradation. Overexpression of GOPC downregulated the total levels of MUC3, an effect that was reversed by introducing CFTR. The results suggest that CFTR and MUC3 compete for binding to GOPC, which in turn can regulate levels of these two proteins. For the first time a direct coupling between mucins and the CFTR channel is demonstrated, a finding that will shed further light on the still poorly understood relationship between cystic fibrosis and the mucus phenotype of this disease.

  2. How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

    Science.gov (United States)

    Zwick, Matthias; Esposito, Cinzia; Hellstern, Manuel; Seelig, Anna

    2016-07-01

    The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators. PMID:27226582

  3. Regulated trafficking of the CFTR chloride channel

    NARCIS (Netherlands)

    Braakman, L.J.; Kleizen, B.; Jonge, H.R. de

    2000-01-01

    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. Wh

  4. Differential expression of gill Na+,K+-ATPaseα - and β-subunits, Na+,K+,2Cl- cotransporter and CFTR anion channel in juvenile anadromous and landlocked Atlantic salmon Salmo salar

    Science.gov (United States)

    Nilsen, Tom O.; Ebbesson, Lars O.E.; Madsen, Steffen S.; McCormick, Stephen D.; Andersson, Eva; Bjornsson, Bjorn Thrandur; Prunet, Patrick; Stefansson, Sigurd O.

    2007-01-01

    differences in NKA, both during preparatory development and during salinity adjustments in salmon. Furthermore, landlocked salmon have lost some of the unique preparatory upregulation of gill NKA, NKCC and, to some extent, CFTR anion channel associated with the development of hypo-osmoregulatory ability in anadromous salmon.

  5. Functional reconstitution and channel activity measurements of purified wildtype and mutant CFTR protein.

    Science.gov (United States)

    Eckford, Paul D W; Li, Canhui; Bear, Christine E

    2015-03-09

    The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

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

    OpenAIRE

    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 epi...

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

    Science.gov (United States)

    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

  8. Endocytic Trafficking of CFTR in Health and Disease

    OpenAIRE

    Ameen, Nadia; Silvis, Mark; Bradbury, Neil A.

    2006-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl-selective anion channel expressed in epithelial tissues. Mutations in CFTR lead to the debilitating genetic disease cystic fibrosis (CF). Within each epithelial cell, CFTR interacts with a large number of transient macromolecular complexes, many of which are involved in the trafficking and targeting of CFTR. Understanding how these complexes regulate the trafficking and fate of CFTR, provides a singular insight not only in...

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

    Science.gov (United States)

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

    2013-05-01

    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.

  10. Influence of salinity on the localization of Na+/K +-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and CFTR anion channel in chloride cells of the Hawaiian goby (Stenogobius hawaiiensis)

    Science.gov (United States)

    McCormick, S.D.; Sundell, K.; Bjornsson, Bjorn Thrandur; Brown, C.L.; Hiroi, J.

    2003-01-01

    Na+/K+-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR) are the three major transport proteins thought to be involved in chloride secretion in teleost fish. If this is the case, the levels of these transporters should be high in chloride cells of seawater-acclimated fish. We therefore examined the influence of salinity on immunolocalization of Na +/K+-ATPase, NKCC and CFTR in the gills of the Hawaiian goby (Stenogobius hawaiiensis). Fish were acclimated to freshwater and 20??? and 30??? seawater for 10 days. Na+/K +-ATPase and NKCC were localized specifically to chloride cells and stained throughout most of the cell except for the nucleus and the most apical region, indicating a basolateral/tubular distribution. All Na+/K +-ATPase-positive chloride cells were also positive for NKCC in all salinities. Salinity caused a slight increase in chloride cell number and size and a slight decrease in staining intensity for Na+/K +-ATPase and NKCC, but the basic pattern of localization was not altered. Gill Na+/K+-ATPase activity was also not affected by salinity. CFTR was localized to the apical surface of chloride cells, and only cells staining positive for Na+/K+-ATPase were CFTR-positive. CFTR-positive cells greatly increased in number (5-fold), area stained (53%) and intensity (29%) after seawater acclimation. In freshwater, CFTR immunoreactivity was light and occurred over a broad apical surface on chloride cells, whereas in seawater there was intense immunoreactivity around the apical pit (which was often punctate in appearance) and a light subapical staining. The results indicate that Na+/K +-ATPase, NKCC and CFTR are all present in chloride cells and support current models that all three are responsible for chloride secretion by chloride cells of teleost fish.

  11. Steviol reduces MDCK Cyst formation and growth by inhibiting CFTR channel activity and promoting proteasome-mediated CFTR degradation.

    Directory of Open Access Journals (Sweden)

    Chaowalit Yuajit

    Full Text Available Cyst enlargement in polycystic kidney disease (PKD involves cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR chloride channel. This study aimed to investigate an inhibitory effect and detailed mechanisms of steviol and its derivatives on cyst growth using a cyst model in Madin-Darby canine kidney (MDCK cells. Among 4 steviol-related compounds tested, steviol was found to be the most potent at inhibiting MDCK cyst growth. Steviol inhibition of cyst growth was dose-dependent; steviol (100 microM reversibly inhibited cyst formation and cyst growth by 72.53.6% and 38.2±8.5%, respectively. Steviol at doses up to 200 microM had no effect on MDCK cell viability, proliferation and apoptosis. However, steviol acutely inhibited forskolin-stimulated apical chloride current in MDCK epithelia, measured with the Ussing chamber technique, in a dose-dependent manner. Prolonged treatment (24 h with steviol (100 microM also strongly inhibited forskolin-stimulated apical chloride current, in part by reducing CFTR protein expression in MDCK cells. Interestingly, proteasome inhibitor, MG-132, abolished the effect of steviol on CFTR protein expression. Immunofluorescence studies demonstrated that prolonged treatment (24 h with steviol (100 microM markedly reduced CFTR expression at the plasma membrane. Taken together, the data suggest that steviol retards MDCK cyst progression in two ways: first by directly inhibiting CFTR chloride channel activity and second by reducing CFTR expression, in part, by promoting proteasomal degradation of CFTR. Steviol and related compounds therefore represent drug candidates for treatment of polycystic kidney disease.

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

    OpenAIRE

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

    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...

  13. CFTR channel in oocytes from Xenopus laevis and its regulation by xShroom1 protein.

    Science.gov (United States)

    Palma, Alejandra G; Galizia, Luciano; Kotsias, Basilio A; Marino, Gabriela I

    2016-05-01

    Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in Xenopus laevis oocytes, and it is required for the expression of the epithelial sodium channel (ENaC). As there is a close relationship between ENaC and the cystic fibrosis transmembrane regulator (CFTR), we examined the action of xShroom1 on CFTR expression and activity. Biotinylation was used to measure CFTR surface expression, and currents were registered with voltage clamp when stimulated with forskolin and 3-isobutyl-1-methylxanthine. Oocytes were coinjected with CFTR complementary RNAs (cRNAs) and xShroom1 sense or antisense oligonucleotides. We observed an increment in CFTR currents and CFTR surface expression in oocytes coinjected with CFTR and xShroom1 antisense oligonucleotides. MG-132, a proteasome inhibitor, did not prevent the increment in currents when xShroom1 was suppressed by antisense oligonucleotides. In addition, we inhibited the delivery of newly synthesized proteins to the plasma membrane with BFA and we found that the half-life of plasma membrane CFTR was prolonged when coinjected with the xShroom1 antisense oligonucleotides. Chloroquine, an inhibitor of the late endosome/lysosome, did not significantly increase CFTR currents when xShroom1 expression was inhibited. The higher expression of CFTR when xShroom1 is suppressed is in concordance with the functional studies suggesting that the suppression of the xShroom1 protein resulted in an increment in CFTR currents by promoting the increase of the half-life of CFTR in the plasma membrane. The role of xShroom1 in regulating CFTR expression could be relevant in the understanding of the channel malfunction in several diseases.

  14. Functional role of anion channels in cardiac diseases

    Institute of Scientific and Technical Information of China (English)

    Da-yue DUAN; Luis LH LIU; Nathan BOZEAT; Z Maggie HUANG; Sunny Y XIANG; Guan-lei WANG; Linda YE; Joseph R HUME

    2005-01-01

    In comparison to cation (K+, Na+, and Ca2+) channels, much less is currently known about the functional role of anion (Cl-) channels in cardiovascular physiology and pathophysiology. Over the past 15 years, various types of Cl- currents have been recorded in cardiac cells from different species including humans. All cardiac Cl- channels described to date may be encoded by five different Cl- channel genes: the PKA- and PKC-activated cystic fibrosis tansmembrane conductance regulator (CFTR), the volume-regulated ClC-2 and ClC-3, and the Ca2+-activated CLCA or Bestrophin. Recent studies using multiple approaches to examine the functional role of Cl- channels in the context of health and disease have demonstrated that Cl- channels might contribute to: 1) arrhythmogenesis in myocardial injury; 2) cardiac ischemic preconditioning; and 3) the adaptive remodeling of the heart during myocardial hypertrophy and heart failure. Therefore,anion channels represent very attractive novel targets for therapeutic approaches to the treatment of heart diseases. Recent evidence suggests that Cl- channels,like cation channels, might function as a multiprotein complex or functional module.In the post-genome era, the emergence of functional proteomics has necessitated a new paradigm shift to the structural and functional assessment of integrated Cl- channel multiprotein complexes in the heart, which could provide new insight into our understanding of the underlying mechanisms responsible for heart disease and protection.

  15. Rattlesnake Phospholipase A2 Increases CFTR-Chloride Channel Current and Corrects ∆F508CFTR Dysfunction: Impact in Cystic Fibrosis.

    Science.gov (United States)

    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

  16. Activation Effect of Cathartic Natural Compound Rhein to CFTR Chloride Channel

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed in intestinal exocrine glands, which plays a key role in intestinal fluid secretion. A natural anthraquinone activator of CFTR Cl- channel, rhein, was identified by screening 217 single compounds from Chinese herbs via a cellbased halide-sensitive fluorescent assay. Rhein activates CFTR Cl- transportation in a dose-dependent manner in the presence of cAMP with a physiological concentration. This study provides a novel molecular pharmacological mechanism for the laxative drugs in Traditional Chinese Medicine such as aloe, cascara and senna.

  17. Characterization of a critical role for CFTR chloride channels in cardioprotection against ischemia/reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Sunny Yang XIANG; Linda L YE; LI-lu Marie DUAN; Li-hui LIU; Zhi-dong GE; John A AUCHAMPACH; Garrett J GROSS; Dayue Darrel DUAN

    2011-01-01

    Aim: To further characterize the functional role of cystic fibrosis transmembrane conductance regulator (CFTR) in early and late (second window) ischemic preconditioning (IPC)- and postcondtioning (POC)-mediated cardioprotection against ischemia/reperfusion (I/R) injury.Methods: CFTR knockout (CFTR-/-) mice and age- and gender-matched wild-type (CFTR+/+) and heterozygous (CFTR+/-) mice were used.In in vivo studies, the animals were subjected to a 30-min coronary occlusion followed by a 40-min reperfusion. In ex vivo (isolate heart) studies, a 45-min global ischemia was applied. To evaluate apoptosis, the level of activated caspase 3 and TdT-mediated dUTP-X nick end labeling (TUNEL) were examined.Results: In the in vivo I/R models, early IPC significantly reduced the myocardial infarct size in wild-type (CFTR+/+) (from 40.4%±5.3% to 10.4%±2.0%, n=8, P<0.001) and heterozygous (CFTR+/-) littermates (from 39.4%±2.4% to 15.4%±5.1%, n=6, P<0.001) but failed to protect CFTR knockout (CFTR-/-) mice from I/R induced myocardial infarction (46.9%±6.2% vs 55.5%±7.8%, n=6, P>0.5). Similar results were observed in the in vivo late IPC experiments. Furthermore, in both in vivo and ex vivo I/R models, POC significantly reduced myocardial infarction in wild-type mice, but not in CFTR knockout mice. In ex vivo I/R models, targeted inactivation of CFTRgene abolished the protective effects of IPC against I/R-induced apoptosis.Conclusion: These results provide compelling evidence for a critical role for CFTR Cl- channels in IPC- and POC-mediated cardioprotection against I/R-induced myocardial injury.

  18. Functional classification of mitochondrion-rich cells in euryhaline Mozambique tilapia (Oreochromis mossambicus) embryos, by means of triple immunofluorescence staining for Na+/K+-ATPase, Na +/K+/2Cl- cotransporter and CFTR anion channel

    Science.gov (United States)

    Hiroi, J.; McCormick, S.D.; Ohtani-Kaneko, R.; Kaneko, T.

    2005-01-01

    Mozambique tilapia Oreochromis mossambicus embryos were transferred from freshwater to seawater and vice versa, and short-term changes in the localization of three major ion transport proteins, Na+/K +-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR) were examined within mitochondrion-rich cells (MRCs) in the embryonic yolk-sac membrane. Triple-color immunofluorescence staining allowed us to classify MRCs into four types: type I, showing only basolateral Na+/K +-ATPase staining; type II, basolateral Na+/K +-ATPase and apical NKCC; type III, basolateral Na+/K +-ATPase and basolateral NKCC; type IV, basolateral Na +/K+-ATPase, basolateral NKCC and apical CFTR. In freshwater, type-I, type-II and type-III cells were observed. Following transfer from freshwater to seawater, type-IV cells appeared at 12 h and showed a remarkable increase in number between 24 h and 48 h, whereas type-III cells disappeared. When transferred from seawater back to freshwater, type-IV cells decreased and disappeared at 48 h, type-III cells increased, and type-II cells, which were not found in seawater, appeared at 12 h and increased in number thereafter. Type-I cells existed consistently irrespective of salinity changes. These results suggest that type I is an immature MRC, type II is a freshwater-type ion absorptive cell, type III is a dormant type-IV cell and/or an ion absorptive cell (with a different mechanism from type II), and type IV is a seawater-type ion secretory cell. The intracellular localization of the three ion transport proteins in type-IV cells is completely consistent with a widely accepted model for ion secretion by MRCs. A new model for ion absorption is proposed based on type-II cells possessing apical NKCC.

  19. A High-affinity Activator of G551D-CFTR Chloride Channel Identified By High Throughput Screening

    Institute of Scientific and Technical Information of China (English)

    ZHAO Lu; HE Cheng-yan; LIU Yan-li; ZHOU Hong-lan; ZHOU Jin-song; SHANG De-jing; YANG Hong

    2004-01-01

    A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(Kd=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation.

  20. Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop.

    Science.gov (United States)

    Ehrhardt, Annette; Chung, W Joon; Pyle, Louise C; Wang, Wei; Nowotarski, Krzysztof; Mulvihill, Cory M; Ramjeesingh, Mohabir; Hong, Jeong; Velu, Sadanandan E; Lewis, Hal A; Atwell, Shane; Aller, Steve; Bear, Christine E; Lukacs, Gergely L; Kirk, Kevin L; Sorscher, Eric J

    2016-01-22

    In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating.

  1. CFTR chloride channel as a molecular target of anthraquinone compounds in herbal laxatives

    Institute of Scientific and Technical Information of China (English)

    Hong YANG; Li-na XU; Cheng-yan HE; Xin LIU; Rou-yu FANG; Tong-hui MA

    2011-01-01

    Aim: To clarify whether CFTR is a molecular target of intestinal fluid secretion caused by the anthraquinone compounds from laxative herbal plants.Methods: A cell-based fluorescent assay to measure I- influx through CFTR chloride channel. A short-circuit current assay to measure transcellular Cl- current across single layer FRT cells and freshly isolated colon mucosa. A closed loop experiment to measure colon fluid secretion in vivo.Results: Anthraquinone compounds rhein, aloe-emodin and 1,8-dihydroxyanthraquinone (DHAN) stimulated l- influx through CFTR chloride channel in a dose-dependent manner in the presence of physiological concentration of cAMP. In the short-circuit current assay,the three compound enhanced Cl- currents in epithelia formed by CFTR-expressing FRT cells with EC5o values of 73±1.4, 56±1.7, and 50±0.5 μmol/L, respectively, and Rhein also enhanced Cl- current in freshly isolated rat colonic mucosa with a similar potency. These effects were completely reversed by the CFTR selective blocker CFTRinh-172. In in vivo closed loop experiments, rhein 2 mmol/L stimu-lated colonic fluid accumulation that was largely blocked by CFTRinh-172. The anthraquinone compounds did not elevate cAMP level in cultured FRT cells and rat colonic mucosa, suggesting a direct effect on CFTR activity.Conclusion: Natural anthraquinone compounds in vegetable laxative drugs are CFTR potsntiators that stimulated colonic chloride and fluid secretion. Thus CFTR chloride channel is a molecular target of vegetable laxative drugs.

  2. Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.

    OpenAIRE

    Jessica LaRusch; Jinsei Jung; General, Ignacio J.; Lewis, Michele D; Hyun Woo Park; Brand, Randall E.; Andres Gelrud; Anderson, Michelle A.; Banks, Peter A; Darwin Conwell; Christopher Lawrence; Joseph Romagnuolo; John Baillie; Samer Alkaade; Gregory Cote

    2014-01-01

    CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev ) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize tha...

  3. Synthesis and Characterization of A Small Molecule CFTR Chloride Channel Inhibitor

    Institute of Scientific and Technical Information of China (English)

    HE Cheng-yan; ZHANG Heng-jun; SU Zhong-min; ZHOU Jin-song; YANG Hong; MA Tong-hui

    2004-01-01

    A thiazolidinone CFTR inhibitor(CFTRinh-172) was synthesized by a three-step procedure with trifluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally by means of 1H NMR and functionally in a CFTR-expressing cell line FRT/hCFTR/EYFP-H148Q by both fluorescent and electrophysiological methods. A large amount(100 g) of high-quality small molecule thiazolidinone CFTR chloride channel inhibitor, CFTRinh-172, can be produced with this simple three-step synthetic procedure. The structure of the final product 2-thioxo-3-(3-trifluromethylphenyl)-5-[4-carboxyphenyl-methylene]-4-thiazolidinone was confirmed by 1H NMR. The overall yield was 58% with a purity over 99% as analyzed by HPLC. The synthesized CFTRinh-172 specifically inhibited CFTR chloride channel function in a cell-based fluorescence assay(Kd≈1.5 μmol/L) and in a Ussing chamber-based short-circuit current assay(Kd≈0.2 μmol/L), indicating better quality than that of the commercial combinatorial compound. The synthesized inhibitor is nontoxic to cultured cells at a high concentration and to mouse at a high dose. The synthetic procedure developed here can be used to produce a large amount of the high-quality CFTRinh-172 suitable for antidiarrheal studies and for creation of cystic fibrosis models in large animals. The procedure can be used to synthesize radiolabled CFTRinh-172 for in vivo pharmacokinetics studies.

  4. The K+ channel opener 1-EBIO potentiates residual function of mutant CFTR in rectal biopsies from cystic fibrosis patients.

    Directory of Open Access Journals (Sweden)

    Eva K Roth

    Full Text Available BACKGROUND: The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF. Previous studies demonstrated that the K⁺ channel opener 1-ethyl-2-benzimidazolone (1-EBIO potentiates CFTR-mediated Cl⁻ secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown. METHODS: We studied the effects of 1-EBIO on CFTR-mediated Cl⁻ secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl⁻ secretion. RESULTS: Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl⁻ secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl⁻ secretion by 39.2±6.7% (P<0.001 via activation of basolateral Ca²⁺-activated and clotrimazole-sensitive KCNN4 K⁺ channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl⁻ secretion in tissues with residual CFTR function by 44.4±11.5% (P<0.001, but had no effect on tissues lacking CFTR-mediated Cl⁻ conductance. CONCLUSIONS: We conclude that 1-EBIO potentiates Cl⁻secretion in native CF tissues expressing CFTR mutants with residual Cl⁻ channel function by activation of basolateral KCNN4 K⁺ channels that increase the driving force for luminal Cl⁻ exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF.

  5. Molecular physiology of EAAT anion channels.

    Science.gov (United States)

    Fahlke, Christoph; Kortzak, Daniel; Machtens, Jan-Philipp

    2016-03-01

    Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. After release from presynaptic nerve terminals, glutamate is quickly removed from the synaptic cleft by a family of five glutamate transporters, the so-called excitatory amino acid transporters (EAAT1-5). EAATs are prototypic members of the growing number of dual-function transport proteins: they are not only glutamate transporters, but also anion channels. Whereas the mechanisms underlying secondary active glutamate transport are well understood at the functional and at the structural level, mechanisms and cellular roles of EAAT anion conduction have remained elusive for many years. Recently, molecular dynamics simulations combined with simulation-guided mutagenesis and experimental analysis identified a novel anion-conducting conformation, which accounts for all experimental data on EAAT anion currents reported so far. We here review recent findings on how EAATs accommodate a transporter and a channel in one single protein. PMID:26687113

  6. Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis.

    Science.gov (United States)

    Liang, Xiubin; Da Paula, Ana Carina; Bozóky, Zoltán; Zhang, Hui; Bertrand, Carol A; Peters, Kathryn W; Forman-Kay, Julie D; Frizzell, Raymond A

    2012-03-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)-regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3β, γ, and ε isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3β and ε binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3β increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3β knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3β interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3β interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.

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

    Science.gov (United States)

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

    2015-10-01

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

  8. Purinergic regulation of CFTR and Ca2+ -activated Cl- channels and K+ channels in human pancreatic duct epithelium

    DEFF Research Database (Denmark)

    Wang, Jing; Haanes, Kristian A; Novak, Ivana

    2013-01-01

    dependent on intracellular Ca(2+). Apically applied ATP/UTP stimulated CF transmembrane conductance regulator (CFTR) and Ca(2+)-activated Cl(-) (CaCC) channels, which were inhibited by CFTRinh-172 and niflumic acid, respectively. The basolaterally applied ATP stimulated CFTR. In CFPAC-1 cells, which have...... mutated CFTR, basolateral ATP and UTP had negligible effects. In addition to Cl(-) transport in Capan-1 cells, the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DC-EBIO) and clotrimazole indicated functional expression of the intermediate conductance K(+) channels (IK, KCa3.1). The...... receptors both Cl(-) channels (TMEM16A/ANO1 and CFTR) and K(+) channels (IK). The K(+) channels provide the driving force for Cl(-)-channel-dependent secretion, and luminal ATP provided locally or secreted from acini may potentiate secretory processes. Future strategies in augmenting pancreatic duct...

  9. Regulation of CFTR Cl− channel gating by ATP binding and hydrolysis

    OpenAIRE

    Ikuma, Mutsuhiro; Welsh, Michael J.

    2000-01-01

    Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg2+ prevents hydrolysis. We found that even in the absence of Mg2+, ATP could support cha...

  10. Thermal stability of purified and reconstituted CFTR in a locked open channel conformation.

    Science.gov (United States)

    Aleksandrov, Luba A; Jensen, Timothy J; Cui, Liying; Kousouros, Joseph N; He, Lihua; Aleksandrov, Andrei A; Riordan, John R

    2015-12-01

    CFTR is unique among ABC transporters as the only one functioning as an ion channel and from a human health perspective because mutations in its gene cause cystic fibrosis. Although considerable advances have been made towards understanding CFTR's mechanism of action and the impact of mutations, the lack of a high-resolution 3D structure has hindered progress. The large multi-domain membrane glycoprotein is normally present at low copy number and when over expressed at high levels it aggregates strongly, limiting the production of stable mono-disperse preparations. While the reasons for the strong self-association are not fully understood, its relatively low thermal stability seems likely to be one. The major CF causing mutation, ΔF508, renders the protein very thermally unstable and therefore a great deal of attention has been paid to this property of CFTR. Multiple second site mutations of CFTR in NBD1 where F508 normally resides and small molecule binders of the domain increase the thermal stability of the mutant. These manipulations also stabilize the wild-type protein. Here we have applied ΔF508-stabilizing changes and other modifications to generate wild-type constructs that express at much higher levels in scaled-up suspension cultures of mammalian cells. After purification and reconstitution into liposomes these proteins are active in a locked-open conformation at temperatures as high as 50 °C and remain monodisperse at 4 °C in detergent or lipid for at least a week. The availability of adequate amounts of these and related stable active preparations of homogeneous CFTR will enable stalled structural and ligand binding studies to proceed.

  11. Identification of Herbal Compound lmperatorin with Adverse Effects on ANO1 and CFTR Chloride Channels

    Institute of Scientific and Technical Information of China (English)

    HAO Feng; YI Fei; ZHANG Di; NING Yan; SU Wei-heng; FENG Xue-chao; YANG Hong; MA Tong-hui

    2011-01-01

    Calcium-activated chloride channels(CaCCs) are the crucial regulators of transepithelial fluid secretion,smooth muscle contraction and sensory transduction. Recently, compelling evidence has indicated that TMEM 16A(ANO 1 or anoctamin-i ) is a bona fide calcium-acvtivated chloride channel. A few small molecule CaCCs regulators are available for functional and therapeutic studies. We screened 126 natural compounds from Chinese herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells to coexpress ANOI and an iodide-sensitive fluorescent indicator(EYFP-HI48Q/I152L). lmperatorin, a coumarin compound, was identifled to inhibit ANOl-mediated chloride transport activated by multiple calcium-elevating agonists. The inhibitory effect is dose-dependent with IC50 ~14.63 μmol/L. Interestingly, imperatorin activated CFTR chloride channel with EC50 ~35.52 μmol/L. The adverse effects of imperatorin on CaCC and CFTR chloride channels will make it useful in pharmacological dissection of chloride transport in airway and intestinal epithelium. Further studies are required to evaluate the therapeutic effects of imperatorin on hypertension, asthma and certain tumors.

  12. ∆F508 CFTR interactome remodelling promotes rescue of cystic fibrosis.

    Science.gov (United States)

    Pankow, Sandra; Bamberger, Casimir; Calzolari, Diego; Martínez-Bartolomé, Salvador; Lavallée-Adam, Mathieu; Balch, William E; Yates, John R

    2015-12-24

    Deletion of phenylalanine 508 of the cystic fibrosis transmembrane conductance regulator (∆F508 CFTR) is the major cause of cystic fibrosis, one of the most common inherited childhood diseases. The mutated CFTR anion channel is not fully glycosylated and shows minimal activity in bronchial epithelial cells of patients with cystic fibrosis. Low temperature or inhibition of histone deacetylases can partly rescue ∆F508 CFTR cellular processing defects and function. A favourable change of ∆F508 CFTR protein-protein interactions was proposed as a mechanism of rescue; however, CFTR interactome dynamics during temperature shift and inhibition of histone deacetylases are unknown. Here we report the first comprehensive analysis of the CFTR and ∆F508 CFTR interactome and its dynamics during temperature shift and inhibition of histone deacetylases. By using a novel deep proteomic analysis method, we identify 638 individual high-confidence CFTR interactors and discover a ∆F508 deletion-specific interactome, which is extensively remodelled upon rescue. Detailed analysis of the interactome remodelling identifies key novel interactors, whose loss promote ∆F508 CFTR channel function in primary cystic fibrosis epithelia or which are critical for CFTR biogenesis. Our results demonstrate that global remodelling of ∆F508 CFTR interactions is crucial for rescue, and provide comprehensive insight into the molecular disease mechanisms of cystic fibrosis caused by deletion of F508.

  13. Differential expression of gill Na+,K+-ATPase alpha- and beta-subunits, Na+,K+,2Cl- cotransporter and CFTR anion channel in juvenile anadromous and landlocked Atlantic salmon Salmo salar

    DEFF Research Database (Denmark)

    Nilsen, Tom O.; Ebbesson, Lars O. E.; Madsen, Steffen S.;

    2007-01-01

    This study examines changes in gill Na(+),K(+)-ATPase (NKA) alpha- and beta-subunit isoforms, Na(+),K(+),2Cl(-) cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR I and II) in anadromous and landlocked strains of Atlantic salmon during parr-smolt transformation, and...... observed in landlocked salmon in May, increasing to peak levels in June. Gill CFTR I mRNA levels increased significantly from February to April in both strains, followed by a slight, though not significant increase in May and June. CFTR I mRNA levels were significantly lower in landlocked than anadromous...... salmon in April/June. Gill CFTR II mRNA levels did not change significantly in either strain. Our findings demonstrates that differential expression of gill NKA-alpha1a, -alpha1b and -alpha3 isoforms may be important for potential functional differences in NKA, both during preparatory development and...

  14. ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites*

    OpenAIRE

    Randak, Christoph O.; Dong, Qian; Ver Heul, Amanda R.; Elcock, Adrian H.; Welsh, Michael J.

    2013-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, ...

  15. Activation of CFTR by ASBT-mediated bile salt absorption

    NARCIS (Netherlands)

    Bijvelds, MJC; Jorna, H; Verkade, HJ; Bot, AGM; Hofmann, F; Agellon, LB; Sinaasappel, M; de Jonge, HR

    2005-01-01

    In cholangiocytes, bile salt (BS) uptake via the apical sodium-dependent bile acid transporter (ASBT) may evoke ductular flow by enhancing cAMP-mediated signaling to the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. We considered that ASBT-mediated BS uptake in the distal

  16. Simple image-based no-wash method for quantitative detection of surface expressed CFTR.

    Science.gov (United States)

    Larsen, Mads Breum; Hu, Jennifer; Frizzell, Raymond A; Watkins, Simon C

    2016-03-01

    Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians. It is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, which encodes an apical membrane anion channel that is required for regulating the volume and composition of epithelial secretions. The most common CFTR mutation, present on at least one allele in >90% of CF patients, deletes phenylalanine at position 508 (F508del), which causes the protein to misfold. Endoplasmic reticulum (ER) quality control elicits the degradation of mutant CFTR, compromising its trafficking to the epithelial cell apical membrane. The absence of functional CFTR leads to depletion of airway surface liquid, impaired clearance of mucus and bacteria from the lung, and predisposes to recurrent infections. Ultimately, respiratory failure results from inflammation and bronchiectasis. Although high throughput screening has identified small molecules that can restore the anion transport function of F508del CFTR, they correct less than 15% of WT CFTR activity, yielding insufficient clinical benefit. To date, most primary CF drug discovery assays have employed measurements of CFTR's anion transport function, a method that depends on the recruitment of a functional CFTR to the cell surface, involves multiple wash steps, and relies on a signal that saturates rapidly. Screening efforts have also included assays for detection of extracellularly HA-tagged or HRP-tagged CFTR, which require multiple washing steps. We have recently developed tools and cell lines that report the correction of mutant CFTR trafficking by currently available small molecules, and have extended this assay to the 96-well format. This new and simple no-wash assay of F508del CFTR at the cell surface may permit the discovery of more efficacious drugs, and hopefully thereby prevent the catastrophic effects of this disease. In addition, the modular design of this platform should make it useful for other diseases where loss

  17. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor.

    Science.gov (United States)

    Watson, Michael J; Lee, Shernita L; Marklew, Abigail J; Gilmore, Rodney C; Gentzsch, Martina; Sassano, Maria F; Gray, Michael A; Tarran, Robert

    2016-01-01

    CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR's function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR's PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs. PMID:27278076

  18. Synonymous codon usage affects the expression of wild type and F508del CFTR.

    Science.gov (United States)

    Shah, Kalpit; Cheng, Yi; Hahn, Brian; Bridges, Robert; Bradbury, Neil A; Mueller, David M

    2015-03-27

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel composed of 1480 amino acids. The major mutation responsible for cystic fibrosis results in loss of amino acid residue, F508 (F508del). Loss of F508 in CFTR alters the folding pathway resulting in endoplasmic-reticulum-associated degradation. This study investigates the role of synonymous codon in the expression of CFTR and CFTR F508del in human HEK293 cells. DNA encoding the open reading frame (ORF) for CFTR containing synonymous codon replacements was expressed using a heterologous vector integrated into the genome. The results indicate that the codon usage greatly affects the expression of CFTR. While the promoter strength driving expression of the ORFs was largely unchanged and the mRNA half-lives were unchanged, the steady-state levels of the mRNA varied by as much as 30-fold. Experiments support that this apparent inconsistency is attributed to nonsense mediated decay independent of exon junction complex. The ratio of CFTR/mRNA indicates that mRNA containing native codons was more efficient in expressing mature CFTR as compared to mRNA containing synonymous high-expression codons. However, when F508del CFTR was expressed after codon optimization, a greater percentage of the protein escaped endoplasmic-reticulum-associated degradation resulting in considerable levels of mature F508del CFTR on the plasma membrane, which showed channel activity. These results indicate that codon usage has an effect on mRNA levels and protein expression, for CFTR, and likely on chaperone-assisted folding pathway, for F508del CFTR.

  19. Maxi-anion channel as a candidate pathway for osmosensitive ATP release from mouse astrocytes in primary culture

    Institute of Scientific and Technical Information of China (English)

    Hong-Tao Liu; Abduqodir H Toychiev; Nobuyuki Takahashi; Ravshan Z Sabirov; Yasunobu Okada

    2008-01-01

    In the present study,we aimed to evaluate the pathways contributing to ATP release from mouse astrocytes during hypoosmotic stress.We first examined the expression of mRNAs for proteins constituting possible ATPreleasing pathways that have been suggested over the past several years.In RT-PCR analysis using both control and osmotically swollen astrocytes,amplification of cDNA fragments of expected size was seen for connexins (Cx32,Cx37,Cx43),pannexin 1 (Pxl),the P2X7 receptor,MRP1 and MDRI,but not CFTR.Inhibitors of exocytotie vesicular release,gap junction hemi-channels,CFTR,MRPI,MDR1,the P2X7 receptor,and volume-sensitive outwardly rectifying chloride channels had no significant effects on the massive ATP release from astrocytes.In contrast,the hypotonicity-induced ATP release from astrocytes was most effectively inhibited by gadolinium release from several other cell types.Thus,we propose that the maxi-anion channel constitutes a major pathway for swelling-induced ATP release from cultured mouse astrocytes as well.

  20. Investigating CFTR and KCa3.1 Protein/Protein Interactions.

    Science.gov (United States)

    Klein, Hélène; Abu-Arish, Asmahan; Trinh, Nguyen Thu Ngan; Luo, Yishan; Wiseman, Paul W; Hanrahan, John W; Brochiero, Emmanuelle; Sauvé, Rémy

    2016-01-01

    In epithelia, Cl- channels play a prominent role in fluid and electrolyte transport. Of particular importance is the cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) with mutations of the CFTR encoding gene causing cystic fibrosis. The bulk transepithelial transport of Cl- ions and electrolytes needs however to be coupled to an increase in K+ conductance in order to recycle K+ and maintain an electrical driving force for anion exit across the apical membrane. In several epithelia, this K+ efflux is ensured by K+ channels, including KCa3.1, which is expressed at both the apical and basolateral membranes. We show here for the first time that CFTR and KCa3.1 can physically interact. We first performed a two-hybrid screen to identify which KCa3.1 cytosolic domains might mediate an interaction with CFTR. Our results showed that both the N-terminal fragment M1-M40 of KCa3.1 and part of the KCa3.1 calmodulin binding domain (residues L345-A400) interact with the NBD2 segment (G1237-Y1420) and C- region of CFTR (residues T1387-L1480), respectively. An association of CFTR and F508del-CFTR with KCa3.1 was further confirmed in co-immunoprecipitation experiments demonstrating the formation of immunoprecipitable CFTR/KCa3.1 complexes in CFBE cells. Co-expression of KCa3.1 and CFTR in HEK cells did not impact CFTR expression at the cell surface, and KCa3.1 trafficking appeared independent of CFTR stimulation. Finally, evidence is presented through cross-correlation spectroscopy measurements that KCa3.1 and CFTR colocalize at the plasma membrane and that KCa3.1 channels tend to aggregate consequent to an enhanced interaction with CFTR channels at the plasma membrane following an increase in intracellular Ca2+ concentration. Altogether, these results suggest 1) that the physical interaction KCa3.1/CFTR can occur early during the biogenesis of both proteins and 2) that KCa3.1 and CFTR form a dynamic complex, the formation of which depends on

  1. Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

    Science.gov (United States)

    Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

    2014-07-18

    ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.

  2. Relationships among CFTR expression, HCO3- secretion, and host defense may inform gene- and cell-based cystic fibrosis therapies.

    Science.gov (United States)

    Shah, Viral S; Ernst, Sarah; Tang, Xiao Xiao; Karp, Philip H; Parker, Connor P; Ostedgaard, Lynda S; Welsh, Michael J

    2016-05-10

    Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Airway disease is the major source of morbidity and mortality. Successful implementation of gene- and cell-based therapies for CF airway disease requires knowledge of relationships among percentages of targeted cells, levels of CFTR expression, correction of electrolyte transport, and rescue of host defense defects. Previous studies suggested that, when ∼10-50% of airway epithelial cells expressed CFTR, they generated nearly wild-type levels of Cl(-) secretion; overexpressing CFTR offered no advantage compared with endogenous expression levels. However, recent discoveries focused attention on CFTR-mediated HCO3 (-) secretion and airway surface liquid (ASL) pH as critical for host defense and CF pathogenesis. Therefore, we generated porcine airway epithelia with varying ratios of CF and wild-type cells. Epithelia with a 50:50 mix secreted HCO3 (-) at half the rate of wild-type epithelia. Likewise, heterozygous epithelia (CFTR(+/-) or CFTR(+/∆F508)) expressed CFTR and secreted HCO3 (-) at ∼50% of wild-type values. ASL pH, antimicrobial activity, and viscosity showed similar relationships to the amount of CFTR. Overexpressing CFTR increased HCO3 (-) secretion to rates greater than wild type, but ASL pH did not exceed wild-type values. Thus, in contrast to Cl(-) secretion, the amount of CFTR is rate-limiting for HCO3 (-) secretion and for correcting host defense abnormalities. In addition, overexpressing CFTR might produce a greater benefit than expressing CFTR at wild-type levels when targeting small fractions of cells. These findings may also explain the risk of airway disease in CF carriers. PMID:27114540

  3. Analysis of conventional and unconventional trafficking of CFTR and other membrane proteins.

    Science.gov (United States)

    Gee, Heon Yung; Kim, Joo Young; Lee, Min Goo

    2015-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic transmembrane protein that functions as a cAMP-activated anion channel at the apical membrane of epithelial cells. Mutations in CFTR cause cystic fibrosis and are also associated with monosymptomatic diseases in the lung, pancreas, intestines, and vas deferens. Many disease-causing CFTR mutations, including the deletion of a single phenylalanine residue at position 508 (ΔF508-CFTR), result in protein misfolding and trafficking defects. Therefore, intracellular trafficking of wild-type and mutant CFTR has been studied extensively, and results from these studies significantly contribute to our general understanding of mechanisms involved in the cell-surface trafficking of membrane proteins. CFTR is a glycoprotein that undergoes complex N-glycosylation as it passes through Golgi-mediated conventional exocytosis. Interestingly, results from recent studies revealed that CFTR and other membrane proteins can reach the plasma membrane via an unconventional alternative route that bypasses Golgi in specific cellular conditions. Here, we describe methods that have been used to investigate the conventional and unconventional surface trafficking of CFTR. With appropriate modifications, the protocols described in this chapter can also be applied to studies investigating the intracellular trafficking of other plasma membrane proteins.

  4. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

    Science.gov (United States)

    Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

    2015-05-29

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

  5. Activation of CFTR by ASBT-mediated bile salt absorption.

    Science.gov (United States)

    Bijvelds, Marcel J C; Jorna, Huub; Verkade, Henkjan J; Bot, Alice G M; Hofmann, Franz; Agellon, Luis B; Sinaasappel, Maarten; de Jonge, Hugo R

    2005-11-01

    In cholangiocytes, bile salt (BS) uptake via the apical sodium-dependent bile acid transporter (ASBT) may evoke ductular flow by enhancing cAMP-mediated signaling to the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. We considered that ASBT-mediated BS uptake in the distal ileum might also modulate intestinal fluid secretion. Taurocholate (TC) induced a biphasic rise in the short circuit current across ileal tissue, reflecting transepithelial electrogenic ion transport. This response was sensitive to bumetanide and largely abrogated in Cftr-null mice, indicating that it predominantly reflects CFTR-mediated Cl- secretion. The residual response in Cftr-null mice could be attributed to electrogenic ASBT activity, as it matched the TC-coupled absorptive Na+ flux. TC-evoked Cl- secretion required ASBT-mediated TC uptake, because it was blocked by a selective ASBT inhibitor and was restricted to the distal ileum. Suppression of neurotransmitter or prostaglandin release, blocking of the histamine H1 receptor, or pretreatment with 5-hydroxytryptamine did not abrogate the TC response, suggesting that neurocrine or immune mediators of Cl- secretion are not involved. Responses to TC were retained after carbachol treatment and after permeabilization of the basolateral membrane with nystatin, indicating that BS modulate CFTR channel gating rather than the driving force for Cl- exit. TC-induced Cl- secretion was maintained in cGMP-dependent protein kinase II-deficient mice and only partially inhibited by the cAMP-dependent protein kinase inhibitor H89, suggesting a mechanism of CFTR activation different from cAMP or cGMP signaling. We conclude that active BS absorption in the ileum triggers CFTR activation and, consequently, local salt and water secretion, which may serve to prevent intestinal obstruction in the postprandial state. PMID:16037545

  6. Relationships among CFTR expression, HCO3− secretion, and host defense may inform gene- and cell-based cystic fibrosis therapies

    Science.gov (United States)

    Shah, Viral S.; Ernst, Sarah; Tang, Xiao Xiao; Karp, Philip H.; Parker, Connor P.; Ostedgaard, Lynda S.; Welsh, Michael J.

    2016-01-01

    Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Airway disease is the major source of morbidity and mortality. Successful implementation of gene- and cell-based therapies for CF airway disease requires knowledge of relationships among percentages of targeted cells, levels of CFTR expression, correction of electrolyte transport, and rescue of host defense defects. Previous studies suggested that, when ∼10–50% of airway epithelial cells expressed CFTR, they generated nearly wild-type levels of Cl− secretion; overexpressing CFTR offered no advantage compared with endogenous expression levels. However, recent discoveries focused attention on CFTR-mediated HCO3− secretion and airway surface liquid (ASL) pH as critical for host defense and CF pathogenesis. Therefore, we generated porcine airway epithelia with varying ratios of CF and wild-type cells. Epithelia with a 50:50 mix secreted HCO3− at half the rate of wild-type epithelia. Likewise, heterozygous epithelia (CFTR+/− or CFTR+/∆F508) expressed CFTR and secreted HCO3− at ∼50% of wild-type values. ASL pH, antimicrobial activity, and viscosity showed similar relationships to the amount of CFTR. Overexpressing CFTR increased HCO3− secretion to rates greater than wild type, but ASL pH did not exceed wild-type values. Thus, in contrast to Cl− secretion, the amount of CFTR is rate-limiting for HCO3− secretion and for correcting host defense abnormalities. In addition, overexpressing CFTR might produce a greater benefit than expressing CFTR at wild-type levels when targeting small fractions of cells. These findings may also explain the risk of airway disease in CF carriers. PMID:27114540

  7. Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.

    Directory of Open Access Journals (Sweden)

    Jessica LaRusch

    2014-07-01

    Full Text Available CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev cause complete loss of CFTR function and result in cystic fibrosis (CF, a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002. Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005 and male infertility (OR 395, p<<0.0001. WNK1-SPAK pathway-activated increases in

  8. MAST205 competes with cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand for binding to CFTR to regulate CFTR-mediated fluid transport.

    Science.gov (United States)

    Ren, Aixia; Zhang, Weiqiang; Yarlagadda, Sunitha; Sinha, Chandrima; Arora, Kavisha; Moon, Chang-Suk; Naren, Anjaparavanda P

    2013-04-26

    The PDZ (postsynaptic density-95/discs large/zona occludens-1) domain-based interactions play important roles in regulating the expression and function of the cystic fibrosis transmembrane conductance regulator (CFTR). Several PDZ domain-containing proteins (PDZ proteins for short) have been identified as directly or indirectly interacting with the C terminus of CFTR. To better understand the regulation of CFTR processing, we conducted a genetic screen and identified MAST205 (a microtubule-associated serine/threonine kinase with a molecular mass of 205 kDa) as a new CFTR regulator. We found that overexpression of MAST205 increased the expression of CFTR and augmented CFTR-mediated fluid transport in a dose-dependent manner. Conversely, knockdown of MAST205 inhibited CFTR function. The PDZ motif of CFTR is required for the regulatory role of MAST205 in CFTR expression and function. We further demonstrated that MAST205 and the CFTR-associated ligand competed for binding to CFTR, which facilitated the processing of CFTR and consequently up-regulated the expression and function of CFTR at the plasma membrane. More importantly, we found that MAST205 could facilitate the processing of F508del-CFTR mutant and augment its quantity and channel function at the plasma membrane. Taken together, our data suggest that MAST205 plays an important role in regulating CFTR expression and function. Our findings have important clinical implications for treating CFTR-associated diseases such as cystic fibrosis and secretory diarrheas.

  9. A truncated CFTR protein rescues endogenous ΔF508-CFTR and corrects chloride transport in mice

    OpenAIRE

    Cormet-Boyaka, Estelle; Jeong S Hong; Berdiev, Bakhram K.; James A Fortenberry; Rennolds, Jessica; Clancy, J. P.; Benos, Dale J.; Boyaka, Prosper N.; Sorscher, Eric J.

    2009-01-01

    Cystic fibrosis (CF) is most frequently associated with deletion of phenylalanine at position 508 (ΔF508) in the CF transmembrane conductance regulator (CFTR) protein. The ΔF508-CFTR mutant protein exhibits a folding defect that affects its processing and impairs chloride-channel function. This study aimed to determine whether CFTR fragments approximately half the size of wild-type CFTR and complementary to the portion of CFTR bearing the mutation can specifically rescue the processing of end...

  10. Conductance hysteresis in the voltage-dependent anion channel.

    Science.gov (United States)

    Rappaport, Shay M; Teijido, Oscar; Hoogerheide, David P; Rostovtseva, Tatiana K; Berezhkovskii, Alexander M; Bezrukov, Sergey M

    2015-09-01

    Hysteresis in the conductance of voltage-sensitive ion channels is observed when the transmembrane voltage is periodically varied with time. Although this phenomenon has been used in studies of gating of the voltage-dependent anion channel, VDAC, from the outer mitochondrial membrane for nearly four decades, full hysteresis curves have never been reported, because the focus was solely on the channel opening branches of the hysteresis loops. We studied the hysteretic response of a multichannel VDAC system to a triangular voltage ramp the frequency of which was varied over three orders of magnitude, from 0.5 mHz to 0.2 Hz. We found that in this wide frequency range the area encircled by the hysteresis curves changes by less than a factor of three, suggesting broad distribution of the characteristic times and strongly non-equilibrium behavior. At the same time, quasi-equilibrium two-state behavior is observed for hysteresis branches corresponding to VDAC opening. This enables calculation of the usual equilibrium gating parameters, gating charge and voltage of equipartitioning, which were found to be almost insensitive to the ramp frequency. To rationalize this peculiarity, we hypothesize that during voltage-induced closure and opening the system explores different regions of the complex free energy landscape, and, in the opening branch, follows quasi-equilibrium paths.

  11. THE ROLE OF CHLORIDE ANION AND CFTR IN KILLING OF PSEUDOMONAS AERUGINOSA BY NORMAL AND CF NEUTROPHILS

    OpenAIRE

    Painter, Richard G.; Bonvillain, Ryan W.; Valentine, Vincent G.; Lombard, Gisele A.; LaPlace, Stephanie G.; Nauseef, William M.; Wang, Guoshun

    2008-01-01

    Chloride anion is essential for myeloperoxidase to produce hypochlorous acid (HOCl) in neutrophils (PMNs). To define whether chloride availability to PMNs affects their HOCl production and microbicidal capacity, we examined how extracellular chloride concentration affects killing of Pseudomonas aeruginosa (PsA) by normal neutrophils. PMN-mediated bacterial killing was strongly dependent on extracellular chloride concentration. Neutrophils in a chloride-deficient medium killed PsA poorly. Howe...

  12. 公丁香提取物抑制CFTR氯离子通道的发现与研究%The extract of clove inhibits CFTR chloride channel

    Institute of Scientific and Technical Information of China (English)

    栾剑; 张耀方; 杨红

    2015-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride chan‐nel .In recent years ,the blockers of CFTR become the new hot spot in the treatment of secretory di‐arrhea .The aim of this research is using high‐throughput screening techniques screened blockers of CFTR chloride channel from traditional Chinese medicine .In this study ,after 40000 fractions of Chi‐nese herbal medicine have been screened ,clove extract was found .In cell‐based fluorescence assays and voltage clamp experiments ,the best active fraction‐E06 significantly blocks CFTR chloride chan‐nel .Therefore ,clove extract screened from traditional Chinese medicine blocks CFTR chloride chan‐nel and provides a theoretical basis for the in‐depth study of anti‐diarrheal drugs .%囊性纤维化跨膜电导调节因子(CFTR)是一种上皮细胞顶膜中表达的氯离子通道,是近年来治疗分泌型腹泻的新热点。利用高通量筛选技术,自中国传统中药中筛选能够抑制CFTR氯离子通道的中药组分。结果显示,自500种中草药的40000种中药组分中筛选到公丁香。经细胞荧光实验和电压膜片钳实验验证公丁香最佳活性孔———E06对CFTR具有明显的抑制作用,IC50=103 mg/L 。本研究结果为深入探讨公丁香的抗泻药物研发提供理论依据。

  13. Regulation of Plasma Membrane Recycling by CFTR

    Science.gov (United States)

    Bradbury, Neil A.; Jilling, Tamas; Berta, Gabor; Sorscher, Eric J.; Bridges, Robert J.; Kirk, Kevin L.

    1992-04-01

    The gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) is defective in patients with cystic fibrosis. Although the protein product of the CFTR gene has been proposed to function as a chloride ion channel, certain aspects of its function remain unclear. The role of CFTR in the adenosine 3',5'-monophosphate (cAMP)-dependent regulation of plasma membrane recycling was examined. Adenosine 3',5'-monophosphate is known to regulate endocytosis and exocytosis in chloride-secreting epithelial cells that express CFTR. However, mutant epithelial cells derived from a patient with cystic fibrosis exhibited no cAMP-dependent regulation of endocytosis and exocytosis until they were transfected with complementary DNA encoding wild-type CFTR. Thus, CFTR is critical for cAMP-dependent regulation of membrane recycling in epithelial tissues, and this function of CFTR could explain in part the pleiotropic nature of cystic fibrosis.

  14. Defective CFTR-regulated granulosa cell proliferation in polycystic ovarian syndrome.

    Science.gov (United States)

    Chen, Hui; Guo, Jing Hui; Zhang, Xiao Hu; Chan, Hsiao Chang

    2015-05-01

    Polycystic ovarian syndrome (PCOS) is one of the most frequent causes of female infertility, featured by abnormal hormone profile, chronic oligo/anovulation, and presence of multiple cystic follicles in the ovary. However, the mechanism underlying the abnormal folliculogenesis remains obscure. We have previously demonstrated that CFTR, a cAMP-dependent Cl(-) and HCO3 (-) conducting anion channel, is expressed in the granulosa cells and its expression is downregulated in PCOS rat models and human patients. In this study, we aimed to investigate the possible involvement of downregulation of CFTR in the impaired follicle development in PCOS using two rat PCOS models and primary culture of granulosa cells. Our results indicated that the downregulation of CFTR in the cystic follicles was accompanied by reduced expression of proliferating cell nuclear antigen (PCNA), in rat PCOS models. In addition, knockdown or inhibition of CFTR in granulosa cell culture resulted in reduced cell viability and downregulation of PCNA. We further demonstrated that CFTR regulated both basal and FSH-stimulated granulosa cell proliferation through the HCO3 (-)/sAC/PKA pathway leading to ERK phosphorylation and its downstream target cyclin D2 (Ccnd2) upregulation. Reduced ERK phosphorylation and CCND2 were found in ovaries of rat PCOS model compared with the control. This study suggests that CFTR is required for normal follicle development and that its downregulation in PCOS may inhibit granulosa cell proliferation, resulting in abnormal follicle development in PCOS.

  15. A host defense mechanism involving CFTR-mediated bicarbonate secretion in bacterial prostatitis.

    Directory of Open Access Journals (Sweden)

    Chen Xie

    Full Text Available BACKGROUND: Prostatitis is associated with a characteristic increase in prostatic fluid pH; however, the underlying mechanism and its physiological significance have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: In this study a primary culture of rat prostatic epithelial cells and a rat prostatitis model were used. Here we reported the involvement of CFTR, a cAMP-activated anion channel conducting both Cl(- and HCO(3(-, in mediating prostate HCO(3(- secretion and its possible role in bacterial killing. Upon Escherichia coli (E. coli-LPS challenge, the expression of CFTR and carbonic anhydrase II (CA II, along with several pro-inflammatory cytokines was up-regulated in the primary culture of rat prostate epithelial cells. Inhibiting CFTR function in vitro or in vivo resulted in reduced bacterial killing by prostate epithelial cells or the prostate. High HCO(3(- content (>50 mM, rather than alkaline pH, was found to be responsible for bacterial killing. The direct action of HCO(3(- on bacterial killing was confirmed by its ability to increase cAMP production and suppress bacterial initiation factors in E. coli. The relevance of the CFTR-mediated HCO(3(- secretion in humans was demonstrated by the upregulated expression of CFTR and CAII in human prostatitis tissues. CONCLUSIONS/SIGNIFICANCE: The CFTR and its mediated HCO(3(- secretion may be up-regulated in prostatitis as a host defense mechanism.

  16. Swelling-Activated Anion Channels Are Essential for Volume Regulation of Mouse Thymocytes

    Directory of Open Access Journals (Sweden)

    Ravshan Z. Sabirov

    2011-12-01

    Full Text Available Channel-mediated trans-membrane chloride movement is a key process in the active cell volume regulation under osmotic stress in most cells. However, thymocytes were hypothesized to regulate their volume by activating a coupled K-Cl cotransport mechanism. Under the patch-clamp, we found that osmotic swelling activates two types of macroscopic anion conductance with different voltage-dependence and pharmacology. At the single-channel level, we identified two types of events: one corresponded to the maxi-anion channel, and the other one had characteristics of the volume-sensitive outwardly rectifying (VSOR chloride channel of intermediate conductance. A VSOR inhibitor, phloretin, significantly suppressed both macroscopic VSOR-type conductance and single-channel activity of intermediate amplitude. The maxi-anion channel activity was largely suppressed by Gd3+ ions but not by phloretin. Surprisingly, [(dihydroindenyloxy] alkanoic acid (DIOA, a known antagonist of K-Cl cotransporter, was found to significantly suppress the activity of the VSOR-type single-channel events with no effect on the maxi-anion channels at 10 μM. The regulatory volume decrease (RVD phase of cellular response to hypotonicity was mildly suppressed by Gd3+ ions and was completely abolished by phloretin suggesting a major impact of the VSOR chloride channel and modulatory role of the maxi-anion channel. The inhibitory effect of DIOA was also strong, and, most likely, it occurred via blocking the VSOR Cl− channels.

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

    Science.gov (United States)

    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.

  18. Activation of CFTR-mediated Cl- Transport by Magnolin

    Institute of Scientific and Technical Information of China (English)

    JIN Ling-ling; LIU Xin; SUN Yan; LIN Sen; ZHOU Na; XU Li-na; YU BO; HOU Shu-guang; YANG Hong

    2008-01-01

    Magnolin is a herbal compound from Magnolia biondii Pamp.It possesses numerous biological activities.Cystic fibrosis transmembrane conductance regulator(CFTR)is all epithelial chloride channel that plays a key role in the fluid secretion of various exocrine organs.In the present study,the activation of CFTR-mediated chloride transport by magnolin is indentified and characterized.In CFTR stably trailsfected FRT cells.magnolin increases CFTR Cl- currents in a concentration-dependent manner.The activation of magnolin on CFTR is rapid,reversible,and cAMP-dependent.Magnolin does not elevate cellular cAMP level.indicating that it activates CFTR by direct binding and interaction with CFTR protein.Magnolin selectively activates wildtype CFTR rather than mutant CFTIL Magnolin may present a novel class of therapeutic lead compound for the treatment of diseases associated with reduced CFTR function such as keratoconjunctivitis sicca,idiopathic chronic pancreatiti,and chromc constipation.

  19. Pseudomonas aeruginosa triggers CFTR-mediated airway surface liquid secretion in swine trachea

    OpenAIRE

    Luan, Xiaojie; Campanucci, Verónica A; Nair, Manoj; YILMAZ, Orhan; Belev, George; Machen, Terry E.; Chapman, Dean; Ianowski, Juan P.

    2014-01-01

    Cystic fibrosis (CF) is a genetic disorder caused by mutations in the gene encoding for the anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Several organs are affected in CF, but most of the morbidity and mortality comes from lung disease caused by the failure to clear bacteria. Bacterial clearance depends on a layer of airway surface liquid (ASL) covering the airways, rich in antimicrobial compounds and mucins, that removes bacteria from the airway through mucocilia...

  20. Purification and Characterization of Two Voltage-Dependent Anion Channel Isoforms from Plant Seeds1

    Science.gov (United States)

    Abrecht, Helge; Wattiez, Ruddy; Ruysschaert, Jean-Marie; Homblé, Fabrice

    2000-01-01

    Mitochondria were isolated from imbibed seeds of lentil (Lens culinaris) and Phaseolus vulgaris. We copurified two voltage-dependent anion channel from detergent solubilized mitochondria in a single purification step using hydroxyapatite. The two isoforms from P. vulgaris were separated by chromatofocusing chromatography in 4 m urea without any loss of channel activity. Channel activity of each isoform was characterized upon reconstitution into diphytanoyl phosphatidylcholine planar lipid bilayers. Both isoforms form large conductance channels that are slightly anion selective and display cation selective substates. PMID:11080295

  1. Purification and characterization of two voltage-dependent anion channel isoforms from plant seeds.

    Science.gov (United States)

    Abrecht, H; Wattiez, R; Ruysschaert, J M; Homblé, F

    2000-11-01

    Mitochondria were isolated from imbibed seeds of lentil (Lens culinaris) and Phaseolus vulgaris. We copurified two voltage-dependent anion channel from detergent solubilized mitochondria in a single purification step using hydroxyapatite. The two isoforms from P. vulgaris were separated by chromatofocusing chromatography in 4 M urea without any loss of channel activity. Channel activity of each isoform was characterized upon reconstitution into diphytanoyl phosphatidylcholine planar lipid bilayers. Both isoforms form large conductance channels that are slightly anion selective and display cation selective substates.

  2. Altered plasmodial surface anion channel activity and in vitro resistance to permeating antimalarial compounds

    OpenAIRE

    Lisk, Godfrey; Pain, Margaret; Sellers, Morgan; Gurnev, Philip A.; Pillai, Ajay D.; Bezrukov, Sergey M.; Desai, Sanjay A.

    2010-01-01

    Erythrocytes infected with malaria parasites have increased permeability to various solutes. These changes may be mediated by an unusual small conductance ion channel known as the plasmodial surface anion channel (PSAC). While channel activity benefits the parasite by permitting nutrient acquisition, it can also be detrimental because water-soluble antimalarials may more readily access their parasite targets via this channel. Recently, two such toxins, blasticidin S and leupeptin, were used t...

  3. Calcium-regulated anion channels in the plasma membrane of Lilium longiflorum pollen protoplasts.

    Science.gov (United States)

    Tavares, Bárbara; Dias, Pedro Nuno; Domingos, Patrícia; Moura, Teresa Fonseca; Feijó, José Alberto; Bicho, Ana

    2011-10-01

    • Currents through anion channels in the plasma membrane of Lilium longiflorum pollen grain protoplasts were studied under conditions of symmetrical anionic concentrations by means of patch-clamp whole-cell configuration. • With Cl(-) -based intra- and extracellular solutions, three outward-rectifying anion conductances, I(Cl1) , I(Cl2) and I(Cl3) , were identified. These three activities were discriminated by differential rundown behaviour and sensitivity to 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), which could not be attributed to one or more channel types. All shared strong outward rectification, activated instantaneously and displayed a slow time-dependent activation for positive potentials. All showed modulation by intracellular calcium ([Ca(2+) ](in) ), increasing intensity from 6.04 nM up to 0.5 mM (I(Cl1) ), or reaching a maximum value with 8.50 μM (I(Cl2) and I(Cl3) ). • After rundown, the anionic currents measured using NO(3) (-) -based solutions were indistinguishable, indicating that the permeabilities of the channels for Cl(-) and NO(3) (-) are similar. Additionally, unitary anionic currents were measured from outside-out excised patches, confirming the presence of individual anionic channels. • This study shows for the first time the presence of a large anionic conductance across the membrane of pollen protoplasts, resulting from the presence of Ca(2+) -regulated channels. A similar conductance was also found in germinated pollen. We hypothesize that these putative channels may be responsible for the large anionic fluxes previously detected by means of self-referencing vibrating probes. PMID:21668885

  4. COMMD1-mediated ubiquitination regulates CFTR trafficking.

    Directory of Open Access Journals (Sweden)

    Loïc Drévillon

    Full Text Available The CFTR (cystic fibrosis transmembrane conductance regulator protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.

  5. Anion channels in Chara corallina tonoplast membrane: Calcium dependence and rectification

    NARCIS (Netherlands)

    Berecki, G.; Varga, Z.; Iren, F. van; Duijn, B. van

    1999-01-01

    Tonoplast K+ channels of Chara corallina are well characterized but only a few reports mention anion channels, which are likely to play an important role in the tonoplast action potential and osmoregulation of this plant. For experiments internodal cells were isolated. Cytoplasmic droplets were form

  6. G551D-CFTR needs more bound actin than wild-type CFTR to maintain its presence in plasma membranes.

    Science.gov (United States)

    Trouvé, Pascal; Kerbiriou, Mathieu; Teng, Ling; Benz, Nathalie; Taiya, Mehdi; Le Hir, Sophie; Férec, Claude

    2015-08-01

    Cystic Fibrosis is due to mutations in the CFTR gene. The missense mutation G551D (approx. 5% of cases) encodes a CFTR chloride channel with normal cell surface expression but with an altered chloride channel activity, leading to a severe phenotype. Our aim was to identify specific interacting proteins of G551D-CFTR which could explain the channel defect. Wild-type CFTR (Wt-CFTR) was co-immunoprecipitated from stably transfected HeLa cells and resolved by 2D gel electrophoresis. Among the detected spots, one was expressed at a high level. Mass Spectrometry revealed that it corresponded to actin which is known to be involved in the CFTR's channel function. To assess whether actin could be involved in the altered G551D-CFTR function, its basal expression was studied. Because actin expression was the same in wt- and in G551D-CFTR expressing cells, its interaction with both wt- and G551D-CFTR was studied by co-immunoprecipitation, and we found that a higher amount of actin was bound onto G551D-CFTR than onto Wt-CFTR. The role of actin upon wt- and G551D-CFTR function was further studied by patch-clamp experiments after cytochalasin D treatment of the cells. We found a decrease of the very weak currents in G551D-CFTR expressing cells. Because a higher amount of actin is bound onto G551D-CFTR than onto Wt-CFTR, it is likely to be not involved in the mutated CFTR's defect. Nevertheless, because actin is necessary to maintain the very weak global currents observed in G551D-CFTR expressing HeLa cells, we conclude that more actin is necessary to maintain G551D-CFTR in the plasma membrane than for Wt-CFTR.

  7. NEUROSCIENCE. Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics.

    Science.gov (United States)

    Govorunova, Elena G; Sineshchekov, Oleg A; Janz, Roger; Liu, Xiaoqin; Spudich, John L

    2015-08-01

    Light-gated rhodopsin cation channels from chlorophyte algae have transformed neuroscience research through their use as membrane-depolarizing optogenetic tools for targeted photoactivation of neuron firing. Photosuppression of neuronal action potentials has been limited by the lack of equally efficient tools for membrane hyperpolarization. We describe anion channel rhodopsins (ACRs), a family of light-gated anion channels from cryptophyte algae that provide highly sensitive and efficient membrane hyperpolarization and neuronal silencing through light-gated chloride conduction. ACRs strictly conducted anions, completely excluding protons and larger cations, and hyperpolarized the membrane of cultured animal cells with much faster kinetics at less than one-thousandth of the light intensity required by the most efficient currently available optogenetic proteins. Natural ACRs provide optogenetic inhibition tools with unprecedented light sensitivity and temporal precision.

  8. Increased anion channel activity is an unavoidable event in ozone-induced programmed cell death.

    Directory of Open Access Journals (Sweden)

    Takashi Kadono

    Full Text Available BACKGROUND: Ozone is a major secondary air pollutant often reaching high concentrations in urban areas under strong daylight, high temperature and stagnant high-pressure systems. Ozone in the troposphere is a pollutant that is harmful to the plant. PRINCIPAL FINDINGS: By exposing cells to a strong pulse of ozonized air, an acute cell death was observed in suspension cells of Arabidopsis thaliana used as a model. We demonstrated that O(3 treatment induced the activation of a plasma membrane anion channel that is an early prerequisite of O(3-induced cell death in A. thaliana. Our data further suggest interplay of anion channel activation with well known plant responses to O(3, Ca(2+ influx and NADPH-oxidase generated reactive oxygen species (ROS in mediating the oxidative cell death. This interplay might be fuelled by several mechanisms in addition to the direct ROS generation by O(3; namely, H(2O(2 generation by salicylic and abscisic acids. Anion channel activation was also shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during programmed cell death. SIGNIFICANCE: Collectively, our data indicate that anion efflux is an early key component of morphological and biochemical events leading to O(3-induced programmed cell death. Because ion channels and more specifically anion channels assume a crucial position in cells, an understanding about the underlying role(s for ion channels in the signalling pathway leading to programmed cell death is a subject that warrants future investigation.

  9. The ΔF508-CFTR mutation inhibits wild-type CFTR processing and function when co-expressed in human airway epithelia and in mouse nasal mucosa

    Directory of Open Access Journals (Sweden)

    Tucker Torry A

    2012-09-01

    Full Text Available Abstract Background Rescue or correction of CFTR function in native epithelia is the ultimate goal of CF therapeutics development. Wild-type (WT CFTR introduction and replacement is also of particular interest. Such therapies may be complicated by possible CFTR self-assembly into an oligomer or multimer. Results Surprisingly, functional CFTR assays in native airway epithelia showed that the most common CFTR mutant, ΔF508-CFTR (ΔF-CFTR, inhibits WT-CFTR when both forms are co-expressed. To examine more mechanistically, both forms of CFTR were transfected transiently in varying amounts into IB3-1 CF human airway epithelial cells and HEK-293 human embryonic kidney cells null for endogenous CFTR protein expression. Increasing amounts of ΔF-CFTR inhibited WT-CFTR protein processing and function in CF human airway epithelial cells but not in heterologous HEK-293 cells. Stably expressed ΔF-CFTR in clones of the non-CF human airway epithelial cell line, CALU-3, also showed reduction in cAMP-stimulated anion secretion and in WT-CFTR processing. An ultimate test of this dominant negative-like effect of ΔF-CFTR on WT-CFTR was the parallel study of two different CF mouse models: the ΔF-CFTR mouse and the bitransgenic CFTR mouse corrected in the gut but null in the lung and airways. WT/ΔF heterozygotes had an intermediate phenotype with regard to CFTR agonist responses in in vivo nasal potential difference (NPD recordings and in Ussing chamber recordings of short-circuit current (ISC in vitro on primary tracheal epithelial cells isolated from the same mice. In contrast, CFTR bitransgenic +/− heterozygotes had no difference in their responses versus +/+ wild-type mice. Conclusions Taken altogether, these data suggest that ΔF-CFTR and WT-CFTR co-assemble into an oligomeric macromolecular complex in native epithelia and share protein processing machinery and regulation at the level of the endoplasmic reticulum (ER. As a consequence, ΔF-CFTR slows WT-CFTR

  10. Molecular Motors and Apical CFTR Traffic in Epithelia

    Directory of Open Access Journals (Sweden)

    Dmitri V. Kravtsov

    2013-05-01

    Full Text Available Intracellular protein traffic plays an important role in the regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR chloride channels. Microtubule and actin-based motor proteins direct CFTR movement along trafficking pathways. As shown for other regulatory proteins such as adaptors, the involvement of protein motors in CFTR traffic is cell-type specific. Understanding motor specificity provides insight into the biology of the channel and opens opportunity for discovery of organ-specific drug targets for treating CFTR-mediated diseases.

  11. Evidence for functional interaction of plasma membrane electron transport, voltage-dependent anion channel and volume-regulated anion channel in frog aorta

    Indian Academy of Sciences (India)

    Rashmi P Rao; J Prakasa Rao

    2010-12-01

    Frog aortic tissue exhibits plasma membrane electron transport (PMET) owing to its ability to reduce ferricyanide even in the presence of mitochondrial poisons, such as cyanide and azide. Exposure to hypotonic solution (108 mOsmol/kg H2O) enhanced the reduction of ferricyanide in excised aortic tissue of frog. Increment in ferricyanide reductase activity was also brought about by the presence of homocysteine (100 M dissolved in isotonic frog Ringer solution), a redox active compound and a potent modulator of PMET. Two plasma-membrane-bound channels, the volume regulated anion channel (VRAC) and the voltage-dependent anion channel (VDAC), are involved in the response to hypotonic stress. The presence of VRAC and VDAC antagonists–tamoxifen, glibenclamide, fluoxetine and verapamil, and 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS), respectively–inhibited this enhanced activity brought about by either hypotonic stress or homocysteine. The blockers do not affect the ferricyanide reductase activity under isotonic conditions. Taken together, these findings indicate a functional interaction of the three plasma membrane proteins, namely, ferricyanide reductase (PMET), VDAC and VRAC.

  12. CFTR activity and mitochondrial function

    Directory of Open Access Journals (Sweden)

    Angel Gabriel Valdivieso

    2013-01-01

    Full Text Available Cystic Fibrosis (CF is a frequent and lethal autosomal recessive disease, caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR. Before the discovery of the CFTR gene, several hypotheses attempted to explain the etiology of this disease, including the possible role of a chloride channel, diverse alterations in mitochondrial functions, the overexpression of the lysosomal enzyme α-glucosidase and a deficiency in the cytosolic enzyme glucose 6-phosphate dehydrogenase. Because of the diverse mitochondrial changes found, some authors proposed that the affected gene should codify for a mitochondrial protein. Later, the CFTR cloning and the demonstration of its chloride channel activity turned the mitochondrial, lysosomal and cytosolic hypotheses obsolete. However, in recent years, using new approaches, several investigators reported similar or new alterations of mitochondrial functions in Cystic Fibrosis, thus rediscovering a possible role of mitochondria in this disease. Here, we review these CFTR-driven mitochondrial defects, including differential gene expression, alterations in oxidative phosphorylation, calcium homeostasis, oxidative stress, apoptosis and innate immune response, which might explain some characteristics of the complex CF phenotype and reveals potential new targets for therapy.

  13. Function and regulation of TRPM7, as well as intracellular magnesium content, are altered in cells expressing ΔF508-CFTR and G551D-CFTR.

    Science.gov (United States)

    Huguet, F; Calvez, M L; Benz, N; Le Hir, S; Mignen, O; Buscaglia, P; Horgen, F D; Férec, C; Kerbiriou, M; Trouvé, P

    2016-09-01

    Cystic fibrosis (CF), one of the most common fatal hereditary disorders, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene product is a multidomain adenosine triphosphate-binding cassette (ABC) protein that functions as a chloride (Cl(-)) channel that is regulated by intracellular magnesium [Mg(2+)]i. The most common mutations in CFTR are a deletion of a phenylalanine residue at position 508 (ΔF508-CFTR, 70-80 % of CF phenotypes) and a Gly551Asp substitution (G551D-CFTR, 4-5 % of alleles), which lead to decreased or almost abolished Cl(-) channel function, respectively. Magnesium ions have to be finely regulated within cells for optimal expression and function of CFTR. Therefore, the melastatin-like transient receptor potential cation channel, subfamily M, member 7 (TRPM7), which is responsible for Mg(2+) entry, was studies and [Mg(2+)]i measured in cells stably expressing wildtype CFTR, and two mutant proteins (ΔF508-CFTR and G551D-CFTR). This study shows for the first time that [Mg(2+)]i is decreased in cells expressing ΔF508-CFTR and G551D-CFTR mutated proteins. It was also observed that the expression of the TRPM7 protein is increased; however, membrane localization was altered for both ΔF508del-CFTR and G551D-CFTR. Furthermore, both the function and regulation of the TRPM7 channel regarding Mg(2+) is decreased in the cells expressing the mutated CFTR. Ca(2+) influx via TRPM7 were also modified in cells expressing a mutated CFTR. Therefore, there appears to be a direct involvement of TRPM7 in CF physiopathology. Finally, we propose that the TRPM7 activator Naltriben is a new potentiator for G551D-CFTR as the function of this mutant increases upon activation of TRPM7 by Naltriben.

  14. From CFTR biology toward combinatorial pharmacotherapy: expanded classification of cystic fibrosis mutations.

    Science.gov (United States)

    Veit, Gudio; Avramescu, Radu G; Chiang, Annette N; Houck, Scott A; Cai, Zhiwei; Peters, Kathryn W; Hong, Jeong S; Pollard, Harvey B; Guggino, William B; Balch, William E; Skach, William R; Cutting, Garry R; Frizzell, Raymond A; Sheppard, David N; Cyr, Douglas M; Sorscher, Eric J; Brodsky, Jeffrey L; Lukacs, Gergely L

    2016-02-01

    More than 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been described that confer a range of molecular cell biological and functional phenotypes. Most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis (CF) with variable disease severity. Based on the molecular phenotypic complexity of CFTR mutants and their susceptibility to pharmacotherapy, it has been recognized that mutations may impose combinatorial defects in CFTR channel biology. This notion led to the conclusion that the combination of pharmacotherapies addressing single defects (e.g., transcription, translation, folding, and/or gating) may show improved clinical benefit over available low-efficacy monotherapies. Indeed, recent phase 3 clinical trials combining ivacaftor (a gating potentiator) and lumacaftor (a folding corrector) have proven efficacious in CF patients harboring the most common mutation (deletion of residue F508, ΔF508, or Phe508del). This drug combination was recently approved by the U.S. Food and Drug Administration for patients homozygous for ΔF508. Emerging studies of the structural, cell biological, and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different CFTR alleles. Establishment of a set of combinatorial categories of the previously defined basic defects in CF alleles will aid the design of even more efficacious therapeutic interventions for CF patients.

  15. Pharmacological correctors of mutant CFTR mistrafficking

    Directory of Open Access Journals (Sweden)

    Nicoletta ePedemonte

    2012-10-01

    Full Text Available The lack of phenylalanine 508 (∆F508 mutation in the CFTR Cl- channel represents the most frequent cause of cystic fibrosis (CF, a genetic disease affecting multiple organs such lung, pancreas, and liver. ∆F508 causes instability and misfolding of CFTR protein leading to early degradation in the endoplasmic reticulum and accelerated removal from the plasma membrane. Pharmacological correctors of mutant CFTR protein have been identified by high-throughput screening of large chemical libraries, by in silico docking of virtual compounds on CFTR structure models, or by using compounds that affect the whole proteome (e.g. histone deacetylase inhibitors or a single CFTR-interacting protein. The presence of multiple defects caused at the CFTR protein level by ∆F508 mutation and the redundancy of quality control mechanisms detecting ∆F508-CFTR as a defective protein impose a ceiling to the maximal effect that a single compound (corrector may obtain. Therefore, treatment of patients with the most frequent CF mutation may require the optimized combination of two drugs having additive or synergic effects.

  16. Plasma membrane CFTR regulates RANTES expression via its C-terminal PDZ-interacting motif.

    Science.gov (United States)

    Estell, Kim; Braunstein, Gavin; Tucker, Torry; Varga, Karoly; Collawn, James F; Schwiebert, Lisa M

    2003-01-01

    Despite the identification of 1,000 mutations in the cystic fibrosis gene product CFTR, there remains discordance between CFTR genotype and lung disease phenotype. The study of CFTR, therefore, has expanded beyond its chloride channel activity into other possible functions, such as its role as a regulator of gene expression. Findings indicate that CFTR plays a role in the expression of RANTES in airway epithelia. RANTES is a chemokine that has been implicated in the regulation of mucosal immunity and the pathogenesis of airway inflammatory diseases. Results demonstrate that CFTR triggers RANTES expression via a mechanism that is independent of CFTR's chloride channel activity. Neither pharmacological inhibition of CFTR nor activation of alternative chloride channels, including hClC-2, modulated RANTES expression. Through the use of CFTR disease-associated and truncation mutants, experiments suggest that CFTR-mediated transcription factor activation and RANTES expression require (i) insertion of CFTR into the plasma membrane and (ii) an intact CFTR C-terminal PDZ-interacting domain. Expression of constructs encoding wild-type or dominant-negative forms of the PDZ-binding protein EBP50 suggests that EBP50 may be involved in CFTR-dependent RANTES expression. Together, these data suggest that CFTR modulates gene expression in airway epithelial cells while located in a macromolecular signaling complex at the plasma membrane. PMID:12509457

  17. Divergent signaling via SUMO modification: potential for CFTR modulation.

    Science.gov (United States)

    Ahner, Annette; Gong, Xiaoyan; Frizzell, Raymond A

    2016-02-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is generally responsible for the cAMP/PKA regulated anion conductance at the apical membranes of secretory epithelial cells. Mutations in CFTR underlie cystic fibrosis (CF), in which the most common variant, F508del, causes protein misfolding and its proteasome-mediated degradation. A new pathway that contributes to mutant CFTR degradation is mediated by the small heat shock protein, Hsp27, which cooperates with Ubc9, the E2 enzyme for SUMOylation, to selectively conjugate mutant CFTR with SUMO-2/3. This SUMO paralog can form polychains, which are recognized by the ubiquitin E3 enzyme, RNF4, leading to CFTR ubiquitylation and recognition by the proteasome. We found also that F508del CFTR could be modified by SUMO-1, a paralog that does not support SUMO polychain formation. The use of different SUMO paralogs to modify and target a single substrate for divergent purposes is not uncommon. In this short review we discuss the possibility that conjugation with SUMO-1 could protect mutant CFTR from disposal by RNF4 and similar ubiquitin ligases. We hypothesize that such a pathway could contribute to therapeutic efforts to stabilize immature mutant CFTR and thereby enhance the action of therapeutics that correct CFTR trafficking to the apical membranes.

  18. Proteomic identification of calumenin as a G551D-CFTR associated protein.

    Directory of Open Access Journals (Sweden)

    Ling Teng

    Full Text Available Cystic fibrosis (CF is the most common lethal autosomal recessive disease in the Caucasian population. It is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR gene. To date, over 1910 mutations have been identified in the CFTR gene. Among these mutations, the CF-causing missense mutation G551D-CFTR (approx. 5% of cases encodes for a CFTR chloride channel with normal expression on the cell surface. Nevertheless, it is associated with severe disease due to its altered channel activation. The aim of the present study was to identify specific interacting proteins of G551D-CFTR. Co-immunoprecipitated proteins with G551D-CFTR were resolved by 2D-gel electrophoresis (2-DE. Mass Spectrometry revealed that calumenin was present in the protein complex linked to G551D-CFTR. Despite its basal expression was not modified in G551D-CFTR expressing cells when compared to Wt-CFTR expressing cells, it was more abundant in the G551D-CFTR complex detected by immunoprecipitation. The calumenin-CFTR interaction was also shown by Surface Plasmon Resonance and further confirmed by computational analysis of the predicted calumenin's partners. Because in our cellular model calumenin was found in the endoplasmic reticulum (ER by immunofluorescence experiments, we suggest that calumenin is likely involved in the mutated CFTR's maturation. In conclusion, we showed for the first time that calumenin binds to CFTR and that it is increased in the G551D-CFTR complex. We suggest that it may be involved in the physiopathology of G551D-CFTR and that G551D-CFTR may follow a specific maturation and trafficking pathway. We also hypothesize that UPR may be triggered independently of the retention of G551D-CFTR in the ER because Grp78/Bip expression is increased in the cells. Finally, we propose here that Calumenin is a new CFTR chaperone.

  19. Proteomic identification of calumenin as a G551D-CFTR associated protein.

    Science.gov (United States)

    Teng, Ling; Kerbiriou, Mathieu; Taiya, Mehdi; Le Hir, Sophie; Mignen, Olivier; Benz, Nathalie; Trouvé, Pascal; Férec, Claude

    2012-01-01

    Cystic fibrosis (CF) is the most common lethal autosomal recessive disease in the Caucasian population. It is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To date, over 1910 mutations have been identified in the CFTR gene. Among these mutations, the CF-causing missense mutation G551D-CFTR (approx. 5% of cases) encodes for a CFTR chloride channel with normal expression on the cell surface. Nevertheless, it is associated with severe disease due to its altered channel activation. The aim of the present study was to identify specific interacting proteins of G551D-CFTR. Co-immunoprecipitated proteins with G551D-CFTR were resolved by 2D-gel electrophoresis (2-DE). Mass Spectrometry revealed that calumenin was present in the protein complex linked to G551D-CFTR. Despite its basal expression was not modified in G551D-CFTR expressing cells when compared to Wt-CFTR expressing cells, it was more abundant in the G551D-CFTR complex detected by immunoprecipitation. The calumenin-CFTR interaction was also shown by Surface Plasmon Resonance and further confirmed by computational analysis of the predicted calumenin's partners. Because in our cellular model calumenin was found in the endoplasmic reticulum (ER) by immunofluorescence experiments, we suggest that calumenin is likely involved in the mutated CFTR's maturation. In conclusion, we showed for the first time that calumenin binds to CFTR and that it is increased in the G551D-CFTR complex. We suggest that it may be involved in the physiopathology of G551D-CFTR and that G551D-CFTR may follow a specific maturation and trafficking pathway. We also hypothesize that UPR may be triggered independently of the retention of G551D-CFTR in the ER because Grp78/Bip expression is increased in the cells. Finally, we propose here that Calumenin is a new CFTR chaperone.

  20. Anion channels and the stimulation of anthocyanin accumulation by blue light in Arabidopsis seedlings

    Science.gov (United States)

    Noh, B.; Spalding, E. P.; Evans, M. H. (Principal Investigator)

    1998-01-01

    Activation of anion channels by blue light begins within seconds of irradiation in seedlings and is related to the ensuing growth inhibition. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) is a potent, selective, and reversible blocker of these anion channels in Arabidopsis thaliana. Here we show that 20 microM NPPB blocked 72% of the blue-light-induced accumulation of anthocyanin pigments in seedlings. Feeding biosynthetic intermediates to wild-type and tt5 seedlings provided evidence that NPPB prevented blue light from up-regulating one or more steps between and including phenylalanine ammonia lyase and chalcone isomerase. NPPB was found to have no significant effect on the blue-light-induced increase in transcript levels of PAL1, CHS, CHI, or DFR, which are genes that encode anthocyanin-biosynthetic enzymes. Immunoblots revealed that NPPB also did not inhibit the accumulation of the chalcone synthase, chalcone isomerase, or flavanone-3-hydroxylase proteins. This is in contrast to the reduced anthocyanin accumulation displayed by a mutant lacking the HY4 blue-light receptor, as hy4 displayed reduced expression of the above enzymes. Taken together, the data indicate that blue light acting through HY4 leads to an increase in the amount of biosynthetic enzymes but blue light must also act through a separate, anion-channel-dependent system to create a fully functional biosynthetic pathway.

  1. The progress in CFTR chloride ion channel%CFTR型氯离子通道研究进展

    Institute of Scientific and Technical Information of China (English)

    郭晓强

    2007-01-01

    囊性纤维化跨膜传导调节因子(CFTR)是一种重要的氯离子通道,突变易引起囊性纤维化病变,故得名.一系列研究表明,CFTR由5个结构域组成:两个跨膜结构域形成氯离子通道;两个核苷酸结合结构域调节通道的开闭;一个调节结构域主要影响氯通道的活动.这些结构域通过协同作用共同控制了氯离子的跨膜流动,而一些突变可以影响细胞功能而导致囊性纤维化的发生.本文通过介绍CFTR基本结构、调节机制、与囊性纤维化病变的关系及针对CFTR的治疗而对CFTR型氯离子通道有一个的全面的理解.

  2. Endosomal SNARE proteins regulate CFTR activity and trafficking in epithelial cells.

    Science.gov (United States)

    Bilan, Frédéric; Nacfer, Magali; Fresquet, Fleur; Norez, Caroline; Melin, Patricia; Martin-Berge, Alice; Costa de Beauregard, Marie-Alyette; Becq, Frédéric; Kitzis, Alain; Thoreau, Vincent

    2008-07-01

    The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein is a chloride channel localized at the apical plasma membrane of epithelial cells. We previously described that syntaxin 8, an endosomal SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) protein, interacts with CFTR and regulates its trafficking to the plasma membrane and hence its channel activity. Syntaxin 8 belongs to the endosomal SNARE complex which also contains syntaxin 7, vti1b and VAMP8. Here, we report that these four endosomal SNARE proteins physically and functionally interact with CFTR. In LLC-PK1 cells transfected with CFTR and in Caco-2 cells endogenously expressing CFTR, we demonstrated that endosomal SNARE protein overexpression inhibits CFTR activity but not swelling- or calcium-activated iodide efflux, indicating a specific effect upon CFTR activity. Moreover, co-immunoprecipitation experiments in LLC-PK1-CFTR cells showed that CFTR and SNARE proteins belong to a same complex and pull-down assays showed that VAMP8 and vti1b preferentially interact with CFTR N-terminus tail. By cell surface biotinylation and immunofluorescence experiments, we evidenced that endosomal SNARE overexpression disturbs CFTR apical targeting. Finally, we found a colocalization of CFTR and endosomal SNARE proteins in Rab11-positive recycling endosomes, suggesting a new role for endosomal SNARE proteins in CFTR trafficking in epithelial cells.

  3. LMTK2-mediated phosphorylation regulates CFTR endocytosis in human airway epithelial cells.

    Science.gov (United States)

    Luz, Simão; Cihil, Kristine M; Brautigan, David L; Amaral, Margarida D; Farinha, Carlos M; Swiatecka-Urban, Agnieszka

    2014-05-23

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-)-selective ion channel expressed in fluid-transporting epithelia. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser(737) in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl(-) secretion mediated by the wild-type and rescued ΔF508-CFTR. Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-Ser(737) mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl(-) channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl(-) channels and improve stability of pharmacologically rescued ΔF508-CFTR in patients with cystic fibrosis.

  4. EFFECTS OF MONOCARBOXYLIC ACID DERIVATIVES ON CARDIAC VENTRICULAR CFTR Cl-CHANNELS IN GUINEA PIG%单羧酸类Cl-通道阻断剂对心室肌CFTR Cl-通道的影响

    Institute of Scientific and Technical Information of China (English)

    周士胜; 臧益民

    1999-01-01

    本文采用全细胞膜片箝与细胞内灌注技术,观察了单羧酸类 Cl-通道阻断剂对豚鼠心室肌囊性纤维变性膜透性调节蛋白(CFTR)Cl-电流的影响,细胞外9-AC以可逆方式增强异丙肾上腺素(ISO)激发的CFTR Cl-的外向电流成分,5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB)和二苯胺羧酸(DPC)对ISO发的CFTR Cl-电流的作用呈现先增强后抑制的双相效应.细胞内NPPB表现为增强ISO激发作用.结果表明,单羧酸类Cl-通道阻断剂在细胞上有不同的作用位点,该类药物作用效果的差异可能与此有关.

  5. Clusters of Cl- channels in CFTR-expressing Sf9 cells switch spontaneously between slow and fast gating modes

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Price, E. M.; Gabriel, S. E.;

    1996-01-01

    The Sf9 insect Spodoptora frugiperda cell line was used for heterologous expression of the cloned human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, or the cloned ß-galactosidase gene, using the baculovirus Autographa califonica as the infection vector. Using application of the...... patch-clamp technique, evidence for functional expression of CFTR was obtained according to the following three criteria. Firstly, whole-cell currents recorded 2 days after infection with CFTR revealed a statistically significant increase of membrane conductance, ˜25 times above that of mock......-infected control cells, with the reversal potential of the major current component being governed by the chloride equilibrium potential (E Cl). Secondly, in contrast to uninfected cells and cells infected with ß-galactosidase, the membrane conductance to chloride of CFTR-injected cells was stimulated by cytosolic...

  6. Modulation of CFTR gating by permeant ions.

    Science.gov (United States)

    Yeh, Han-I; Yeh, Jiunn-Tyng; Hwang, Tzyh-Chang

    2015-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is unique among ion channels in that after its phosphorylation by protein kinase A (PKA), its ATP-dependent gating violates microscopic reversibility caused by the intimate involvement of ATP hydrolysis in controlling channel closure. Recent studies suggest a gating model featuring an energetic coupling between opening and closing of the gate in CFTR's transmembrane domains and association and dissociation of its two nucleotide-binding domains (NBDs). We found that permeant ions such as nitrate can increase the open probability (Po) of wild-type (WT) CFTR by increasing the opening rate and decreasing the closing rate. Nearly identical effects were seen with a construct in which activity does not require phosphorylation of the regulatory domain, indicating that nitrate primarily affects ATP-dependent gating steps rather than PKA-dependent phosphorylation. Surprisingly, the effects of nitrate on CFTR gating are remarkably similar to those of VX-770 (N-(2,4-Di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide), a potent CFTR potentiator used in clinics. These include effects on single-channel kinetics of WT CFTR, deceleration of the nonhydrolytic closing rate, and potentiation of the Po of the disease-associated mutant G551D. In addition, both VX-770 and nitrate increased the activity of a CFTR construct lacking NBD2 (ΔNBD2), indicating that these gating effects are independent of NBD dimerization. Nonetheless, whereas VX-770 is equally effective when applied from either side of the membrane, nitrate potentiates gating mainly from the cytoplasmic side, implicating a common mechanism for gating modulation mediated through two separate sites of action. PMID:25512598

  7. Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators protect G551D but not ΔF508 CFTR from thermal instability.

    Science.gov (United States)

    Liu, Xuehong; Dawson, David C

    2014-09-01

    The G551D cystic fibrosis transmembrane conductance regulator (CFTR) mutation is associated with severe disease in ∼5% of cystic fibrosis patients worldwide. This amino acid substitution in NBD1 results in a CFTR chloride channel characterized by a severe gating defect that can be at least partially overcome in vitro by exposure to a CFTR potentiator. In contrast, the more common ΔF508 mutation is associated with a severe protein trafficking defect, as well as impaired channel function. Recent clinical trials demonstrated a beneficial effect of the CFTR potentiator, Ivacaftor (VX-770), on lung function of patients bearing at least one copy of G551D CFTR, but no comparable effect on ΔF508 homozygotes. This difference in efficacy was not surprising in view of the established difference in the molecular phenotypes of the two mutant channels. Recently, however, it was shown that the structural defect introduced by the deletion of F508 is associated with the thermal instability of ΔF508 CFTR channel function in vitro. This additional mutant phenotype raised the possibility that the differences in the behavior of ΔF508 and G551D CFTR, as well as the disparate efficacy of Ivacaftor, might be a reflection of the differing thermal stabilities of the two channels at 37 °C. We compared the thermal stability of G551D and ΔF508 CFTR in Xenopus oocytes in the presence and absence of CTFR potentiators. G551D CFTR exhibited a thermal instability that was comparable to that of ΔF508 CFTR. G551D CFTR, however, was protected from thermal instability by CFTR potentiators, whereas ΔF508 CFTR was not. These results suggest that the efficacy of VX-770 in patients bearing the G551D mutation is due, at least in part, to the ability of the small molecule to protect the mutant channel from thermal instability at human body temperature.

  8. Synergistic Malaria Parasite Killing by Two Types of Plasmodial Surface Anion Channel Inhibitors.

    Directory of Open Access Journals (Sweden)

    Margaret Pain

    Full Text Available Malaria parasites increase their host erythrocyte's permeability to a broad range of ions and organic solutes. The plasmodial surface anion channel (PSAC mediates this uptake and is an established drug target. Development of therapies targeting this channel is limited by several problems including interactions between known inhibitors and permeating solutes that lead to incomplete channel block. Here, we designed and executed a high-throughput screen to identify a novel class of PSAC inhibitors that overcome this solute-inhibitor interaction. These new inhibitors differ from existing blockers and have distinct effects on channel-mediated transport, supporting a model of two separate routes for solute permeation though PSAC. Combinations of inhibitors specific for the two routes had strong synergistic action against in vitro parasite propagation, whereas combinations acting on a single route produced only additive effects. The magnitude of synergism depended on external nutrient concentrations, consistent with an essential role of the channel in parasite nutrient acquisition. The identified inhibitors will enable a better understanding of the channel's structure-function and may be starting points for novel combination therapies that produce synergistic parasite killing.

  9. Structure of wild type and mutant F508del CFTR: A small-angle X-ray scattering study of the protein-detergent complexes.

    Science.gov (United States)

    Pollock, Naomi L; Satriano, Letizia; Zegarra-Moran, Olga; Ford, Robert C; Moran, Oscar

    2016-04-01

    CFTR is an anionic channel expressed in epithelia whose mutations cause cystic fibrosis. Wild (WT) and mutated (F508del) types were over-expressed in yeast, solubilised in the detergent LPG-14 and purified. The detergent-CFTR complexes were studied by SAXS techniques using a solvent of variable density. The final result of the study is the numerical value of a set of parameters: molecular mass, volume and radius of gyration, average electron density and second moment of the electron density fluctuations inside the particles. It is also shown that in the complex the centres of gravity of CFTR and of the detergent are displaced relative to each other. The analysis of these parameters led to the determination of the size and shape of the volumes occupied by protein and by detergent in the complex. WT-CFTR to be an elongated molecule (maximum diameter ∼12.4nm) which spans a flat detergent micelle. The distance distribution function, P(r) confirms that the WT-CFTR is elongated and with an inhomogeneous electronic density. The F508del-CFTR molecule is also elongated (maximum diameter ∼13.2nm), but the associated detergent micelle hides a larger surface, plausibly related to an increased exposure of hydrophobic portions of the mutated protein. The corresponding P(r) is consistent with the presence of well defined domains, probably linked by flexible regions. These differences suggest that the full-length mutant F508del-CFTR has a detectably different conformation, in contrast to the minor differences observed for the isolated F508-containing domain. We interpret the data in terms of an incomplete post-translational assembly of the protein domains.

  10. Structure of wild type and mutant F508del CFTR: A small-angle X-ray scattering study of the protein-detergent complexes.

    Science.gov (United States)

    Pollock, Naomi L; Satriano, Letizia; Zegarra-Moran, Olga; Ford, Robert C; Moran, Oscar

    2016-04-01

    CFTR is an anionic channel expressed in epithelia whose mutations cause cystic fibrosis. Wild (WT) and mutated (F508del) types were over-expressed in yeast, solubilised in the detergent LPG-14 and purified. The detergent-CFTR complexes were studied by SAXS techniques using a solvent of variable density. The final result of the study is the numerical value of a set of parameters: molecular mass, volume and radius of gyration, average electron density and second moment of the electron density fluctuations inside the particles. It is also shown that in the complex the centres of gravity of CFTR and of the detergent are displaced relative to each other. The analysis of these parameters led to the determination of the size and shape of the volumes occupied by protein and by detergent in the complex. WT-CFTR to be an elongated molecule (maximum diameter ∼12.4nm) which spans a flat detergent micelle. The distance distribution function, P(r) confirms that the WT-CFTR is elongated and with an inhomogeneous electronic density. The F508del-CFTR molecule is also elongated (maximum diameter ∼13.2nm), but the associated detergent micelle hides a larger surface, plausibly related to an increased exposure of hydrophobic portions of the mutated protein. The corresponding P(r) is consistent with the presence of well defined domains, probably linked by flexible regions. These differences suggest that the full-length mutant F508del-CFTR has a detectably different conformation, in contrast to the minor differences observed for the isolated F508-containing domain. We interpret the data in terms of an incomplete post-translational assembly of the protein domains. PMID:26850167

  11. Capturing the Direct Binding of CFTR Correctors to CFTR by Using Click Chemistry.

    Science.gov (United States)

    Sinha, Chandrima; Zhang, Weiqiang; Moon, Chang Suk; Actis, Marcelo; Yarlagadda, Sunitha; Arora, Kavisha; Woodroofe, Koryse; Clancy, John P; Lin, Songbai; Ziady, Assem G; Frizzell, Raymond; Fujii, Naoaki; Naren, Anjaparavanda P

    2015-09-21

    Cystic fibrosis (CF) is a lethal genetic disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel. F508del is the most prevalent mutation of the CFTR gene and encodes a protein defective in folding and processing. VX-809 has been reported to facilitate the folding and trafficking of F508del-CFTR and augment its channel function. The mechanism of action of VX-809 has been poorly understood. In this study, we sought to answer a fundamental question underlying the mechanism of VX-809: does it bind CFTR directly in order to exert its action? We synthesized two VX-809 derivatives, ALK-809 and SUL-809, that possess an alkyne group and retain the rescue capacity of VX-809. By using Cu(I) -catalyzed click chemistry, we provide evidence that the VX-809 derivatives bind CFTR directly in vitro and in cells. Our findings will contribute to the elucidation of the mechanism of action of CFTR correctors and the design of more potent therapeutics to combat CF.

  12. A slow anion channel in guard cells, activating at large hyperpolarization, may be principal for stomatal closing.

    Science.gov (United States)

    Linder, B; Raschke, K

    1992-11-16

    Slowly activating anion channel currents were discovered at micromolar 'cytoplasmic' Ca2+ during patch-clamp measurements on guard-cell protoplasts of Vicia faba and Xanthium strumarium. They activated at potentials as low as -200 mV, with time constants between 5 and 60 s, and no inactivation. The broad voltage dependence exhibited a current maximum near -40 mV. The single-channel open time was in the order of seconds, and the unitary conductance was 33 ps, similar to that of the already described 'quick' anion channel of guard cells. Because of its activity at low potentials, the slow anion channel may be essential for the depolarization of the plasmalemma that is required for salt efflux during stomatal closing. PMID:1385219

  13. CFTR: A New Horizon in the Pathomechanism and Treatment of Pancreatitis.

    Science.gov (United States)

    Hegyi, Péter; Wilschanski, Michael; Muallem, Shmuel; Lukacs, Gergely L; Sahin-Tóth, Miklós; Uc, Aliye; Gray, Michael A; Rakonczay, Zoltán; Maléth, József

    2016-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts chloride and bicarbonate ions across epithelial cell membranes. Mutations in the CFTR gene diminish the ion channel function and lead to impaired epithelial fluid transport in multiple organs such as the lung and the pancreas resulting in cystic fibrosis. Heterozygous carriers of CFTR mutations do not develop cystic fibrosis but exhibit increased risk for pancreatitis and associated pancreatic damage characterized by elevated mucus levels, fibrosis, and cyst formation. Importantly, recent studies demonstrated that pancreatitis causing insults, such as alcohol, smoking, or bile acids, strongly inhibit CFTR function. Furthermore, human studies showed reduced levels of CFTR expression and function in all forms of pancreatitis. These findings indicate that impairment of CFTR is critical in the development of pancreatitis; therefore, correcting CFTR function could be the first specific therapy in pancreatitis. In this review, we summarize recent advances in the field and discuss new possibilities for the treatment of pancreatitis. PMID:26856995

  14. Lumacaftor alone and combined with ivacaftor: preclinical and clinical trial experience of F508del CFTR correction.

    Science.gov (United States)

    Brewington, John J; McPhail, Gary L; Clancy, John P

    2016-01-01

    Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR), leading to significant morbidity and mortality. CFTR is a chloride and bicarbonate channel at the epithelial cell membrane. The most common CFTR mutation is F508del, resulting in minimal CFTR at the plasma membrane. Current disease management is supportive, whereas an ultimate goal is to develop therapies to restore CFTR activity. We summarize experience with lumacaftor, a small molecule that increases F508del-CFTR levels at the plasma membrane. Lumacaftor in combination with ivacaftor, a modulator of CFTR gating defects, improves clinical outcome measures in patients homozygous for the F508del mutation. Lumacaftor represents a significant advancement in the treatment of biochemical abnormalities in CF. Further development of CFTR modulators will improve upon current therapies, although it remains unclear whether this approach will provide therapies for all CFTR mutations. PMID:26581802

  15. Mechanosensitive activation of CFTR by increased cell volume and hydrostatic pressure but not shear stress.

    Science.gov (United States)

    Vitzthum, Constanze; Clauss, Wolfgang G; Fronius, Martin

    2015-11-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel that is essential for electrolyte and fluid homeostasis. Preliminary evidence indicates that CFTR is a mechanosensitive channel. In lung epithelia, CFTR is exposed to different mechanical forces such as shear stress (Ss) and membrane distention. The present study questioned whether Ss and/or stretch influence CFTR activity (wild type, ∆F508, G551D). Human CFTR (hCFTR) was heterologously expressed in Xenopus oocytes and the response to the mechanical stimulus and forskolin/IBMX (FI) was measured by two-electrode voltage-clamp experiments. Ss had no influence on hCFTR activity. Injection of an intracellular analogous solution to increase cell volume alone did not affect hCFTR activity. However, hCFTR activity was augmented by injection after pre-stimulation with FI. The response to injection was similar in channels carrying the common mutations ∆F508 and G551D compared to wild type hCFTR. Stretch-induced CFTR activation was further assessed in Ussing chamber measurements using Xenopus lung preparations. Under control conditions increased hydrostatic pressure (HP) decreased the measured ion current including activation of a Cl(-) secretion that was unmasked by the CFTR inhibitor GlyH-101. These data demonstrate activation of CFTR in vitro and in a native pulmonary epithelium in response to mechanical stress. Mechanosensitive regulation of CFTR is highly relevant for pulmonary physiology that relies on ion transport processes facilitated by pulmonary epithelial cells. PMID:26357939

  16. Photoelectron anisotropy and channel branching ratios in the detachment of solvated iodide cluster anions

    International Nuclear Information System (INIS)

    Photoelectron spectra and angular distributions in 267 nm detachment of the I-·Ar, I-·H2O, I-·CH3I, and I-·CH3CN cluster anions are examined in comparison with bare I- using velocity-map photoelectron imaging. In all cases, features are observed that correlate to two channels producing either I(2P3/2) or I(2P1/2). In the photodetachment of I- and I-·Ar, the branching ratios of the 2P1/2 and 2P3/2 channels are observed to be ≅0.4, in both cases falling short of the statistical ratio of 0.5. For I-·H2O and I-·CH3I, the 2P1/2 to 2P3/2 branching ratios are greater by a factor of 1.6 compared to the bare iodide case. The relative enhancement of the 2P1/2 channel is attributed to dipole effects on the final-state continuum wave function in the presence of polar solvents. For I-·CH3CN the 2P1/2 to 2P3/2 ratio falls again, most likely due to the proximity of the detachment threshold in the excited spin-orbit channel. The photoelectron angular distributions in the photodetachment of I-, I-·Ar, I-·H2O, and I-·CH3CN are understood within the framework of direct detachment from I-. Hence, the corresponding anisotropy parameters are modeled using variants of the Cooper-Zare central-potential model for atomic-anion photodetachment. In contrast, I-·CH3I yields nearly isotropic photoelectron angular distributions in both detachment channels. The implications of this anomalous behavior are discussed with reference to alternative mechanisms, affording the solvent molecule an active role in the electron ejection process

  17. Adrenaline-induced colonic K+ secretion is mediated by KCa1.1 (BK) channels

    DEFF Research Database (Denmark)

    Sørensen, Mads Vaarby; Sausbier, Matthias; Ruth, Peter;

    2010-01-01

    secretory K(+) channel in the apical membrane of the murine distal colon. The BK channel is responsible for both resting and Ca(2+)-activated colonic K(+) secretion and is up-regulated by aldosterone. Agonists (e.g. adrenaline) that elevate cAMP are potent activators of distal colonic K(+) secretion....... However, the secretory K(+) channel responsible for cAMP-induced K(+) secretion remains to be defined. In this study we used the Ussing chamber to identify adrenaline-induced electrogenic K(+) secretion. We found that the adrenaline-induced electrogenic ion secretion is a compound effect dominated...... by anion secretion and a smaller electrically opposing K(+) secretion. Using tissue from (i) BK wildtype (BK(+/+)) and knockout (BK(/)) and (ii) cystic fibrosis transmembrane regulator (CFTR) wildtype (CFTR(+/+)) and knockout (CFTR(/)) mice we were able to isolate the adrenaline-induced K(+) secretion. We...

  18. Islet-intrinsic effects of CFTR mutation.

    Science.gov (United States)

    Koivula, Fiona N Manderson; McClenaghan, Neville H; Harper, Alan G S; Kelly, Catriona

    2016-07-01

    Cystic fibrosis-related diabetes (CFRD) is the most significant extra-pulmonary comorbidity in cystic fibrosis (CF) patients, and accelerates lung decline. In addition to the traditional view that CFRD is a consequence of fibrotic destruction of the pancreas as a whole, emerging evidence may implicate a role for cystic fibrosis transmembrane-conductance regulator (CFTR) in the regulation of insulin secretion from the pancreatic islet. Impaired first-phase insulin responses and glucose homeostasis have also been reported in CF patients. CFTR expression in both human and mouse beta cells has been confirmed, and recent studies have shown differences in endocrine pancreatic morphology from birth in CF. Recent experimental evidence suggests that functional CFTR channels are required for insulin exocytosis and the regulation of membrane potential in the pancreatic beta cell, which may account for the impairments in insulin secretion observed in many CF patients. These novel insights suggest that the pathogenesis of CFRD is more complicated than originally thought, with implications for diabetes treatment and screening in the CF population. This review summarises recent emerging evidence in support of a primary role for endocrine pancreatic dysfunction in the development of CFRD. Summary • CF is an autosomal recessive disorder caused by mutations in the CFTR gene • The vast majority of morbidity and mortality in CF results from lung disease. However CFRD is the largest extra-pulmonary co-morbidity and rapidly accelerates lung decline • Recent experimental evidence shows that functional CFTR channels are required for normal patterns of first phase insulin secretion from the pancreatic beta cell • Current clinical recommendations suggest that insulin is more effective than oral glucose-lowering drugs for the treatment of CFRD. However, the emergence of CFTR corrector and potentiator drugs may offer a personalised approach to treating diabetes in the CF population

  19. Pseudomonas aeruginosa triggers CFTR-mediated airway surface liquid secretion in swine trachea.

    Science.gov (United States)

    Luan, Xiaojie; Campanucci, Verónica A; Nair, Manoj; Yilmaz, Orhan; Belev, George; Machen, Terry E; Chapman, Dean; Ianowski, Juan P

    2014-09-01

    Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the gene encoding for the anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Several organs are affected in CF, but most of the morbidity and mortality comes from lung disease. Recent data show that the initial consequence of CFTR mutation is the failure to eradicate bacteria before the development of inflammation and airway remodeling. Bacterial clearance depends on a layer of airway surface liquid (ASL) consisting of both a mucus layer that traps, kills, and inactivates bacteria and a periciliary liquid layer that keeps the mucus at an optimum distance from the underlying epithelia, to maximize ciliary motility and clearance of bacteria. The airways in CF patients and animal models of CF demonstrate abnormal ASL secretion and reduced antimicrobial properties. Thus, it has been proposed that abnormal ASL secretion in response to bacteria may facilitate the development of the infection and inflammation that characterize CF airway disease. Whether the inhalation of bacteria triggers ASL secretion, and the role of CFTR, have never been tested, however. We developed a synchrotron-based imaging technique to visualize the ASL layer and measure the effect of bacteria on ASL secretion. We show that the introduction of Pseudomonas aeruginosa and other bacteria into the lumen of intact isolated swine tracheas triggers CFTR-dependent ASL secretion by the submucosal glands. This response requires expression of the bacterial protein flagellin. In patients with CF, the inhalation of bacteria would fail to trigger ASL secretion, leading to infection and inflammation. PMID:25136096

  20. Inter-channel effects in monosolvated atomic iodide cluster anion detachment: correlation of the anisotropy parameter with solvent dipole moment.

    Science.gov (United States)

    Mbaiwa, Foster; Dao, Diep; Holtgrewe, Nicholas; Lasinski, Joshua; Mabbs, Richard

    2012-03-21

    Photoelectron imaging results are presented for I(-)[middle dot]X cluster anions (X = CO(2), C(4)H(5)N [pyrrole], (CH(3))(2)CO, CH(3)NO(2)). The available detachment channels are labeled according to the neutral iodine atom states produced (channel I ≡ (2)P(3/2) and channel II ≡ (2)P(1/2)). At photon energies in the vicinity of the channel II threshold these data are compared to previously reported results for I(-)[middle dot]X (X = CH(3)CN, CH(3)Cl, CH(3)Br, and H(2)O). In particular, these results show a strong connection between the dipole moment of the solvent molecule and the behavior of the channel I photoelectron angular distributions in this region, which is consistent with an electronic autodetachment process. The evolution of the channel II:channel I branching ratios in this excitation regime supports this contention.

  1. Inter-channel effects in monosolvated atomic iodide cluster anion detachment: Correlation of the anisotropy parameter with solvent dipole moment

    Science.gov (United States)

    Mbaiwa, Foster; Dao, Diep; Holtgrewe, Nicholas; Lasinski, Joshua; Mabbs, Richard

    2012-03-01

    Photoelectron imaging results are presented for I-.X cluster anions (X = CO2, C4H5N [pyrrole], (CH3)2CO, CH3NO2). The available detachment channels are labeled according to the neutral iodine atom states produced (channel I ≡ 2P3/2 and channel II ≡ 2P1/2). At photon energies in the vicinity of the channel II threshold these data are compared to previously reported results for I-.X (X = CH3CN, CH3Cl, CH3Br, and H2O). In particular, these results show a strong connection between the dipole moment of the solvent molecule and the behavior of the channel I photoelectron angular distributions in this region, which is consistent with an electronic autodetachment process. The evolution of the channel II:channel I branching ratios in this excitation regime supports this contention.

  2. Potentiators exert distinct effects on human, murine, and Xenopus CFTR.

    Science.gov (United States)

    Cui, Guiying; Khazanov, Netaly; Stauffer, Brandon B; Infield, Daniel T; Imhoff, Barry R; Senderowitz, Hanoch; McCarty, Nael A

    2016-08-01

    VX-770 (Ivacaftor) has been approved for clinical usage in cystic fibrosis patients with several CFTR mutations. Yet the binding site(s) on CFTR for this compound and other small molecule potentiators are unknown. We hypothesize that insight into this question could be gained by comparing the effect of potentiators on CFTR channels from different origins, e.g., human, mouse, and Xenopus (frog). In the present study, we combined this comparative molecular pharmacology approach with that of computer-aided drug discovery to identify and characterize new potentiators of CFTR and to explore possible mechanism of action. Our results demonstrate that 1) VX-770, NPPB, GlyH-101, P1, P2, and P3 all exhibited ortholog-specific behavior in that they potentiated hCFTR, mCFTR, and xCFTR with different efficacies; 2) P1, P2, and P3 potentiated hCFTR in excised macropatches in a manner dependent on the degree of PKA-mediated stimulation; 3) P1 and P2 did not have additive effects, suggesting that these compounds might share binding sites. Also 4) using a pharmacophore modeling approach, we identified three new potentiators (IOWH-032, OSSK-2, and OSSK-3) that have structures similar to GlyH-101 and that also exhibit ortholog-specific potentiation of CFTR. These could potentially serve as lead compounds for development of new drugs for the treatment of cystic fibrosis. The ortholog-specific behavior of these compounds suggest that a comparative pharmacology approach, using cross-ortholog chimeras, may be useful for identification of binding sites on human CFTR. PMID:27288484

  3. Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance

    DEFF Research Database (Denmark)

    Hoffmann, Else Kay; Sørensen, Belinda Halling; Sauter, Daniel Rafael Peter;

    2015-01-01

    Volume-regulated channels for anions (VRAC) / organic osmolytes (VSOAC) play essential roles in cell volume regulation and other cellular functions, e.g. proliferation, cell migration and apoptosis. LRRC8A, which belongs to the leucine rich-repeat containing protein family, was recently shown to ...

  4. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    Science.gov (United States)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  5. Functional interaction of endothelial nitric oxide synthase with a voltage-dependent anion channel

    Science.gov (United States)

    Sun, Jianxin; Liao, James K.

    2002-01-01

    Endothelium-derived nitric oxide (NO) is an important regulator of vascular function. NO is produced by endothelial NO synthase (eNOS) whose function is modulated, in part, by specific protein interactions. By coimmunoprecipitation experiments followed by MS analyses, we identified a human voltage-dependent anion/cation channel or porin as a binding partner of eNOS. The interaction between porin and eNOS was demonstrated by coimmunoprecipitation studies in nontransfected human endothelial cells and Cos-7 cells transiently transfected with eNOS and porin cDNAs. In vitro binding studies with glutathione S-transferase–porin indicated that porin binds directly to eNOS and that this interaction augmented eNOS activity. The calcium ionophore, A23187, and bradykinin, which are known to activate eNOS, markedly increased porin–eNOS interaction, suggesting a potential role of intracellular Ca2+ in mediating this interaction. Theses results indicate that the interaction between a voltage-dependent membrane channel and eNOS may be important for regulating eNOS activity. PMID:12228731

  6. Modulation of the voltage-dependent anion channel of mitochondria by elaidic acid.

    Science.gov (United States)

    Tewari, Debanjan; Bera, Amal Kanti

    2016-08-26

    Dietary trans fatty acids (TFAs) are known to increase the risk of cardiovascular diseases by altering plasma lipid profile and activating various inflammatory signaling pathways. Here we show that elaidic acid (EA), the most abundant TFA in diet, alters the electrophysiological properties of voltage-dependent anion channel (VDAC) of mitochondria. Purified bovine brain VDAC, when incorporated in the planar lipid bilayer (PLB) composed of 1,2-diphytanoyl-sn-glycero-3 phosphatidyl choline (DPhPC) and EA in a 9 to 1 ratio (wt/wt), exhibited complete closing events at different voltages. The closing events were observed at even -10 mV, a voltage at which VDAC usually remains fully open all the time. Additionally, the voltage sensitivity of VDAC was lost in presence of EA; the channel conductance did not decrease with increasing voltages. In identical experimental conditions, membrane containing oleic acid (OA), the cis isomer of EA did not produce any such effect. We propose that EA possibly exerts its adverse effect by modulating VDAC. PMID:27318085

  7. Structural basis of control of inward rectifier Kir2 channel gating by bulk anionic phospholipids.

    Science.gov (United States)

    Lee, Sun-Joo; Ren, Feifei; Zangerl-Plessl, Eva-Maria; Heyman, Sarah; Stary-Weinzinger, Anna; Yuan, Peng; Nichols, Colin G

    2016-09-01

    Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP2) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL(-)) with a distinct second site is required for high PIP2 sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP2 sensitivity, even in the absence of PL(-) Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP2 (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL(-) binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP2 site and explaining the positive allostery between PL(-) binding and PIP2 sensitivity. PMID:27527100

  8. Regulation of Mitochondrial Function by Voltage Dependent Anion Channels in Ethanol Metabolism and the Warburg Effect

    Science.gov (United States)

    Lemasters, John J.; Holmuhamedov, Ekhson L.; Czerny, Christoph; Zhong, Zhi; Maldonado, Eduardo N.

    2012-01-01

    Voltage dependent anion channels (VDAC) are highly conserved proteins that are responsible for permeability of the mitochondrial outer membrane to hydrophilic metabolites like ATP, ADP and respiratory substrates. Although previously assumed to remain open, VDAC closure is emerging as an important mechanism for regulation of global mitochondrial metabolism in apoptotic cells and also in cells that are not dying. During hepatic ethanol oxidation to acetaldehyde, VDAC closure suppresses exchange of mitochondrial metabolites, resulting in inhibition of ureagenesis. In vivo, VDAC closure after ethanol occurs coordinately with mitochondrial uncoupling. Since acetaldehyde passes through membranes independently of channels and transporters, VDAC closure and uncoupling together foster selective and more rapid oxidative metabolism of toxic acetaldehyde to nontoxic acetate by mitochondrial aldehyde dehydrogenase. In single reconstituted VDAC, tubulin decreases VDAC conductance, and in HepG2 hepatoma cells, free tubulin negatively modulates mitochondrial membrane potential, an effect enhanced by protein kinase A. Tubulin-dependent closure of VDAC in cancer cells contributes to suppression of mitochondrial metabolism and may underlie the Warburg phenomenon of aerobic glycolysis. PMID:22172804

  9. Activation of G551D-CFTR by Bicyclooctane Compounds Is cAMP-dependent and Exhibits Low Sensitivity to Thiazolidinone CFTR Inhibitor CFTRinh-172

    Institute of Scientific and Technical Information of China (English)

    WANG Ying; ZHAO Lu; HE Cheng-yan; XU Li-na; YANG Hong

    2005-01-01

    The G551D-CFTR mutation causing cystic fibrosis (CF) results from a missense mutation at codon 551(G551D) in the gene encoding of the cystic fibrosis transmembrane conductance regulator (CFTR). The G551D mutation in CFTR results in a reduced functional channel but G551D-CFTR is appropriately inserted in the apical membrane. In previous studies we discovered a class of high-affinity bicyclooctane (BCO)G551D-CFTR activators(G551DBCOs) with Kd down to 1μmol/L. In this study, we analyzed the pharmacological activation of G551D-CFTR by the G551DBcos by means of short circuit current analysis and cell-based fluorescence quenching assay. The G551DBCOs-induced G551D-CFTR activation is cAMP-dependent and is less sensitive to thiazolidinone CFTR inhibitor CFTRinh-172. These data suggest that (1) the phosphorylation of G551D-CFTR by protein kinase A is required for the activation by G551DBcos; (2) G551DBCos and CFTRinh-172 may act at the same site on the G551D-CFTR molecule.

  10. CFTR: a hub for kinases and crosstalk of cAMP and Ca2+.

    Science.gov (United States)

    Kunzelmann, Karl; Mehta, Anil

    2013-09-01

    Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR). The resulting disease is pleiotropic consistent with the idea that CFTR acts as a node within a network of signalling proteins. CFTR is not only a regulator of multiple transport proteins and controlled by numerous kinases but also participates in many signalling pathways that are disrupted after expression of its commonest mutant (F508del-CFTR). It operates in membrane compartments creating a scaffold for cytoskeletal elements, surface receptors, kinases and phosphodiesterases. CFTR is exposed to membrane-local second messengers such that a CFTR-interacting, low cellular energy sensor kinase (AMP- and ADP-activated kinase, AMPK) signals through a high energy phosphohistidine protein kinase (nucleoside diphosphate kinase, NDPK). CFTR also translocates a Ca(2+)-dependent adenylate cyclase to its proximity so that a rigid separation between cAMP-dependent and Ca(2+)-dependent regulation of Cl(-) transport becomes obsolete. In the presence of wild-type CFTR, parallel activation of CFTR and outwardly rectifying anoctamin 6 Cl(-) channels is observed, while the Ca(2+)-activated anoctamin 1 Cl(-) channel is inhibited. In contrast, in CF cells, CFTR is missing/mislocalized and the outwardly rectifying chloride channel is attenuated while Ca(2+)-dependent Cl(-) secretion (anoctamin 1) appears upregulated. Additionally, we consider the idea that F508del-CFTR when trapped in the endoplasmic reticulum augments IP3-mediated Ca(2+) release by providing a shunt pathway for Cl(-). CFTR and the IP3 receptor share the characteristic that they both assemble their partner proteins to increase the plasticity of their hub responses. In CF, the CFTR hub fails to form at the plasma membrane, with widespread detrimental consequences for cell signalling.

  11. Functional interaction between TRP4 and CFTR in mouse aorta endothelial cells

    Directory of Open Access Journals (Sweden)

    Droogmans Guy

    2001-05-01

    Full Text Available Abstract Background This study describes the functional interaction between the putative Ca2+ channel TRP4 and the cystic fibrosis transmembrane conductance regulator, CFTR, in mouse aorta endothelium (MAEC. Results MAEC cells express CFTR transcripts as shown by RT-PCR analysis. Application of a phosphorylating cocktail activated a Cl- current with characteristics similar to those of CFTR mediated currents in other cells types (slow activation by cAMP, absence of rectification, block by glibenclamide. The current is present in trp4 +/+ MAEC, but not in trp4 -/- cells, although the expression of CFTR seems unchanged in the trp4 deficient cells as judged from RT-PCR analysis. Conclusions It is concluded that TRP4 is necessary for CFTR activation in endothelium, possibly by providing a scaffold for the formation of functional CFTR channels.

  12. Over Expression of Voltage Dependent Anion Channel 2 (VDAC2 in Muscles of Electrically Stunned Chickens

    Directory of Open Access Journals (Sweden)

    Norshahida Abu Samah, Azura Amid, and Faridah Yusof

    2011-12-01

    Full Text Available Water bath stunning is a common practice in commercial slaughterhouses. Such treatment is economic and in line with animal welfare practice. However, the conditions applied for the stunning process may vary from a slaughterhouse to another slaughterhouse. Such a loose regulation on the stunning procedure has opened up doors for food adulteration such as over dose stunning. In this study, a simple and reliable approach using proteomics have been developed to study the effect of different currents and voltages in stunning on the protein expression of the chickens. Protein profiles of the chickens were constructed in order to detect any differences in protein expression and modifications. The different voltage studied were 10 V, 40 V and 70 V while the values for current studied were 0.25 A, 0.5 A, and 0.75 A. After the proteomics analyses using 2D Platinum ImageMaster 6.0 and Matrix-assisted laser desorption ionization- time of flight (MALDI TOF spectrometry identification, Voltage dependent anion channel 2 (VDAC2 was identified to be over expressed in the muscle sample of over stunned chicken. The over expression of VDAC2 was confirmed at the transcriptional level of RNA expression. Real Time PCR showed that all over stunned samples contained higher mRNA expression level for VDAC2 genes. The mRNA level of VDAC2 was up-regulated by 59.87 fold change when normalized with housekeeping gene. In conclusion, VDAC2 could serve as potential biomarkers for identification of electrically stimulated chickens. The existence of these biomarkers will help to monitor the slaughtering and stunning process in the future. It will revolutionize the food authentication field and give a new breathe to the meat industry.ABSTRAK: Kaedah "waterbath stunning" merupakan amalan biasa di pusat-pusat penyembelihan. Kaedah ini adalah ekonomik dan selari dengan amalan kebajikan haiwan. Walaubagaimanapun, syarat-syarat yang digunakan untuk proses kejutan tersebut mungkin

  13. [Cystic fibrosis: new treatments targeting the CFTR protein].

    Science.gov (United States)

    Fajac, I; Sermet-Gaudelus, I

    2013-04-01

    Cystic fibrosis is an autosomal recessive genetic disease due to mutations in the (cystic fibrosis transmembrane conductance regulator) CFTR gene. The CFTR protein is a chloride channel expressed at the surface of several epithelial cells. Defective function of the CFTR protein leads to a severe disease in which lung disease is the leading cause of death. Current treatments are symptomatic. Nevertheless, with specialist and holistic care in dedicated cystic fibrosis centres, the median survival has improved. But the average age of death remains 29 years. Innovative molecules aiming to correct the CFTR protein itself are under development. These will be personalised treatments depending on the genotype or the type of CFTR dysfunction. The first molecule, ivacaftor, has just been approved in Europe and the USA. Adults and children treated with ivacaftor in clinical trials had a 10% improvement in FEV1 that was maintained for more than a year. Although at present ivacaftor is approved for only a small percentage of patients, the therapeutic strategy of correcting CFTR protein has been proved a valid approach. Other molecules targeting other defects in the CFTR protein are under evaluation.

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

    Science.gov (United States)

    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

  15. Targeting F508del-CFTR to develop rational new therapies for cystic fibrosis

    Institute of Scientific and Technical Information of China (English)

    Zhi-wei CAI; Jia LIU; Hong-yu LI; David N SHEPPARD

    2011-01-01

    The mutation F508del is the commonest cause of the genetic disease cystic fibrosis (CF). CF disrupts the function of many organs in the body, most notably the lungs, by perturbing salt and water transport across epithelial surfaces. F508del causes harm in two principal ways. First,the mutation prevents delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) to its correct cellular location,the apical(lumen-facing) membrane of epithelial cells. Second, F508del perturbs the Cl- channel function of CFTR by disrupting channel gating. Here, we discuss the development of rational new therapies for CF that target F508del-CFTR.We highlight how structural studies provide new insight into the role of F508 in the regulation of channel gating by cycles of ATP binding and hydrolysis. We emphasize the use of high-throughput screening to identify lead compounds for therapy development.These compounds include CFTR correctors that restore the expression of F508del-CFTR at the apical membrane of epithelial cells and CFTR potentiators that rescue the F508del-CFTR gating defect. Initial results from clinical trials of CFTR correctors and potentiators augur well for the development of small molecule therapies that target the root cause of CF: mutations in CFTR.

  16. RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels.

    Science.gov (United States)

    Yagoda, Nicholas; von Rechenberg, Moritz; Zaganjor, Elma; Bauer, Andras J; Yang, Wan Seok; Fridman, Daniel J; Wolpaw, Adam J; Smukste, Inese; Peltier, John M; Boniface, J Jay; Smith, Richard; Lessnick, Stephen L; Sahasrabudhe, Sudhir; Stockwell, Brent R

    2007-06-14

    Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs. Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS-RAF-MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)--a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS-RAF-MEK pathway.

  17. Cloning and Expressional Studies of the Voltage-dependent Anion Channel Gene from Brassica rapa L.

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The voltage-dependent anion channel (VDAC) plays an essential role in the permeability of mitochondrial membrane. In the present study, we isolated a novel VDAC gene (brvdac) based on the assembly of expressed sequence tag sequences from Brassica rapa L. and explored its differential expression patterns in growth,tissues, abiotic stress, and stress recovery. Results of a tissue-specific expression study in young seedlings indicated that, of all tissues tested, brvdac expression was the highest in the leaves. Under cold, drought, and salt stresses, brvdac expression showed a transient increase, and then returned to normal levels when the stress was removed. When plants were exposed to heat shock, there was no increase in brvdac expression,whereas during recovery a quick and considerable increase in expression was observed. These observations indicate that dissimilar modulations of brvdac transcription may occur when plant cells encounter heat shock and the other three types of stress. In addition, phylogenetic analysis implied that an earlier duplication of vdac probably occurred before the divergence between monocotyledons and dicotyledons.

  18. The Cystic Fibrosis Transmembrane Regulator (CFTR in the kidney

    Directory of Open Access Journals (Sweden)

    MORALES MARCELO M.

    2000-01-01

    Full Text Available The cystic fibrosis transmembrane regulator (CFTR is a Cl- channel. Mutations of this transporter lead to a defect of chloride secretion by epithelial cells causing the Cystic Fibrosis disease (CF. In spite of the high expression of CFTR in the kidney, patients with CF do not show major renal dysfunction, but it is known that both the urinary excretion of drugs and the renal capacity to concentrate and dilute urine is deficient. CFTR mRNA is expressed in all nephron segments and its protein is involved with chloride secretion in the distal tubule, and the principal cells of the cortical (CCD and medullary (IMCD collecting ducts. Several studies have demonstrated that CFTR does not only transport Cl- but also secretes ATP and, thus, controls other conductances such as Na+ (ENaC and K+ (ROMK2 channels, especially in CCD. In the polycystic kidney the secretion of chloride through CFTR contributes to the cyst enlargement. This review is focused on the role of CFTR in the kidney and the implications of extracellular volume regulators, such as hormones, on its function and expression.

  19. Targeted Correction and Restored Function of the CFTR Gene in Cystic Fibrosis Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Ana M. Crane

    2015-04-01

    Full Text Available Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources—potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC lines. We then utilized zinc-finger nucleases (ZFNs, designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR. We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.

  20. Duplicated CFTR isoforms in eels diverged in regulatory structures and osmoregulatory functions.

    Science.gov (United States)

    Wong, Marty Kwok-Shing; Pipil, Supriya; Kato, Akira; Takei, Yoshio

    2016-09-01

    Two cystic fibrosis transmembrane conductance regulator (CFTR) isoforms, CFTRa and CFTRb, were cloned in Japanese eel and their structures and functions were studied in different osmoregulatory tissues in freshwater (FW) and seawater (SW) eels. Molecular phylogenetic results suggested that the CFTR duplication in eels occurred independently of the duplication event in salmonid. CFTRa was expressed in the intestine and kidney and downregulated in both tissues in SW eels, while CFTRb was specifically expressed in the gill and greatly upregulated in SW eels. Structurally, the CFTR isoforms are similar in most functional domains except the regulatory R domain, where the R domain of CFTRa is similar to that of human CFTR but the R domain of CFTRb is unique in having high intrinsic negative charges and fewer phosphorylation sites, suggesting divergence of isoforms in terms of gating properties and hormonal regulation. Immunohistochemical results showed that CFTR was localized on the apical regions of SW ionocytes, suggesting a Cl(-) secretory role as in other teleosts. In intestine and kidney, however, immunoreactive CFTR was mostly found in the cytosolic vesicles in FW eels, indicating that Cl(-) channel activity could be low at basal conditions, but could be rapidly increased by membrane insertion of the stored channels. Guanylin (GN), a known hormone that increases CFTR activity in mammalian intestine, failed to redistribute CFTR and to affect its expression in eel intestine. The results suggested that GN-independent CFTR regulation is present in eel intestine and kidney. PMID:27322796

  1. Stimulation of Airway and Intestinal Mucosal Secretion by Natural Coumarin CFTR Activators

    OpenAIRE

    Hong eYang; Lina eXu; Yujie eSui; Xin eLiu; Chengyan eHe; Rouyu eFang; Jia eLiu; Feng eHao; Tong-Hui eMa

    2011-01-01

    Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) cause lethal hereditary disease cystic fibrosis (CF) that involves extensive destruction and dysfunction of serous epithelium. Possible pharmacological therapy includes correction of defective intracellular processing and abnormal channel gating. In a previous study, we identified five natural coumarin potentiators of Δ508-CFTR including osthole, imperatorin, isopsoralen, praeruptorin A and scoparone. The present study wa...

  2. Neuroprotective effects of volume-regulated anion channel blocker DCPIB on neonatal hypoxic-ischemic injury

    Institute of Scientific and Technical Information of China (English)

    Ammar ALIBRAHIM; Li-yan ZHAO; Christine You-jin BAE; Andrew BARSZCZYK; Christopher LF SUN; Guan-lei WANG; Hong-shuo SUN

    2013-01-01

    Aim:To evaluate the role of swelling-induced activation of volume-regulated anion channels (VRACs) in a neonatal hypoxic-ischemic injury model using the selective VRAC blocker 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on5-yl) oxobutyric acid (DCPIB).Methods:Cerebral hypoxic-ischemic injury was induced in 7-day-old mouse pups with Rice-Vannucci method.Prior to the onset of ischemia,the animals were ip administered DCPIB (10 mg/kg).The animals were sacrificed 24 h afterwards,coronal sections of the brains were cut and the areas of infarct were examined using TTC staining and an image-analysis system.Cultured PC12 cells were subjected to oxygen-glucose deprivation (OGD) for 4 h.The cellular viability was assessed using Cell Counting Kit 8.Intracellular chloride concentration [Clˉ]i was measured using 6-methoxy-N-ethylquinolinium iodide.Results:DCPIB-treated mice showed a significant reduction in hemispheric corrected infarct volume (26.65%+2.23%) compared to that in vehicle-treated mice (45.52%+1.45%,P<O.O01).DCPIB-treated mice also showed better functional recovery as they were more active than vehicle-treated mice at 4 and 24 h post injury.In cultured PC12 cells,DCPIB (10 μmol/L) significantly reduced OGD-induced cell death.Moreover,DCPIB (20 μmol/L) blocked hypotonic-induced decrease in [Clˉ]i in PC12 cells of both control and OGD groups.Conclusion:The results further support the pathophysiological role of VRACs in ischemic brain injury,and suggest DCPIB as a potential,easily administrable agent targeting VRACs in the context of perinatal and neonatal hypoxic-ischemic brain injury.

  3. Voltage dependent anion channel-1 regulates death receptor mediated apoptosis by enabling cleavage of caspase-8

    International Nuclear Information System (INIS)

    Activation of the extrinsic apoptosis pathway by tumour necrosis factor related apoptosis inducing ligand (TRAIL) is a novel therapeutic strategy for treating cancer that is currently under clinical evaluation. Identification of molecular biomarkers of resistance is likely to play an important role in predicting clinical anti tumour activity. The involvement of the mitochondrial type 1 voltage dependent anion channel (VDAC1) in regulating apoptosis has been highly debated. To date, a functional role in regulating the extrinsic apoptosis pathway has not been formally excluded. We carried out stable and transient RNAi knockdowns of VDAC1 in non-small cell lung cancer cells, and stimulated the extrinsic apoptotic pathway principally by incubating cells with the death ligand TRAIL. We used in-vitro apoptotic and cell viability assays, as well as western blot for markers of apoptosis, to demonstrate that TRAIL-induced toxicity is VDAC1 dependant. Confocal microscopy and mitochondrial fractionation were used to determine the importance of mitochondria for caspase-8 activation. Here we show that either stable or transient knockdown of VDAC1 is sufficient to antagonize TRAIL mediated apoptosis in non-small cell lung cancer (NSCLC) cells. Specifically, VDAC1 is required for processing of procaspase-8 to its fully active p18 form at the mitochondria. Loss of VDAC1 does not alter mitochondrial sensitivity to exogenous caspase-8-cleaved BID induced mitochondrial depolarization, even though VDAC1 expression is essential for TRAIL dependent activation of the intrinsic apoptosis pathway. Furthermore, expression of exogenous VDAC1 restores the apoptotic response to TRAIL in cells in which endogenous VDAC1 has been selectively silenced. Expression of VDAC1 is required for full processing and activation of caspase-8 and supports a role for mitochondria in regulating apoptosis signaling via the death receptor pathway

  4. Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Romain Ferru-Clément

    Full Text Available Cystic fibrosis transmembrane conductance regulator (CFTR is a chloride channel that is expressed on the apical plasma membrane (PM of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o- expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

  5. Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

    Science.gov (United States)

    Ferru-Clément, Romain; Fresquet, Fleur; Norez, Caroline; Métayé, Thierry; Becq, Frédéric; Kitzis, Alain; Thoreau, Vincent

    2015-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

  6. The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in maturation stage ameloblasts, odontoblasts and bone cells

    NARCIS (Netherlands)

    A. Bronckers; L. Kalogeraki; H.J.N. Jorna; M. Wilke; T.J. Bervoets; D.M. Lyaruu; B. Zandieh-Doulabi; P. Denbesten; H. de Jonge

    2010-01-01

    Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these patients is CFTR, a Cl− channel involved in transepithelial salt and water transport and bicarbonate secretion. We tested the hypothesis that Cftr channels are present and operating in the plasma membran

  7. Targeted therapies to improve CFTR function in cystic fibrosis.

    Science.gov (United States)

    Brodlie, Malcolm; Haq, Iram J; Roberts, Katie; Elborn, J Stuart

    2015-01-01

    Cystic fibrosis is the most common genetically determined, life-limiting disorder in populations of European ancestry. The genetic basis of cystic fibrosis is well established to be mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that codes for an apical membrane chloride channel principally expressed by epithelial cells. Conventional approaches to cystic fibrosis care involve a heavy daily burden of supportive treatments to combat lung infection, help clear airway secretions and maintain nutritional status. In 2012, a new era of precision medicine in cystic fibrosis therapeutics began with the licensing of a small molecule, ivacaftor, which successfully targets the underlying defect and improves CFTR function in a subgroup of patients in a genotype-specific manner. Here, we review the three main targeted approaches that have been adopted to improve CFTR function: potentiators, which recover the function of CFTR at the apical surface of epithelial cells that is disrupted in class III and IV genetic mutations; correctors, which improve intracellular processing of CFTR, increasing surface expression, in class II mutations; and production correctors or read-through agents, which promote transcription of CFTR in class I mutations. The further development of such approaches offers great promise for future therapeutic strategies in cystic fibrosis. PMID:26403534

  8. Study of the ion-channel behavior on glassy carbon electrode supported bilayer lipid membranes stimulated by perchlorate anion

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Zhiquan; Shi, Jun; Huang, Weimin, E-mail: huangwm@jlu.edu.cn

    2015-10-01

    In this paper, a kind of didodecyldimethylammonium bromide (DDAB) layer membranes was supported on a glassy carbon electrode (GCE). We studied the ion channel behavior of the supported bilayer lipid membrane by scanning electrochemical microscopy (SCEM) in tris(2,2′-bipyridine) ruthenium(II) solution. Perchlorate anion was used as a presence of stimulus and ruthenium(II) complex cations as the probing ions for the measurement of SECM, the lipid membrane channel was opened and exhibited the behavior of distinct SECM positive feedback curve. The channel was in a closed state in the absence of perchlorate anions while reflected the behavior of SECM negative feedback curve. The rates of electron transfer reaction in the lipid membranes surface were detected and it was dependant on the potential of SECM. - Highlights: • The rates of electron transfer reaction in the lipid membranes surface were detected. • Dynamic investigations of ion-channel behavior of supported bilayer lipid membranes by scanning electrochemical microscopy • A novel way to explore the interaction between molecules and supported bilayer lipid membranes.

  9. Targeting the intracellular environment in cystic fibrosis: restoring autophagy as a novel strategy to circumvent the CFTR defect

    Directory of Open Access Journals (Sweden)

    Valeria Rachela Villella

    2013-01-01

    Full Text Available Cystic fibrosis (CF patients harboring the most common deletion mutation of the cystic fibrosis transmembrane conductance regulator (CFTR, F508del, are poor responders to potentiators of CFTR channel activity which can be used to treat a small subset of CF patients who genetically carry plasma membrane-resident CFTR mutants. The misfolded F508del-CFTR protein is unstable in the plasma membrane even if rescued by pharmacological agents that prevent its intracellular retention and degradation. CF is a conformational disease in which defective CFTR induces an impressive derangement of general proteostasis resulting from disabled autophagy. In this review, we discuss how rescuing Beclin 1 (BECN1, a major player of autophagosome formation, either by means of direct gene transfer or indirectly by administration of proteostasis regulators, could stabilize F508del-CFTR at the plasma membrane. We focus on the relationship between the improvement of peripheral proteostasis and CFTR plasma membrane stability in F508del-CFTR homozygous bronchial epithelia or mouse lungs. Moreover, this article reviews recent preclinical evidence indicating that targeting the intracellular environment surrounding the misfolded mutant CFTR instead of protein itself could constitute an attractive therapeutic option to sensitize patients carrying the F508del-CFTR mutation to the beneficial action of CFTR potentiators on lung inflammation.

  10. Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity.

    Science.gov (United States)

    Luz, Simão; Kongsuphol, Patthara; Mendes, Ana Isabel; Romeiras, Francisco; Sousa, Marisa; Schreiber, Rainer; Matos, Paulo; Jordan, Peter; Mehta, Anil; Amaral, Margarida D; Kunzelmann, Karl; Farinha, Carlos M

    2011-11-01

    Previously, the pleiotropic "master kinase" casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.

  11. Targeting the Intracellular Environment in Cystic Fibrosis: Restoring Autophagy as a Novel Strategy to Circumvent the CFTR Defect.

    Science.gov (United States)

    Villella, Valeria Rachela; Esposito, Speranza; Bruscia, Emanuela M; Maiuri, Maria Chiara; Raia, Valeria; Kroemer, Guido; Maiuri, Luigi

    2013-01-01

    Cystic fibrosis (CF) patients harboring the most common deletion mutation of the CF transmembrane conductance regulator (CFTR), F508del, are poor responders to potentiators of CFTR channel activity which can be used to treat a small subset of CF patients who genetically carry plasma membrane (PM)-resident CFTR mutants. The misfolded F508del-CFTR protein is unstable in the PM even if rescued by pharmacological agents that prevent its intracellular retention and degradation. CF is a conformational disease in which defective CFTR induces an impressive derangement of general proteostasis resulting from disabled autophagy. In this review, we discuss how rescuing Beclin 1 (BECN1), a major player of autophagosome formation, either by means of direct gene transfer or indirectly by administration of proteostasis regulators, could stabilize F508del-CFTR at the PM. We focus on the relationship between the improvement of peripheral proteostasis and CFTR PM stability in F508del-CFTR homozygous bronchial epithelia or mouse lungs. Moreover, this article reviews recent pre-clinical evidence indicating that targeting the intracellular environment surrounding the misfolded mutant CFTR instead of protein itself could constitute an attractive therapeutic option to sensitize patients carrying the F508del-CFTR mutation to the beneficial action of CFTR potentiators on lung inflammation. PMID:23346057

  12. Determination of CFTR densities in erythrocyte plasma membranes using recognition imaging

    Energy Technology Data Exchange (ETDEWEB)

    Ebner, Andreas; Hinterdorfer, Peter [Institute for Biophysics, University of Linz, A-4040 Linz (Austria); Nikova, Dessy; Lange, Tobias; Bruns, Reimer; Oberleithner, Hans; Schillers, Hermann [Institute of Physiology II, University of Muenster, D-48149 Muenster (Germany); Haeberle, Johannes; Falk, Sabine; Duebbers, Angelika [Department of Pediatrics, University Hospitals of Muenster, D-48149 Muenster (Germany)], E-mail: schille@uni-muenster.de

    2008-09-24

    CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride (Cl{sup -}) channel that plays an important role in salt and fluid movement across epithelia. Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR. The most predominant mutation, F508del, disturbs CFTR protein trafficking, resulting in a reduced number of CFTR in the plasma membrane. Recent studies indicate that CFTR is not only found in epithelia but also in human erythrocytes. Although considerable attempts have been made to quantify CFTR in cells, conclusions on numbers of CFTR molecules localized in the plasma membrane have been drawn indirectly. AFM has the power to provide the needed information, since both sub-molecular spatial resolution and direct protein recognition via antibody-antigen interaction can be observed. We performed a quantification study of the CFTR copies in erythrocyte membranes at the single molecule level, and compared the difference between healthy donors and CF patients. We detected that the number of CFTR molecules is reduced by 70% in erythrocytes of cystic fibrosis patients.

  13. RNA interference for CFTR attenuates lung fluid absorption at birth in rats

    Directory of Open Access Journals (Sweden)

    Folkesson Hans G

    2008-07-01

    Full Text Available Abstract Background Small interfering RNA (siRNA against αENaC (α-subunit of the epithelial Na channel and CFTR (cystic fibrosis transmembrane conductance regulator was used to explore ENaC and CTFR function in newborn rat lungs. Methods Twenty-four hours after trans-thoracic intrapulmonary (ttip injection of siRNA-generating plasmid DNA (pSi-0, pSi-4, or pSi-C2, we measured CFTR and ENaC expression, extravascular lung water, and mortality. Results αENaC and CFTR mRNA and protein decreased by ~80% and ~85%, respectively, following αENaC and CFTR silencing. Extravascular lung water and mortality increased after αENaC and CFTR-silencing. In pSi-C2-transfected isolated DLE cells there were attenuated CFTR mRNA and protein. In pSi-4-transfected DLE cells αENaC mRNA and protein were both reduced. Interestingly, CFTR-silencing also reduced αENaC mRNA and protein. αENaC silencing, on the other hand, only slightly reduced CFTR mRNA and protein. Conclusion Thus, ENaC and CFTR are both involved in the fluid secretion to absorption conversion around at birth.

  14. Determination of CFTR densities in erythrocyte plasma membranes using recognition imaging

    Science.gov (United States)

    Ebner, Andreas; Nikova, Dessy; Lange, Tobias; Häberle, Johannes; Falk, Sabine; Dübbers, Angelika; Bruns, Reimer; Hinterdorfer, Peter; Oberleithner, Hans; Schillers, Hermann

    2008-09-01

    CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride (Cl-) channel that plays an important role in salt and fluid movement across epithelia. Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR. The most predominant mutation, F508del, disturbs CFTR protein trafficking, resulting in a reduced number of CFTR in the plasma membrane. Recent studies indicate that CFTR is not only found in epithelia but also in human erythrocytes. Although considerable attempts have been made to quantify CFTR in cells, conclusions on numbers of CFTR molecules localized in the plasma membrane have been drawn indirectly. AFM has the power to provide the needed information, since both sub-molecular spatial resolution and direct protein recognition via antibody-antigen interaction can be observed. We performed a quantification study of the CFTR copies in erythrocyte membranes at the single molecule level, and compared the difference between healthy donors and CF patients. We detected that the number of CFTR molecules is reduced by 70% in erythrocytes of cystic fibrosis patients.

  15. RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR.

    Science.gov (United States)

    Trzcińska-Daneluti, Agata M; Chen, Anthony; Nguyen, Leo; Murchie, Ryan; Jiang, Chong; Moffat, Jason; Pelletier, Lawrence; Rotin, Daniela

    2015-06-01

    Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the Cystic fibrosis transmembrane conductance regulator (CFTR). ΔF508-CFTR, the most common disease-causing CF mutant, exhibits folding and trafficking defects and is retained in the endoplasmic reticulum, where it is targeted for proteasomal degradation. To identify signaling pathways involved in ΔF508-CFTR rescue, we screened a library of endoribonuclease-prepared short interfering RNAs (esiRNAs) that target ∼750 different kinases and associated signaling proteins. We identified 20 novel suppressors of ΔF508-CFTR maturation, including the FGFR1. These were subsequently validated by measuring channel activity by the YFP halide-sensitive assay following shRNA-mediated knockdown, immunoblotting for the mature (band C) ΔF508-CFTR and measuring the amount of surface ΔF508-CFTR by ELISA. The role of FGFR signaling on ΔF508-CFTR trafficking was further elucidated by knocking down FGFRs and their downstream signaling proteins: Erk1/2, Akt, PLCγ-1, and FRS2. Interestingly, inhibition of FGFR1 with SU5402 administered to intestinal organoids (mini-guts) generated from the ileum of ΔF508-CFTR homozygous mice resulted in a robust ΔF508-CFTR rescue. Moreover, combination of SU5402 and VX-809 treatments in cells led to an additive enhancement of ΔF508-CFTR rescue, suggesting these compounds operate by different mechanisms. Chaperone array analysis on human bronchial epithelial cells harvested from ΔF508/ΔF508-CFTR transplant patients treated with SU5402 identified altered expression of several chaperones, an effect validated by their overexpression or knockdown experiments. We propose that FGFR signaling regulates specific chaperones that control ΔF508-CFTR maturation, and suggest that FGFRs may serve as important targets for therapeutic intervention for the treatment of CF.

  16. RhoA exerts a permissive effect on volume-regulated anion channels in vascular endothelial cells

    DEFF Research Database (Denmark)

    Carton, Iris; Trouet, Dominique; Hermans, Diane;

    2002-01-01

    pathway reduces the swelling-dependent activation of I(Cl,swell). However, these experiments did not allow us to discriminate between a direct activator role or a permissive effect. We now show that the Rho pathway did not affect VRAC activity if this pathway was activated by transfecting CPAE cells......'-O-(3-thiotriphosphate) or C3 exoenzyme had no effect on VRACs in caveolin-1-expressing Caco-2 cells. We conclude that the Rho pathway exerts a permissive effect on VRACs in CPAE cells, i.e., swelling-induced opening of VRACs requires a functional Rho pathway, but not an activation of the Rho pathway.......Cell swelling triggers in most cell types an outwardly rectifying anion current, I(Cl,swell), via volume-regulated anion channels (VRACs). We have previously demonstrated in calf pulmonary artery endothelial (CPAE) cells that inhibition of the Rho/Rho kinase/myosin light chain phosphorylation...

  17. Potentiation of ΔF508- and G551D-CFTR-Mediated Cl- Current by Novel Hydroxypyrazolines.

    Science.gov (United States)

    Park, Jinhong; Khloya, Poonam; Seo, Yohan; Kumar, Satish; Lee, Ho K; Jeon, Dong-Kyu; Jo, Sungwoo; Sharma, Pawan K; Namkung, Wan

    2016-01-01

    The most common mutation of CFTR, affecting approximately 90% of CF patients, is a deletion of phenylalanine at position 508 (F508del, ΔF508). Misfolding of ΔF508-CFTR impairs both its trafficking to the plasma membrane and its chloride channel activity. To identify small molecules that can restore channel activity of ΔF508-CFTR, we synthesized and evaluated eighteen novel hydroxypyrazoline analogues as CFTR potentiators. To elucidate potentiation activities of hydroxypyrazolines for ΔF508-CFTR, CFTR activity was measured using a halide-sensitive YFP assay, Ussing chamber assay and patch-clamp technique. Compounds 7p, 7q and 7r exhibited excellent potentiation with EC50 value <10 μM. Among the compounds, 7q (a novel CFTR potentiator, CP7q) showed the highest potentiation activity with EC50 values of 0.88 ± 0.11 and 4.45 ± 0.31 μM for wild-type and ΔF508-CFTR, respectively. In addition, CP7q significantly potentiated chloride conductance of G551D-CFTR, a CFTR gating mutant; its maximal potentiation activity was 1.9 fold higher than the well-known CFTR potentiator genistein. Combination treatment with CP7q and VX-809, a corrector of ΔF508-CFTR, significantly enhanced functional rescue of ΔF508-CFTR compared with VX-809 alone. CP7q did not alter the cytosolic cAMP level and showed no cytotoxicity at the concentration showing maximum efficacy. The hydroxypyrazolines may be potential development candidates for drug therapy of cystic fibrosis. PMID:26863533

  18. Potentiation of ΔF508- and G551D-CFTR-Mediated Cl- Current by Novel Hydroxypyrazolines.

    Directory of Open Access Journals (Sweden)

    Jinhong Park

    Full Text Available The most common mutation of CFTR, affecting approximately 90% of CF patients, is a deletion of phenylalanine at position 508 (F508del, ΔF508. Misfolding of ΔF508-CFTR impairs both its trafficking to the plasma membrane and its chloride channel activity. To identify small molecules that can restore channel activity of ΔF508-CFTR, we synthesized and evaluated eighteen novel hydroxypyrazoline analogues as CFTR potentiators. To elucidate potentiation activities of hydroxypyrazolines for ΔF508-CFTR, CFTR activity was measured using a halide-sensitive YFP assay, Ussing chamber assay and patch-clamp technique. Compounds 7p, 7q and 7r exhibited excellent potentiation with EC50 value <10 μM. Among the compounds, 7q (a novel CFTR potentiator, CP7q showed the highest potentiation activity with EC50 values of 0.88 ± 0.11 and 4.45 ± 0.31 μM for wild-type and ΔF508-CFTR, respectively. In addition, CP7q significantly potentiated chloride conductance of G551D-CFTR, a CFTR gating mutant; its maximal potentiation activity was 1.9 fold higher than the well-known CFTR potentiator genistein. Combination treatment with CP7q and VX-809, a corrector of ΔF508-CFTR, significantly enhanced functional rescue of ΔF508-CFTR compared with VX-809 alone. CP7q did not alter the cytosolic cAMP level and showed no cytotoxicity at the concentration showing maximum efficacy. The hydroxypyrazolines may be potential development candidates for drug therapy of cystic fibrosis.

  19. Triple-channel portable capillary electrophoresis instrument with individual background electrolytes for the concurrent separations of anionic and cationic species.

    Science.gov (United States)

    Mai, Thanh Duc; Le, Minh Duc; Sáiz, Jorge; Duong, Hong Anh; Koenka, Israel Joel; Pham, Hung Viet; Hauser, Peter C

    2016-03-10

    The portable capillary electrophoresis instrument is automated and features three independent channels with different background electrolytes to allow the concurrent optimized determination of three different categories of charged analytes. The fluidic system is based on a miniature manifold which is based on mechanically milled channels for injection of samples and buffers. The planar manifold pattern was designed to minimize the number of electronic valves required for each channel. The system utilizes pneumatic pressurization to transport solutions at the grounded as well as the high voltage side of the separation capillaries. The instrument has a compact design, with all components arranged in a briefcase with dimensions of 45 (w) × 35 (d) × 15 cm (h) and a weight of about 15 kg. It can operate continuously for 8 h in the battery-powered mode if only one electrophoresis channel is in use, or for about 2.5 h in the case of simultaneous employment of all three channels. The different operations, i.e. capillary flushing, rinsing of the interfaces at both capillary ends, sample injection and electrophoretic separation, are activated automatically with a control program featuring a graphical user interface. For demonstration, the system was employed successfully for the concurrent separation of different inorganic cations and anions, organic preservatives, additives and artificial sweeteners in various beverage and food matrices. PMID:26893094

  20. Characterization and functional analysis of voltage-dependent anion channel 1 (VDAC1) from orange-spotted grouper (Epinephelus coioides).

    Science.gov (United States)

    Shi, Yan; Zhao, Zhe; Hong, Xiaoyou; Chen, Kunci; Zhu, Xinping

    2014-07-01

    The voltage-dependent anion channel (VDAC) is a highly conserved integral protein of mitochondria in different eukaryotic species. It forms a selective channel in the mitochondrial outer membrane that serves as the controlled pathway for small metabolites and ions. In this study, a VDAC gene, EcVDAC1, was isolated from orange-spotted grouper (Epinephelus coioides). The EcVDAC1 exhibits ubiquitous expression in various tissues of orange-spotted grouper and is upregulated in liver, gill, and spleen after stimulation with lipopolysaccharides (LPS). Subcellular localization analysis shows that the EcVDAC1 protein colocalized with the mitochondria. A caspase-3 assay demonstrates that overexpression of the EcVDAC1 induced apoptotic cell death in fathead minnow cells. The data presented in this study provide new information regarding the relationship between LPS and the EcVDAC1 gene, suggesting that the fish VDAC1 gene may play an important role in antibacterial immune response.

  1. Optogenetics Comes of Age: Novel Inhibitory Light-Gated Anionic Channels Allow Efficient Silencing of Neural Function.

    Science.gov (United States)

    Peralvárez-Marín, Alex; Garriga, Pere

    2016-02-01

    Optogenetics, the developing field of research that uses light-switchable biochemical tools in a sophisticated technological approach to monitor or control neural function, is rapidly evolving with the discovery and development of novel microbial rhodopsins. Light-absorbing membrane proteins, as tools for brain research, are promoting new applications within the discipline of optogenetics. Light-gated rhodopsin ion channels with better intrinsic light sensitivity and improved resolution are needed to overcome some of the current limitations of existing molecules. The recent discovery of light-gated inhibitory anion channels opens new opportunities for studying physiological neural processes and, at the same time, represent a powerful approach for elucidating the mechanisms of neurological and mental disorders that could benefit from this approach.

  2. Bioelectric characterization of epithelia from neonatal CFTR knockout ferrets

    NARCIS (Netherlands)

    J.T. Fisher (John); S.R. Tyler (Scott); Y. Zhang (Yulong); B.J. Lee (Ben); X. Liu (Xiaoming); X. Sun (Xinying); H. Sui (Hongshu); B. Liang (Bo); M. Luo (Ma); W. Xie (Weiliang); I. Yi (Iasson); W. Zhou (Weili); Y. Song (Yiqing); N. Keiser (Nicholas); K. Wang (Kai); H.R. de Jonge (Hugo); J.F. Engelhardt (John)

    2013-01-01

    textabstractCystic fibrosis (CF) is a life-shortening, recessive, multiorgan genetic disorder caused by the loss of CF transmembrane conductance regulator (CFTR) chloride channel function found in many types of epithelia. Animal models that recapitulate the human disease phenotype are critical to un

  3. Involvement of CFTR in Uterine Bicarbonate Secretion and the Fertilizing Capacity of Sperm

    Institute of Scientific and Technical Information of China (English)

    WangXiao,Fei; ZhouChen-Xi; ShiQi-Xian; YuanYu-Ying; YuMei-Kuen; LouisChukwuemekaAjonuma

    2005-01-01

    Cystic fibrosis transmembrane conductance regulator (CFFR)is a cAMP-activated chloride channel expressed in a wide variety of epithelial cells,mutations of which are responsible for the hallmark defective chloride secretion observed in cystic fibrosis(CF).Although CFTR has been implicated in bicarbonate secretion,its ability to directly mediate bicarbonate secretion of any physiological significance has not been shown.We demonstrate here that endometriaI epithelial ceils possess a CFTR-mediated bicarbonate transport mechanism.Co-culture of sperm with endometrial ceils treated with antisense oligonucleotide against CFTR,or with bicarbonate secretion-defective CF epithelial cells,resulted in lower sperm capacitation and egg-fertilizing ability.These results are consistent with a critical role of CFTR in controlling uterine bicarbonate secretion and the fertilizing capacity of sperm,providing a link between defective CFTR and lower female fertility in CF.

  4. Biophysical characterisation of calumenin as a charged F508del-CFTR folding modulator.

    Science.gov (United States)

    Tripathi, Rashmi; Benz, Nathalie; Culleton, Bridget; Trouvé, Pascal; Férec, Claude

    2014-01-01

    The cystic fibrosis transmembrane regulator (CFTR) is a cyclic-AMP dependent chloride channel expressed at the apical surface of epithelial cells lining various organs such as the respiratory tract. Defective processing and functioning of this protein caused by mutations in the CFTR gene results in loss of ionic balance, defective mucus clearance, increased proliferation of biofilms and inflammation of human airways observed in cystic fibrosis (CF) patients. The process by which CFTR folds and matures under the influence of various chaperones in the secretory pathway remains incompletely understood. Recently, calumenin, a secretory protein, belonging to the CREC family of low affinity calcium binding proteins has been identified as a putative CFTR chaperone whose biophysical properties and functions remain uncharacterized. We compared hydropathy, instability, charge, unfoldability, disorder and aggregation propensity of calumenin and other CREC family members with CFTR associated chaperones and calcium binding proteins, wild-type and mutant CFTR proteins and intrinsically disordered proteins (IDPs). We observed that calumenin, along with other CREC proteins, was significantly more charged and less folded compared to CFTR associated chaperones. Moreover like IDPs, calumenin and other CREC proteins were found to be less hydrophobic and aggregation prone. Phylogenetic analysis revealed a close link between calumenin and other CREC proteins indicating how evolution might have shaped their similar biophysical properties. Experimentally, calumenin was observed to significantly reduce F508del-CFTR aggregation in a manner similar to AavLEA1, a well-characterized IDP. Fluorescence microscopy based imaging analysis also revealed altered trafficking of calumenin in bronchial cells expressing F508del-CFTR, indicating its direct role in the pathophysiology of CF. In conclusion, calumenin is characterized as a charged protein exhibiting close similarity with IDPs and is

  5. The human CFTR protein expressed in CHO cells activates aquaporin-3 in a cAMP-dependent pathway: study by digital holographic microscopy

    KAUST Repository

    Jourdain, P.

    2013-12-11

    The transmembrane water movements during cellular processes and their relationship to ionic channel activity remain largely unknown. As an example, in epithelial cells it was proposed that the movement of water could be directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) protein activity through a cAMP-stimulated aqueous pore, or be dependent on aquaporin. Here, we used digital holographic microscopy (DHM) an interferometric technique to quantify in situ the transmembrane water fluxes during the activity of the epithelial chloride channel, CFTR, measured by patch-clamp and iodide efflux techniques. We showed that the water transport measured by DHM is fully inhibited by the selective CFTR blocker CFTRinh172 and is absent in cells lacking CFTR. Of note, in cells expressing the mutated version of CFTR (F508del-CFTR), which mimics the most common genetic alteration encountered in cystic fibrosis, we also show that the water movement is profoundly altered but restored by pharmacological manipulation of F508del-CFTR-defective trafficking. Importantly, whereas activation of this endogenous water channel required a cAMP-dependent stimulation of CFTR, activation of CFTR or F508del-CFTR by two cAMP-independent CFTR activators, genistein and MPB91, failed to trigger water movements. Finally, using a specific small-interfering RNA against the endogenous aquaporin AQP3, the water transport accompanying CFTR activity decreased. We conclude that water fluxes accompanying CFTR activity are linked to AQP3 but not to a cAMP-stimulated aqueous pore in the CFTR protein.

  6. The human CFTR protein expressed in CHO cells activates aquaporin-3 in a cAMP-dependent pathway: study by digital holographic microscopy.

    Science.gov (United States)

    Jourdain, Pascal; Becq, Frédéric; Lengacher, Sylvain; Boinot, Clément; Magistretti, Pierre J; Marquet, Pierre

    2014-02-01

    The transmembrane water movements during cellular processes and their relationship to ionic channel activity remain largely unknown. As an example, in epithelial cells it was proposed that the movement of water could be directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) protein activity through a cAMP-stimulated aqueous pore, or be dependent on aquaporin. Here, we used digital holographic microscopy (DHM) an interferometric technique to quantify in situ the transmembrane water fluxes during the activity of the epithelial chloride channel, CFTR, measured by patch-clamp and iodide efflux techniques. We showed that the water transport measured by DHM is fully inhibited by the selective CFTR blocker CFTRinh172 and is absent in cells lacking CFTR. Of note, in cells expressing the mutated version of CFTR (F508del-CFTR), which mimics the most common genetic alteration encountered in cystic fibrosis, we also show that the water movement is profoundly altered but restored by pharmacological manipulation of F508del-CFTR-defective trafficking. Importantly, whereas activation of this endogenous water channel required a cAMP-dependent stimulation of CFTR, activation of CFTR or F508del-CFTR by two cAMP-independent CFTR activators, genistein and MPB91, failed to trigger water movements. Finally, using a specific small-interfering RNA against the endogenous aquaporin AQP3, the water transport accompanying CFTR activity decreased. We conclude that water fluxes accompanying CFTR activity are linked to AQP3 but not to a cAMP-stimulated aqueous pore in the CFTR protein.

  7. Tubulin tail sequences and post-translational modifications regulate closure of mitochondrial voltage-dependent anion channel (VDAC).

    Science.gov (United States)

    Sheldon, Kely L; Gurnev, Philip A; Bezrukov, Sergey M; Sackett, Dan L

    2015-10-30

    It was previously shown that tubulin dimer interaction with the mitochondrial outer membrane protein voltage-dependent anion channel (VDAC) blocks traffic through the channel and reduces oxidative metabolism and that this requires the unstructured anionic C-terminal tail peptides found on both α- and β-tubulin subunits. It was unclear whether the α- and β-tubulin tails contribute equally to VDAC blockade and what effects might be due to sequence variations in these tail peptides or to tubulin post-translational modifications, which mostly occur on the tails. The nature of the contribution of the tubulin body beyond acting as an anchor for the tails had not been clarified either. Here we present peptide-protein chimeras to address these questions. These constructs allow us to easily combine a tail peptide with different proteins or combine different tail peptides with a particular protein. The results show that a single tail grafted to an inert protein is sufficient to produce channel closure similar to that observed with tubulin. We show that the β-tail is more than an order of magnitude more potent than the α-tail and that the lower α-tail activity is largely due to the presence of a terminal tyrosine. Detyrosination activates the α-tail, and activation is reversed by the removal of the glutamic acid penultimate to the tyrosine. Nitration of tyrosine reverses the tyrosine inhibition of binding and even induces prolonged VDAC closures. Our results demonstrate that small changes in sequence or post-translational modification of the unstructured tails of tubulin result in substantial changes in VDAC closure. PMID:26306046

  8. The adenosine A2B receptor is involved in anion secretion in human pancreatic duct Capan-1 epithelial cells.

    Science.gov (United States)

    Hayashi, M; Inagaki, A; Novak, I; Matsuda, H

    2016-07-01

    Adenosine modulates a wide variety of biological processes via adenosine receptors. In the exocrine pancreas, adenosine regulates transepithelial anion secretion in duct cells and is considered to play a role in acini-to-duct signaling. To identify the functional adenosine receptors and Cl(-) channels important for anion secretion, we herein performed experiments on Capan-1, a human pancreatic duct cell line, using open-circuit Ussing chamber and gramicidin-perforated patch-clamp techniques. The luminal addition of adenosine increased the negative transepithelial potential difference (V te) in Capan-1 monolayers with a half-maximal effective concentration value of approximately 10 μM, which corresponded to the value obtained on whole-cell Cl(-) currents in Capan-1 single cells. The effects of adenosine on V te, an equivalent short-circuit current (I sc), and whole-cell Cl(-) currents were inhibited by CFTRinh-172, a cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel inhibitor. The adenosine A2B receptor agonist, BAY 60-6583, increased I sc and whole-cell Cl(-) currents through CFTR Cl(-) channels, whereas the A2A receptor agonist, CGS 21680, had negligible effects. The A2B receptor antagonist, PSB 603, inhibited the response of I sc to adenosine. Immunohistochemical analysis showed that the A2A and A2B receptors colocalized with Ezrin in the luminal membranes of Capan-1 monolayers and in rat pancreatic ducts. Adenosine elicited the whole-cell Cl(-) currents in guinea pig duct cells. These results demonstrate that luminal adenosine regulates anion secretion by activating CFTR Cl(-) channels via adenosine A2B receptors on the luminal membranes of Capan-1 cells. The present study endorses that purinergic signaling is important in the regulation of pancreatic secretion. PMID:26965147

  9. Central functions of bicarbonate in S-type anion channel activation and OST1 protein kinase in CO 2 signal transduction in guard cell

    KAUST Repository

    Xue, Shaowu

    2011-03-18

    Plants respond to elevated CO(2) via carbonic anhydrases that mediate stomatal closing, but little is known about the early signalling mechanisms following the initial CO(2) response. It remains unclear whether CO(2), HCO(3)(-) or a combination activates downstream signalling. Here, we demonstrate that bicarbonate functions as a small-molecule activator of SLAC1 anion channels in guard cells. Elevated intracellular [HCO(3)(-)](i) with low [CO(2)] and [H(+)] activated S-type anion currents, whereas low [HCO(3)(-)](i) at high [CO(2)] and [H(+)] did not. Bicarbonate enhanced the intracellular Ca(2+) sensitivity of S-type anion channel activation in wild-type and ht1-2 kinase mutant guard cells. ht1-2 mutant guard cells exhibited enhanced bicarbonate sensitivity of S-type anion channel activation. The OST1 protein kinase has been reported not to affect CO(2) signalling. Unexpectedly, OST1 loss-of-function alleles showed strongly impaired CO(2)-induced stomatal closing and HCO(3)(-) activation of anion channels. Moreover, PYR/RCAR abscisic acid (ABA) receptor mutants slowed but did not abolish CO(2)/HCO(3)(-) signalling, redefining the convergence point of CO(2) and ABA signalling. A new working model of the sequence of CO(2) signalling events in gas exchange regulation is presented.

  10. Tgf-β1 inhibits Cftr biogenesis and prevents functional rescue of ΔF508-Cftr in primary differentiated human bronchial epithelial cells.

    Directory of Open Access Journals (Sweden)

    Steven M Snodgrass

    Full Text Available CFTR is an integral transmembrane glycoprotein and a cAMP-activated Cl(- channel. Mutations in the CFTR gene lead to Cystic Fibrosis (CF-an autosomal recessive disease with majority of the morbidity and mortality resulting from airway infection, inflammation, and fibrosis. The most common disease-associated mutation in the CFTR gene-deletion of Phe508 (ΔF508 leads to a biosynthetic processing defect of CFTR. Correction of the defect and delivery of ΔF508-CFTR to the cell surface has been highly anticipated as a disease modifying therapy. Compared to promising results in cultured cell this approach was much less effective in CF patients in an early clinical trial. Although the cause of failure to rescue ΔF508-CFTR in the clinical trial has not been determined, presence of factor(s that interfere with the rescue in vivo could be considered. The cytokine TGF-β1 is frequently elevated in CF patients. TGF-β1 has pleiotropic effects in different disease models and genetic backgrounds and little is known about TGF-β1 effects on CFTR in human airway epithelial cells. Moreover, there are no published studies examining TGF-β1 effects on the functional rescue of ΔF508-CFTR. Here we found that TGF-β1 inhibits CFTR biogenesis by reducing mRNA levels and protein abundance in primary differentiated human bronchial epithelial (HBE cells from non-CF individuals. TGF-β1 inhibits CFTR biogenesis without compromising the epithelial phenotype or integrity of HBE cells. TGF-β1 also inhibits biogenesis and impairs the functional rescue of ΔF508-CFTR in HBE cells from patients homozygous for the ΔF508 mutation. Our data indicate that activation of TGF-β1 signaling may inhibit CFTR function in non-CF individuals and may interfere with therapies directed at correcting the processing defect of ΔF508-CFTR in CF patients.

  11. Predominant constitutive CFTR conductance in small airways

    Directory of Open Access Journals (Sweden)

    Lytle Christian

    2005-01-01

    Full Text Available Abstract Background The pathological hallmarks of chronic obstructive pulmonary disease (COPD are inflammation of the small airways (bronchiolitis and destruction of lung parenchyma (emphysema. These forms of disease arise from chronic prolonged infections, which are usually never present in the normal lung. Despite the fact that primary hygiene and defense of the airways presumably requires a well controlled fluid environment on the surface of the bronchiolar airway, very little is known of the fluid and electrolyte transport properties of airways of less than a few mm diameter. Methods We introduce a novel approach to examine some of these properties in a preparation of minimally traumatized porcine bronchioles of about 1 mm diameter by microperfusing the intact bronchiole. Results In bilateral isotonic NaCl Ringer solutions, the spontaneous transepithelial potential (TEP; lumen to bath of the bronchiole was small (mean ± sem: -3 ± 1 mV; n = 25, but when gluconate replaced luminal Cl-, the bionic Cl- diffusion potentials (-58 ± 3 mV; n = 25 were as large as -90 mV. TEP diffusion potentials from 2:1 NaCl dilution showed that epithelial Cl- permeability was at least 5 times greater than Na+ permeability. The anion selectivity sequence was similar to that of CFTR. The bionic TEP became more electronegative with stimulation by luminal forskolin (5 μM+IBMX (100 μM, ATP (100 μM, or adenosine (100 μM, but not by ionomycin. The TEP was partially inhibited by NPPB (100 μM, GlyH-101* (5–50 μM, and CFTRInh-172* (5 μM. RT-PCR gave identifying products for CFTR, α-, β-, and γ-ENaC and NKCC1. Antibodies to CFTR localized specifically to the epithelial cells lining the lumen of the small airways. Conclusion These results indicate that the small airway of the pig is characterized by a constitutively active Cl- conductance that is most likely due to CFTR.

  12. Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels.

    Science.gov (United States)

    Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry; Lee, C Justin

    2016-04-01

    Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

  13. Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels

    Science.gov (United States)

    Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry

    2016-01-01

    Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

  14. Manipulating proteostasis to repair the F508del-CFTR defect in cystic fibrosis.

    Science.gov (United States)

    Esposito, Speranza; Tosco, Antonella; Villella, Valeria R; Raia, Valeria; Kroemer, Guido; Maiuri, Luigi

    2016-12-01

    Cystic fibrosis (CF) is a lethal monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that entails the (diagnostic) increase in sweat electrolyte concentrations, progressive lung disease with chronic inflammation and recurrent bacterial infections, pancreatic insufficiency, and male infertility. Therapies aimed at restoring the CFTR defect have emerged. Thus, a small molecule which facilitates chloride channel opening, the potentiator Ivacaftor, has been approved for the treatment of CF patients bearing a particular class of rare CFTR mutations. However, small molecules that directly target the most common misfolded CFTR mutant, F508del, and improve its intracellular trafficking in vitro, have been less effective than expected when tested in CF patients, even in combination with Ivacaftor. Thus, new strategies are required to circumvent the F508del-CFTR defect. Airway and intestinal epithelial cells from CF patients bearing the F508del-CFTR mutation exhibit an impressive derangement of cellular proteostasis, with oxidative stress, overactivation of the tissue transglutaminase (TG2), and disabled autophagy. Proteostasis regulators such as cysteamine can rescue and stabilize a functional F508del-CFTR protein through suppressing TG2 activation and restoring autophagy in vivo in F508del-CFTR homozygous mice, in vitro in CF patient-derived cell lines, ex vivo in freshly collected primary patient's nasal cells, as well as in a pilot clinical trial involving homozygous F508del-CFTR patients. Here, we discuss how the therapeutic normalization of defective proteostasis can be harnessed for the treatment of CF patients with the F508del-CFTR mutation. PMID:26976279

  15. miR-16 rescues F508del-CFTR function in native cystic fibrosis epithelial cells.

    Science.gov (United States)

    Kumar, P; Bhattacharyya, S; Peters, K W; Glover, M L; Sen, A; Cox, R T; Kundu, S; Caohuy, H; Frizzell, R A; Pollard, H B; Biswas, R

    2015-11-01

    Cystic fibrosis (CF) is due to mutations in the CFTR gene, which prevents correct folding, trafficking and function of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. The dysfunctional effect of CFTR mutations, principally the F508del-CFTR mutant, is further manifested by hypersecretion of the pro-inflammatory chemokine interleukin-8 into the airway lumen, which further contributes to morbidity and mortality. We have hypothesized that microRNA (miR)-based therapeutics could rescue the dysfunctional consequences of mutant CFTR. Here we report that a miR-16 mimic can effectively rescue F508del-CFTR protein function in airway cell lines and primary cultures, of differentiated human bronchial epithelia from F508del homozygotes, which express mutant CFTR endogenously. We also identify two other miRs, miR-1 and miR-302a, which are also active. Although miR-16 is expressed at basal comparable levels in CF and control cells, miR-1 and miR-302a are undetectable. When miR mimics are expressed in CF lung or pancreatic cells, the expression of the F508del-CFTR protein is significantly increased. Importantly, miR-16 promotes functional rescue of the cyclic AMP-activated apical F508del-CFTR chloride channel in primary lung epithelial cells from CF patients. We interpret these findings to suggest that these miRs may constitute novel targets for CF therapy. PMID:26133785

  16. miR-16 rescues F508del-CFTR function in native cystic fibrosis epithelial cells.

    Science.gov (United States)

    Kumar, P; Bhattacharyya, S; Peters, K W; Glover, M L; Sen, A; Cox, R T; Kundu, S; Caohuy, H; Frizzell, R A; Pollard, H B; Biswas, R

    2015-11-01

    Cystic fibrosis (CF) is due to mutations in the CFTR gene, which prevents correct folding, trafficking and function of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. The dysfunctional effect of CFTR mutations, principally the F508del-CFTR mutant, is further manifested by hypersecretion of the pro-inflammatory chemokine interleukin-8 into the airway lumen, which further contributes to morbidity and mortality. We have hypothesized that microRNA (miR)-based therapeutics could rescue the dysfunctional consequences of mutant CFTR. Here we report that a miR-16 mimic can effectively rescue F508del-CFTR protein function in airway cell lines and primary cultures, of differentiated human bronchial epithelia from F508del homozygotes, which express mutant CFTR endogenously. We also identify two other miRs, miR-1 and miR-302a, which are also active. Although miR-16 is expressed at basal comparable levels in CF and control cells, miR-1 and miR-302a are undetectable. When miR mimics are expressed in CF lung or pancreatic cells, the expression of the F508del-CFTR protein is significantly increased. Importantly, miR-16 promotes functional rescue of the cyclic AMP-activated apical F508del-CFTR chloride channel in primary lung epithelial cells from CF patients. We interpret these findings to suggest that these miRs may constitute novel targets for CF therapy.

  17. The mitochondrial complex I activity is reduced in cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR function.

    Directory of Open Access Journals (Sweden)

    Angel G Valdivieso

    Full Text Available Cystic fibrosis (CF is a frequent and lethal autosomal recessive disease. It results from different possible mutations in the CFTR gene, which encodes the CFTR chloride channel. We have previously studied the differential expression of genes in CF and CF corrected cell lines, and found a reduced expression of MTND4 in CF cells. MTND4 is a mitochondrial gene encoding the MTND4 subunit of the mitochondrial Complex I (mCx-I. Since this subunit is essential for the assembly and activity of mCx-I, we have now studied whether the activity of this complex was also affected in CF cells. By using Blue Native-PAGE, the in-gel activity (IGA of the mCx-I was found reduced in CFDE and IB3-1 cells (CF cell lines compared with CFDE/6RepCFTR and S9 cells, respectively (CFDE and IB3-1 cells ectopically expressing wild-type CFTR. Moreover, colon carcinoma T84 and Caco-2 cells, which express wt-CFTR, either treated with CFTR inhibitors (glibenclamide, CFTR(inh-172 or GlyH101 or transfected with a CFTR-specific shRNAi, showed a significant reduction on the IGA of mCx-I. The reduction of the mCx-I activity caused by CFTR inhibition under physiological or pathological conditions may have a profound impact on mitochondrial functions of CF and non-CF cells.

  18. Signalling pathways in cystic fibrosis: a cross talk between CFTR trafficking and inflammation mechanisms

    OpenAIRE

    Coelho, João Pedro Lourenço

    2015-01-01

    Tese de mestrado, Bioquímica (Bioquímica), Universidade de Lisboa, Faculdade de Ciências, 2015 Cystic Fibrosis (CF) is the most common lethal autosomic recessive disease among the Caucasians. The disease is caused by mutations in the CFTR gene, that encodes a chloride channel expressed at the apical membrane of epithelial cells. CFTR dysfunction causes the electrolyte unbalance found in exocrine epithelia, leading to mucus thickening and bacteria entrapment in the lung. The inflammatory re...

  19. In vitro analysis of PDZ-dependent CFTR macromolecular signaling complexes.

    Science.gov (United States)

    Wu, Yanning; Wang, Shuo; Li, Chunying

    2012-08-13

    Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis(1-3). CFTR has been implicated in two major diseases: cystic fibrosis (CF)(4) and secretory diarrhea(5). In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States(6). Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut(7). Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo(8-19). In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP(20-27). The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)(20). Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities(22). This multivalency with respect to CFTR binding

  20. A Class of High-affinity Bicyclooctane G551D-CFTR Activators Identified by High Throughput Screening

    Institute of Scientific and Technical Information of China (English)

    HE Cheng-yan; ZHAO Lu; LIU Yan-li; XU Li-na; SHANG De-jing; YANG Hong

    2004-01-01

    The glycine-to-aspartic acid missense mutation at the codon 551(G551D) of the cystic fibrosis transmembrane conductance regulator(CFTR) is one of the five most frequent cystic fibrosis(CF) mutations associated with a severe CF phenotype. To explore the feasibility of pharmacological correction of disrupted activation of CFTR chloride channel caused by G551D mutation, we developed a halide-sensitive fluorescence miniassay for G551D-CFTR in Fisher rat thyroid(FRT) epithelial cells for the discovery of novel activators of G551D-CFTR. A class of bicyclooctane small molecule compounds that efficiently stimulate G551D-CFTR chloride channel activity was identified by high throughput screening via the FRT cell-based assay. This class of compounds selectively activates G551D-CFTR with a high affinity, whereas little effect of the compounds on wildtype CFTR can be seen. The discovery of a class of bicyclooctane G551D-CFTR activators will permit the analysis of structure-activity relationship of the compounds to identify ideal leads for in vivo therapeutic studies.

  1. Evidence that CFTR is expressed in rat tracheal smooth muscle cells and contributes to bronchodilation

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    Mettey Yvette

    2006-08-01

    Full Text Available Abstract Background The airway functions are profoundly affected in many diseases including asthma, chronic obstructive pulmonary disease (COPD and cystic fibrosis (CF. CF the most common lethal autosomal recessive genetic disease is caused by mutations of the CFTR gene, which normally encodes a multifunctional and integral membrane protein, the CF transmembrane conductance regulator (CFTR expressed in airway epithelial cells. Methods To demonstrate that CFTR is also expressed in tracheal smooth muscle cells (TSMC, we used iodide efflux assay to analyse the chloride transports in organ culture of rat TSMC, immunofluorescence study to localize CFTR proteins and isometric contraction measurement on isolated tracheal rings to observe the implication of CFTR in the bronchodilation. Results We characterized three different pathways stimulated by the cAMP agonist forskolin and the isoflavone agent genistein, by the calcium ionophore A23187 and by hypo-osmotic challenge. The pharmacology of the cAMP-dependent iodide efflux was investigated in detail. We demonstrated in rat TSMC that it is remarkably similar to that of the epithelial CFTR, both for activation (using three benzo [c]quinolizinium derivatives and for inhibition (glibenclamide, DPC and CFTRinh-172. Using rat tracheal rings, we observed that the activation of CFTR by benzoquinolizinium derivatives in TSMC leads to CFTRinh-172-sensitive bronchodilation after constriction with carbachol. An immunolocalisation study confirmed expression of CFTR in tracheal myocytes. Conclusion Altogether, these observations revealed that CFTR in the airways of rat is expressed not only in the epithelial cells but also in tracheal smooth muscle cells leading to the hypothesis that this ionic channel could contribute to bronchodilation.

  2. Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening.

    Science.gov (United States)

    Molinski, Steven V; Ahmadi, Saumel; Hung, Maurita; Bear, Christine E

    2015-12-01

    There are nearly 2000 mutations in the CFTR gene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mutations in the CFTR gene. This method was used to generate CFTR mutants bearing deletions (p.Gln2_Trp846del, p.Ser700_Asp835del, p.Ile1234_Arg1239del) and truncation with polyhistidine tag insertion (p.Glu1172-3Gly-6-His*), which either recapitulate a disease phenotype or render tools for modulator binding site identification, with subsequent evaluation of drug responses using a high-throughput (384-well) membrane potential-sensitive fluorescence assay of CFTR channel activity within a 1 wk time frame. This proof-of-concept study shows that these methods enable rapid and quantitative comparison of multiple CFTR mutants to emerging drugs, facilitating future large-scale efforts to stratify mutants according to their "theratype" or most promising targeted therapy.

  3. Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening.

    Science.gov (United States)

    Molinski, Steven V; Ahmadi, Saumel; Hung, Maurita; Bear, Christine E

    2015-12-01

    There are nearly 2000 mutations in the CFTR gene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mutations in the CFTR gene. This method was used to generate CFTR mutants bearing deletions (p.Gln2_Trp846del, p.Ser700_Asp835del, p.Ile1234_Arg1239del) and truncation with polyhistidine tag insertion (p.Glu1172-3Gly-6-His*), which either recapitulate a disease phenotype or render tools for modulator binding site identification, with subsequent evaluation of drug responses using a high-throughput (384-well) membrane potential-sensitive fluorescence assay of CFTR channel activity within a 1 wk time frame. This proof-of-concept study shows that these methods enable rapid and quantitative comparison of multiple CFTR mutants to emerging drugs, facilitating future large-scale efforts to stratify mutants according to their "theratype" or most promising targeted therapy. PMID:26385858

  4. The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy.

    Science.gov (United States)

    Smilansky, Angela; Dangoor, Liron; Nakdimon, Itay; Ben-Hail, Danya; Mizrachi, Dario; Shoshan-Barmatz, Varda

    2015-12-25

    The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼ 2-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment.

  5. ホスファチジン酸による CFTR の細胞内輸送制御機構および CFTR 機能破綻により生じる掻痒病態発症機構の解明

    OpenAIRE

    橋本, 泰明; ハシモト, ヤスアキ; Hashimoto, Yasuaki

    2008-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) in which mutation isthe cause of cystic fibrosis (CF) is a polytopic integral membrane protein that mediatestransepithelial chloride transport across epithelial cells in airways, pancreas, intestines andsweat glands. In addition, CFTR also regulates other various molecules (ion channels andreceptors) such as ENaC by forming multimolecular complex called “Transportsome”.Therefore, CFTR molecule is an extremely important molecule fo...

  6. Computational design of a PDZ domain peptide inhibitor that rescues CFTR activity.

    Directory of Open Access Journals (Sweden)

    Kyle E Roberts

    Full Text Available The cystic fibrosis transmembrane conductance regulator (CFTR is an epithelial chloride channel mutated in patients with cystic fibrosis (CF. The most prevalent CFTR mutation, ΔF508, blocks folding in the endoplasmic reticulum. Recent work has shown that some ΔF508-CFTR channel activity can be recovered by pharmaceutical modulators ("potentiators" and "correctors", but ΔF508-CFTR can still be rapidly degraded via a lysosomal pathway involving the CFTR-associated ligand (CAL, which binds CFTR via a PDZ interaction domain. We present a study that goes from theory, to new structure-based computational design algorithms, to computational predictions, to biochemical testing and ultimately to epithelial-cell validation of novel, effective CAL PDZ inhibitors (called "stabilizers" that rescue ΔF508-CFTR activity. To design the "stabilizers", we extended our structural ensemble-based computational protein redesign algorithm K* to encompass protein-protein and protein-peptide interactions. The computational predictions achieved high accuracy: all of the top-predicted peptide inhibitors bound well to CAL. Furthermore, when compared to state-of-the-art CAL inhibitors, our design methodology achieved higher affinity and increased binding efficiency. The designed inhibitor with the highest affinity for CAL (kCAL01 binds six-fold more tightly than the previous best hexamer (iCAL35, and 170-fold more tightly than the CFTR C-terminus. We show that kCAL01 has physiological activity and can rescue chloride efflux in CF patient-derived airway epithelial cells. Since stabilizers address a different cellular CF defect from potentiators and correctors, our inhibitors provide an additional therapeutic pathway that can be used in conjunction with current methods.

  7. Trimethylangelicin promotes the functional rescue of mutant F508del CFTR protein in cystic fibrosis airway cells.

    Science.gov (United States)

    Favia, Maria; Mancini, Maria T; Bezzerri, Valentino; Guerra, Lorenzo; Laselva, Onofrio; Abbattiscianni, Anna C; Debellis, Lucantonio; Reshkin, Stephan J; Gambari, Roberto; Cabrini, Giulio; Casavola, Valeria

    2014-07-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) carrying the F508del mutation is retained in endoplasmic reticulum and fails to traffic to the cell surface where it functions as a protein kinase A (PKA)-activated chloride channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect; however, the rescued F508del CFTR still displays reduced chloride permeability. Therefore, a combined administration of correctors and potentiators of the gating defect is ideal. We recently found that 4,6,4'-trimethylangelicin (TMA), besides inhibiting the expression of the IL-8 gene in airway cells in which the inflammatory response was challenged with Pseudomonas aeruginosa, also potentiates the cAMP/PKA-dependent activation of wild-type CFTR or F508del CFTR that has been restored to the plasma membrane. Here, we demonstrate that long preincubation with nanomolar concentrations of TMA is able to effectively rescue both F508del CFTR-dependent chloride secretion and F508del CFTR cell surface expression in both primary or secondary airway cell monolayers homozygous for F508del mutation. The correction effect of TMA seems to be selective for CFTR and persisted for 24 h after washout. Altogether, the results suggest that TMA, besides its anti-inflammatory and potentiator activities, also displays corrector properties.

  8. Anion-sensitive regions of L-type CaV1.2 calcium channels expressed in HEK293 cells.

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    Norbert Babai

    Full Text Available L-type calcium currents (I(Ca are influenced by changes in extracellular chloride, but sites of anion effects have not been identified. Our experiments showed that CaV1.2 currents expressed in HEK293 cells are strongly inhibited by replacing extracellular chloride with gluconate or perchlorate. Variance-mean analysis of I(Ca and cell-attached patch single channel recordings indicate that gluconate-induced inhibition is due to intracellular anion effects on Ca(2+ channel open probability, not conductance. Inhibition of CaV1.2 currents produced by replacing chloride with gluconate was reduced from approximately 75%-80% to approximately 50% by omitting beta subunits but unaffected by omitting alpha(2delta subunits. Similarly, gluconate inhibition was reduced to approximately 50% by deleting an alpha1 subunit N-terminal region of 15 residues critical for beta subunit interactions regulating open probability. Omitting beta subunits with this mutant alpha1 subunit did not further diminish inhibition. Gluconate inhibition was unchanged with expression of different beta subunits. Truncating the C terminus at AA1665 reduced gluconate inhibition from approximately 75%-80% to approximately 50% whereas truncating it at AA1700 had no effect. Neutralizing arginines at AA1696 and 1697 by replacement with glutamines reduced gluconate inhibition to approximately 60% indicating these residues are particularly important for anion effects. Expressing CaV1.2 channels that lacked both N and C termini reduced gluconate inhibition to approximately 25% consistent with additive interactions between the two tail regions. Our results suggest that modest changes in intracellular anion concentration can produce significant effects on CaV1.2 currents mediated by changes in channel open probability involving beta subunit interactions with the N terminus and a short C terminal region.

  9. Charged residues distribution modulates selectivity of the open state of human isoforms of the voltage dependent anion-selective channel.

    Science.gov (United States)

    Amodeo, Giuseppe Federico; Scorciapino, Mariano Andrea; Messina, Angela; De Pinto, Vito; Ceccarelli, Matteo

    2014-01-01

    Voltage Dependent Anion-selective Channels (VDACs) are pore-forming proteins located in the outer mitochondrial membrane. They are responsible for the access of ions and energetic metabolites into the inner membrane transport systems. Three VDAC isoforms exist in mammalian, but their specific role is unknown. In this work we have performed extensive (overall ∼5 µs) Molecular Dynamics (MD) simulations of the human VDAC isoforms to detect structural and conformational variations among them, possibly related to specific functional roles of these proteins. Secondary structure analysis of the N-terminal domain shows a high similarity among the three human isoforms of VDAC but with a different plasticity. In particular, the N-terminal domain of the hVDAC1 is characterized by a higher plasticity, with a ∼20% occurrence for the 'unstructured' conformation throughout the folded segment, while hVDAC2, containing a peculiar extension of 11 amino acids at the N-terminal end, presents an additional 310-helical folded portion comprising residues 10' to 3, adhering to the barrel wall. The N-terminal sequences of hVDAC isoforms are predicted to have a low flexibility, with possible consequences in the dynamics of the human VDACs. Clear differences were found between hVDAC1 and hVDAC3 against hVDAC2: a significantly modified dynamics with possible important consequence on the voltage-gating mechanism. Charge distribution inside and at the mouth of the pore is responsible for a different preferential localization of ions with opposite charge and provide a valuable rationale for hVDAC1 and hVDAC3 having a Cl-/K+ selectivity ratio of 1.8, whereas hVDAC2 of 1.4. Our conclusion is that hVDAC isoforms, despite sharing a similar scaffold, have modified working features and a biological work is now requested to give evidence to the described dissimilarities. PMID:25084457

  10. Charged residues distribution modulates selectivity of the open state of human isoforms of the voltage dependent anion-selective channel.

    Directory of Open Access Journals (Sweden)

    Giuseppe Federico Amodeo

    Full Text Available Voltage Dependent Anion-selective Channels (VDACs are pore-forming proteins located in the outer mitochondrial membrane. They are responsible for the access of ions and energetic metabolites into the inner membrane transport systems. Three VDAC isoforms exist in mammalian, but their specific role is unknown. In this work we have performed extensive (overall ∼5 µs Molecular Dynamics (MD simulations of the human VDAC isoforms to detect structural and conformational variations among them, possibly related to specific functional roles of these proteins. Secondary structure analysis of the N-terminal domain shows a high similarity among the three human isoforms of VDAC but with a different plasticity. In particular, the N-terminal domain of the hVDAC1 is characterized by a higher plasticity, with a ∼20% occurrence for the 'unstructured' conformation throughout the folded segment, while hVDAC2, containing a peculiar extension of 11 amino acids at the N-terminal end, presents an additional 310-helical folded portion comprising residues 10' to 3, adhering to the barrel wall. The N-terminal sequences of hVDAC isoforms are predicted to have a low flexibility, with possible consequences in the dynamics of the human VDACs. Clear differences were found between hVDAC1 and hVDAC3 against hVDAC2: a significantly modified dynamics with possible important consequence on the voltage-gating mechanism. Charge distribution inside and at the mouth of the pore is responsible for a different preferential localization of ions with opposite charge and provide a valuable rationale for hVDAC1 and hVDAC3 having a Cl-/K+ selectivity ratio of 1.8, whereas hVDAC2 of 1.4. Our conclusion is that hVDAC isoforms, despite sharing a similar scaffold, have modified working features and a biological work is now requested to give evidence to the described dissimilarities.

  11. Characterization of Oyster Voltage-Dependent Anion Channel 2 (VDAC2) Suggests Its Involvement in Apoptosis and Host Defense.

    Science.gov (United States)

    Li, Yingxiang; Zhang, Linlin; Qu, Tao; Li, Li; Zhang, Guofan

    2016-01-01

    Genomic and transcriptomic studies have revealed a sophisticated and powerful apoptosis regulation network in oyster, highlighting its adaptation to sessile life in a highly stressful intertidal environment. However, the functional molecular basis of apoptosis remains largely unexplored in oysters. In this study, we focused on a representative apoptotic gene encoding voltage-dependent anion channel 2 (VDAC2), a porin that abounds at the mitochondrial outer membrane. This is the first report on the identification and characterization of a VDAC gene in the Pacific oyster, Crassostrea gigas (CgVDAC2). The full length of CgVDAC2 was 1,738 bp with an open reading frame of 843 bp that encoded a protein of 281 amino acids. A four-element eukaryotic porin signature motif, a conserved ATP binding motif, and a VKAKV-like sequence were identified in the predicted CgVDAC2. Expression pattern analysis in different tissues and developmental stages as well as upon infection by ostreid herpesvirus 1 revealed the energy supply-related and immunity-related expression of CgVDAC2. CgVDAC2 was co-localized with mitochondria when it was transiently transfected into HeLa cells. Overexpression of CgVDAC2 in HEK293T cells suppressed the UV irradiation-induced apoptosis by inhibiting the pro-apoptotic function of CgBak. RNA interference induced reduction in CgVDAC2 expression showed a promoted apoptosis level upon UV light irradiation in hemocytes. The yeast two-hybrid system and co-immunoprecipitation assay indicated a direct interaction between CgVDAC2 and the pro-apoptotic protein CgBak. This study revealed the function of VDAC2 in oyster and provided new insights into its involvement in apoptosis modulation and host defense in mollusks. PMID:26727366

  12. Mild Alkalization Acutely Triggers the Warburg Effect by Enhancing Hexokinase Activity via Voltage-Dependent Anion Channel Binding

    Science.gov (United States)

    Lee, Jin Hee; Park, Jin Won; Moon, Seung Hwan; Cho, Young Seok; Choe, Yearn Seong; Lee, Kyung-Han

    2016-01-01

    To fully understand the glycolytic behavior of cancer cells, it is important to recognize how it is linked to pH dynamics. Here, we evaluated the acute effects of mild acidification and alkalization on cancer cell glucose uptake and glycolytic flux and investigated the role of hexokinase (HK). Cancer cells exposed to buffers with graded pH were measured for 18F-fluorodeoxyglucose (FDG) uptake, lactate production and HK activity. Subcellular localization of HK protein was assessed by western blots and confocal microscopy. The interior of T47D breast cancer cells was mildly alkalized to pH 7.5 by a buffer pH of 7.8, and this was accompanied by rapid increases of FDG uptake and lactate extrusion. This shift toward glycolytic flux led to the prompt recovery of a reversed pH gradient. In contrast, mild acidification rapidly reduced cellular FDG uptake and lactate production. Mild acidification decreased and mild alkalization increased mitochondrial HK translocation and enzyme activity. Cells transfected with specific siRNA against HK-1, HK-2 and voltage-dependent anion channel (VDAC)1 displayed significant attenuation of pH-induced changes in FDG uptake. Confocal microscopy showed increased co-localization of HK-1 and HK-2 with VDAC1 by alkaline treatment. In isolated mitochondria, acidic pH increased and alkaline pH decreased release of free HK-1 and HK-2 from the mitochondrial pellet into the supernatant. Furthermore, experiments using purified proteins showed that alkaline pH promoted co-immunoprecipitation of HK with VDAC protein. These findings demonstrate that mild alkalization is sufficient to acutely trigger cancer cell glycolytic flux through enhanced activity of HK by promoting its mitochondrial translocation and VDAC binding. This process might serve as a mechanism through which cancer cells trigger the Warburg effect to maintain a dysregulated pH. PMID:27479079

  13. Conservation of CFTR codon frequency through primates suggests synonymous mutations could have a functional effect.

    Science.gov (United States)

    Pizzo, Lucilla; Iriarte, Andrés; Alvarez-Valin, Fernando; Marín, Mónica

    2015-05-01

    Cystic fibrosis is an inherited chronic disease that affects the lungs and digestive system, with a prevalence of about 1:3000 people. Cystic fibrosis is caused by mutations in CFTR gene, which lead to a defective function of the chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Up-to-date, more than 1900 mutations have been reported in CFTR. However for an important proportion of them, their functional effects and the relation to disease are still not understood. Many of these mutations are silent (or synonymous), namely they do not alter the encoded amino acid. These synonymous mutations have been considered as neutral to protein function. However, more recent evidence in bacterial and human proteins has put this concept under revision. With the aim of understanding possible functional effects of synonymous mutations in CFTR, we analyzed human and primates CFTR codon usage and divergence patterns. We report the presence of regions enriched in rare and frequent codons. This spatial pattern of codon preferences is conserved in primates, but this cannot be explained by sequence conservation alone. In sum, the results presented herein suggest a functional implication of these regions of the gene that may be maintained by purifying selection acting to preserve a particular codon usage pattern along the sequence. Overall these results support the idea that several synonymous mutations in CFTR may have functional importance, and could be involved in the disease.

  14. Involvement of the heterodimeric interface region of the nucleotide binding domain-2 (NBD2) in the CFTR quaternary structure and membrane stability.

    Science.gov (United States)

    Micoud, Julien; Chauvet, Sylvain; Scheckenbach, Klaus Ernst Ludwig; Alfaidy, Nadia; Chanson, Marc; Benharouga, Mohamed

    2015-10-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the ATP-binding cassette (ABC) superfamily that functions as a chloride channel. The predicted structure of CFTR protein contains two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD1 and NBD2). The opening of the Cl- channel is directly linked to ATP-driven tight dimerization of CFTR's NBD1 and NBD2 domains. The presence of a heterodimeric interfaces (HI) region in NBD1 and NBD2 generated a head to tail orientation necessary for channel activity. This process was also suggested to promote important conformational changes in the associated transmembrane domains of CFTR, which may impact the CFTR plasma membrane stability. To better understand the role of the individual HI region in this process, we generated recombinant CFTR protein with suppressed HI-NBD1 and HI-NBD2. Our results indicate that HI-NBD2 deletion leads to the loss of the dimerization profile of CFTR that affect its plasma membrane stability. We conclude that, in addition to its role in Cl- transport, HI-NBD2 domain confers membrane stability of CFTR by consolidating its quaternary structure through interactions with HI-NBD1 region.

  15. Aggregates of mutant CFTR fragments in airway epithelial cells of CF lungs: new pathologic observations.

    Science.gov (United States)

    Du, Kai; Karp, Philip H; Ackerley, Cameron; Zabner, Joseph; Keshavjee, Shaf; Cutz, Ernest; Yeger, Herman

    2015-03-01

    Cystic fibrosis (CF) is caused by a mutation in the CF transmembrane conductance regulator (CFTR) gene resulting in a loss of Cl(-) channel function, disrupting ion and fluid homeostasis, leading to severe lung disease with airway obstruction due to mucus plugging and inflammation. The most common CFTR mutation, F508del, occurs in 90% of patients causing the mutant CFTR protein to misfold and trigger an endoplasmic reticulum based recycling response. Despite extensive research into the pathobiology of CF lung disease, little attention has been paid to the cellular changes accounting for the pathogenesis of CF lung disease. Here we report a novel finding of intracellular retention and accumulation of a cleaved fragment of F508del CFTR in concert with autophagic like phagolysosomes in the airway epithelium of patients with F508del CFTR. Aggregates consisting of poly-ubiquitinylated fragments of only the N-terminal domain of F508del CFTR but not the full-length molecule accumulate to appreciable levels. Importantly, these undegraded intracytoplasmic aggregates representing the NT-NBD1 domain of F508del CFTR were found in ciliated, in basal, and in pulmonary neuroendocrine cells. Aggregates were found in both native lung tissues and ex-vivo primary cultures of bronchial epithelial cells from CF donors, but not in normal control lungs. Our findings present a new, heretofore, unrecognized innate CF gene related cell defect and a potential contributing factor to the pathogenesis of CF lung disease. Mutant CFTR intracytoplasmic aggregates could be analogous to the accumulation of misfolded proteins in other degenerative disorders and in pulmonary "conformational protein-associated" diseases. Consequently, potential alterations to the functional integrity of airway epithelium and regenerative capacity may represent a critical new element in the pathogenesis of CF lung disease.

  16. Optimizing nasal potential difference analysis for CFTR modulator development: assessment of ivacaftor in CF subjects with the G551D-CFTR mutation.

    Directory of Open Access Journals (Sweden)

    Steven M Rowe

    Full Text Available Nasal potential difference (NPD is used as a biomarker of the cystic fibrosis transmembrane conductance regulator (CFTR and epithelial sodium channel (ENaC activity. We evaluated methods to detect changes in chloride and sodium transport by NPD based on a secondary analysis of a Phase II CFTR-modulator study. Thirty-nine subjects with CF who also had the G551D-CFTR mutation were randomized to receive ivacaftor (Kalydeco™; also known as VX-770 in four doses or placebo twice daily for at least 14 days. All data were analyzed by a single investigator who was blinded to treatment assignment. We compared three analysis methods to determine the best approach to quantify changes in chloride and sodium transport: (1 the average of both nostrils; (2 the most-polarized nostril at each visit; and (3 the most-polarized nostril at screening carried forward. Parameters of ion transport included the PD change with zero chloride plus isoproterenol (CFTR activity, the basal PD, Ringer's PD, and change in PD with amiloride (measurements of ENaC activity, and the delta NPD (measuring CFTR and ENaC activity. The average and most-polarized nostril at each visit were most sensitive to changes in chloride and sodium transport, whereas the most-polarized nostril at screening carried forward was less discriminatory. Based on our findings, NPD studies should assess both nostrils rather than a single nostril. We also found that changes in CFTR activity were more readily detected than changes in ENaC activity, and that rigorous standardization was associated with relatively good within-subject reproducibility in placebo-treated subjects (± 2.8 mV. Therefore, we have confirmed an assay of reasonable reproducibility for detecting chloride-transport improvements in response to CFTR modulation.

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

    Science.gov (United States)

    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

  18. Analysis of CFTR Gene Mutations in Children with Cystic Fibrosis, First Report from North-East of Iran

    OpenAIRE

    Atieh Mehdizadeh Hakkak; Mohammad Keramatipour; Saeid Talebi; Azam Brook; Jalil Tavakol Afshari; Amin Raazi; Hamid Reza Kianifar

    2013-01-01

     Objective(s):  More than 1500 registered mutations in cystic fibrosis transmembrane regulator (CFTR) gene are responsible for dysfunction of an ion channel protein and a wide spectrum of clinical manifestations in patients with cystic fibrosis (CF). This study was performed to investigate the frequency of a number of well-known CFTR mutations in North Eastern Iranian CF patients. Material and Methods: A total number of 56 documented CF patients participated in this study. Peripheral blood...

  19. Reconstitution of CO2 Regulation of SLAC1 Anion Channel and Function of CO2-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor.

    Science.gov (United States)

    Wang, Cun; Hu, Honghong; Qin, Xue; Zeise, Brian; Xu, Danyun; Rappel, Wouter-Jan; Boron, Walter F; Schroeder, Julian I

    2016-02-01

    Dark respiration causes an increase in leaf CO2 concentration (Ci), and the continuing increases in atmospheric [CO2] further increases Ci. Elevated leaf CO2 concentration causes stomatal pores to close. Here, we demonstrate that high intracellular CO2/HCO3 (-) enhances currents mediated by the Arabidopsis thaliana guard cell S-type anion channel SLAC1 upon coexpression of any one of the Arabidopsis protein kinases OST1, CPK6, or CPK23 in Xenopus laevis oocytes. Split-ubiquitin screening identified the PIP2;1 aquaporin as an interactor of the βCA4 carbonic anhydrase, which was confirmed in split luciferase, bimolecular fluorescence complementation, and coimmunoprecipitation experiments. PIP2;1 exhibited CO2 permeability. Mutation of PIP2;1 in planta alone was insufficient to impair CO2- and abscisic acid-induced stomatal closing, likely due to redundancy. Interestingly, coexpression of βCA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1 in oocytes enabled extracellular CO2 enhancement of SLAC1 anion channel activity. An inactive PIP2;1 point mutation was identified that abrogated water and CO2 permeability and extracellular CO2 regulation of SLAC1 activity. These findings identify the CO2-permeable PIP2;1 as key interactor of βCA4 and demonstrate functional reconstitution of extracellular CO2 signaling to ion channel regulation upon coexpression of PIP2;1, βCA4, SLAC1, and protein kinases. These data further implicate SLAC1 as a bicarbonate-responsive protein contributing to CO2 regulation of S-type anion channels. PMID:26764375

  20. CFTR mediates bicarbonate-dependent activation of miR-125b in preimplantation embryo development

    Institute of Scientific and Technical Information of China (English)

    Yong Chao Lu; Alvin Chun Hang Ma; Anskar Yu Hung Leung; He Feng Huang; Hsiao Chang Chan; Hui Chen; Kin Lam Fok; Lai Ling Tsang; Mei Kuen Yu; Xiao Hu Zhang; Jing Chen; Xiaohua Jiang; Yiu Wa Chung

    2012-01-01

    Although HCO3-is known to be required for early embryo development,its exact role remains elusive.Here we report that HCO3-acts as an environmental cue in regulating miR-125b expression through CFTR-mediated influx during preimplantation embryo development.The results show that the effect of HCO3-on preimplantation embryo development can be suppressed by interfering the function of a HCO3--conducting channel,CFTR,by a specific inhibitor or gene knockout.Removal of extracellular HCO3-or inhibition of CFTR reduces miR-125b expression in 2 cell-stage mouse embryos.Knockdown of miR-125b mimics the effect of HCO3-removal and CFTR inhibition,while injection of miR-125b precursor reverses it.Downregulation of miR-125b upregulates p53 cascade in both human and mouse embryos.The activation of miR-125b is shown to be mediated by sAC/PKA-dependent nuclear shuttling of NF-KB.These results have revealed a critical role of CFTR in signal transduction linking the environmental HCO3-to activation of miR-125b during preimplantation embryo development and indicated the importance of ion channels in regulation of miRNAs.

  1. Obligate coupling of CFTR pore opening to tight nucleotide-binding domain dimerization.

    Science.gov (United States)

    Mihályi, Csaba; Töröcsik, Beáta; Csanády, László

    2016-01-01

    In CFTR, the chloride channel mutated in cystic fibrosis (CF) patients, ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) opens the pore, and dimer disruption following ATP hydrolysis closes it. Spontaneous openings without ATP are rare in wild-type CFTR, but in certain CF mutants constitute the only gating mechanism, stimulated by ivacaftor, a clinically approved CFTR potentiator. The molecular motions underlying spontaneous gating are unclear. Here we correlate energetic coupling between residues across the dimer interface with spontaneous pore opening/closure in single CFTR channels. We show that spontaneous openings are also strictly coupled to NBD dimerization, which may therefore occur even without ATP. Coordinated NBD/pore movements are therefore intrinsic to CFTR: ATP alters the stability, but not the fundamental structural architecture, of open- and closed-pore conformations. This explains correlated effects of phosphorylation, mutations, and drugs on ATP-driven and spontaneous activity, providing insights for understanding CF mutation and drug mechanisms. PMID:27328319

  2. Functional interaction between CFTR and the sodium-phosphate co-transport type 2a in Xenopus laevis oocytes.

    Directory of Open Access Journals (Sweden)

    Naziha Bakouh

    Full Text Available BACKGROUND: A growing number of proteins, including ion transporters, have been shown to interact with Cystic Fibrosis Transmembrane conductance Regulator (CFTR. CFTR is an epithelial chloride channel that is involved in Cystic Fibrosis (CF when mutated; thus a better knowledge of its functional interactome may help to understand the pathophysiology of this complex disease. In the present study, we investigated if CFTR and the sodium-phosphate co-transporter type 2a (NPT2a functionally interact after heterologous expression of both proteins in Xenopus laevis oocytes. METHODOLOGY/FINDINGS: NPT2a was expressed alone or in combination with CFTR in X. laevis oocytes. Using the two-electrode voltage-clamp technique, the inorganic phosphate-induced current (IPi was measured and taken as an index of NPT2a activity. The maximal IPi for NPT2a substrates was reduced when CFTR was co-expressed with NPT2a, suggesting a decrease in its expression at the oolemna. This was consistent with Western blot analysis showing reduced NPT2a plasma membrane expression in oocytes co-expressing both proteins, whereas NPT2a protein level in total cell lysate was the same in NPT2a- and NPT2a+CFTR-oocytes. In NPT2a+CFTR- but not in NPT2a-oocytes, IPi and NPT2a surface expression were increased upon PKA stimulation, whereas stimulation of Exchange Protein directly Activated by cAMP (EPAC had no effect. When NPT2a-oocytes were injected with NEG2, a short amino-acid sequence from the CFTR regulatory domain that regulates PKA-dependent CFTR trafficking to the plasma membrane, IPi values and NPT2a membrane expression were diminished, and could be enhanced by PKA stimulation, thereby mimicking the effects of CFTR co-expression. CONCLUSION/PERSPECTIVES: We conclude that when both CFTR and NPT2a are expressed in X. laevis oocytes, CFTR confers to NPT2a a cAMPi-dependent trafficking to the membrane. This functional interaction raises the hypothesis that CFTR may play a role in

  3. Regulation of CFTR Expression and Arginine Vasopressin Activity Are Dependent on Polycystin-1 in Kidney-Derived Cells

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    Carolina Monteiro de Lemos Barbosa

    2016-01-01

    Full Text Available Background: Autosomal dominant polycystic kidney disease (ADPKD is characterized by the development of multiple, progressive, fluid-filled renal cysts that distort the renal parenchyma, leading to end-stage renal failure, mainly after the fifth decade of life. ADPKD is caused by a mutation in the PKD1 or PKD2 genes that encode polycystin-1 (PC-1 and polycystin-2 (PC-2, respectively. PC-1 is an important regulator of several signaling pathways and PC-2 is a nonselective calcium channel. The CFTR chloride channel is responsible for driving net fluid secretion into the cysts, promoting cyst growth. Arginine vasopressin hormone (AVP, in turn, is capable of increasing cystic intracellular cAMP, contributing to cell proliferation, transepithelial fluid secretion, and therefore to disease progression. The aim of this study was to assess if AVP can modulate CFTR and whether PC-1 plays a role in this potential modulation. Methods: M1 cells, derived from mouse cortical collecting duct, were used in the current work. The cells were treated with 10-7 M AVP hormone and divided into two main groups: transfected cells superexpressing PC-1 (Transf and cells not transfected (Ctrl. CFTR expression was assessed by immunodetection, CFTR mRNA levels were quantified by quantitative reverse transcription-polymerase chain reaction, and CFTR net ion transport was measured using the Ussing chamber technique. Results: AVP treatment increased the levels of CFTR protein and mRNA. CFTR short-circuit currents were also increased. However, when PC-1 was overexpressed in M1 cells, no increase in any of these parameters was detected. Conclusions: CFTR chloride channel expression is increased by AVP in M1 cells and PC-1 is capable of regulating this modulation.

  4. CFTR protein expression in human primary cells

    NARCIS (Netherlands)

    Meegen, M.A. van

    2016-01-01

    Subjects with cystic fibrosis (CF) display a great variability in clinical manifestations, even when they share the same cystic fibrosis transmembrane conductance regulator (CFTR) genotype. CFTR genotyping has enabled the stratification of subjects associated with mild or severe CF disease. However,

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

    Science.gov (United States)

    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

  6. Hypertension-linked mutation of α-adducin increases CFTR surface expression and activity in HEK and cultured rat distal convoluted tubule cells.

    Directory of Open Access Journals (Sweden)

    Anna Mondini

    Full Text Available The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator activity and localization are influenced by the cytoskeleton, in particular by actin and its polymerization state. In this study we investigated whether the expression of the hypertensive mutations of α-adducin (G460W-S586C in humans, F316Y in rats, an actin capping protein, led to a functional modification of CFTR activity and surface expression. The experiments were performed on HEK293 T cells cotransfected with CFTR and the human wild type (WT or G460W mutated α-adducin. In whole-cell patch-clamp experiments, both the CFTR chloride current and the slope of current activation after forskolin addition were significantly higher in HEK cells overexpressing the G460W adducin. A higher plasma membrane density of active CFTR channels was confirmed by cell-attached patch-clamp experiments, both in HEK cells and in cultured primary DCT cells, isolated from MHS (Milan Hypertensive Strain, a Wistar rat (Rattus norvegicus hypertensive model carrying the F316Y adducin mutation, compared to MNS (Milan Normotensive Strain rats. Western blot experiments demonstrated an increase of the plasma membrane CFTR protein expression, with a modification of the channel glycosylation state, in the presence of the mutated adducin. A higher retention of CFTR protein in the plasma membrane was confirmed both by FRAP (Fluorescence Recovery After Photobleaching and photoactivation experiments. The present data indicate that in HEK cells and in isolated DCT cells the presence of the G460W-S586C hypertensive variant of adducin increases CFTR channel activity, possibly by altering its membrane turnover and inducing a retention of the channel in the plasmamembrane. Since CFTR is known to modulate the activity of many others transport systems, the increased surface expression of the channel could have consequences on the whole network of transport in kidney cells.

  7. Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2

    OpenAIRE

    Szollosi, A; P.; Vergani; Csanady, L.

    2010-01-01

    The chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) displays a typical adenosine trisphosphate (ATP)-binding cassette (ABC) protein architecture comprising two transmembrane domains, two intracellular nucleotide-binding domains (NBDs), and a unique intracellular regulatory domain. Once phosphorylated in the regulatory domain, CFTR channels can open and close when supplied with cytosolic ATP. Despite the general agreement that formation of a head-to-tail NBD dim...

  8. Application of High-Resolution Single-Channel Recording to Functional Studies of Cystic Fibrosis Mutants

    Science.gov (United States)

    Cai, Zhiwei; Sohma, Yoshiro; Bompadre, Silvia G.; Sheppard, David N.; Hwang, Tzyh-Chang

    2016-01-01

    The patch-clamp technique is a powerful and versatile method to investigate the cystic fibrosis transmem-brane conductance regulator (CFTR) Cl− channel, its malfunction in disease and modulation by small molecules. Here, we discuss how the molecular behaviour of CFTR is investigated using high-resolution single-channel recording and kinetic analyses of channel gating. We review methods used to quantify how cystic fibrosis (CF) mutants perturb the biophysical properties and regulation of CFTR. By explaining the relationship between macroscopic and single-channel currents, we demonstrate how single-channel data provide molecular explanations for changes in CFTR-mediated transepithelial ion transport elicited by CF mutants. PMID:21594800

  9. A novel outer-membrane anion channel (porin) as part of a putatively two-component transport system for 4-toluenesulphonate in Comamonas testosteroni T-2

    OpenAIRE

    Mampel, Jörg; Maier, Elke; Tralau, Tewes; Ruff, Jürgen; Benz, Roland; Cook, Alasdair M.

    2004-01-01

    Inducible mineralization of TSA (4-toluenesulphonate) by Comamonas testosteroni T-2 is initiated by a secondary transport system, followed by oxygenation and oxidation by TsaMBCD to 4-sulphobenzoate under the regulation of TsaR and TsaQ. Evidence is presented for a novel, presumably two-component transport system (TsaST). It is proposed that TsaT, an outer-membrane porin, formed an anion-selective channel that works in co-operation with the putative secondary transporter, TsaS, located in the...

  10. Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR.

    Science.gov (United States)

    Cui, Guiying; Rahman, Kazi S; Infield, Daniel T; Kuang, Christopher; Prince, Chengyu Z; McCarty, Nael A

    2014-08-01

    The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1-6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5'-(β,γ-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues. PMID:25024266

  11. Critical Role of Cystic Fibrosis Transmembrane Conductance Regulation(CFTR)in Female Reproduction

    Institute of Scientific and Technical Information of China (English)

    Hsiao Chang CHAN

    2003-01-01

    @@ Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl- channel, mutations of which are responsible for defective Cl- and/or HCO-3 secretions seen in cystic fibrosis (CF), a common lethal genetic disease affecting most exocrine glands/organs, including the lungs, intestine, pancreas and reproductive tracts of both sexes.

  12. Potentiators of Defective ΔF508-CFTR Gating that Do Not Interfere with Corrector Action.

    Science.gov (United States)

    Phuan, Puay-Wah; Veit, Guido; Tan, Joseph A; Finkbeiner, Walter E; Lukacs, Gergely L; Verkman, A S

    2015-10-01

    Combination drug therapies under development for cystic fibrosis caused by the ∆F508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) include a "corrector" to improve its cellular processing and a "potentiator" to improve its chloride channel function. Recently, it was reported that the approved potentiator N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (Ivacaftor) reduces ∆F508-CFTR cellular stability and the efficacy of investigational correctors, including 3-(6-[([1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl) amino]-3-methyl-2-pyridinyl)-benzoic acid and 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-(1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(2-hydroxy-1,1-dimethylethyl)-1H-indol-5-yl), which might contribute to the modest reported efficacy of combination therapy in clinical trials. Here, we report the identification and characterization of potentiators that do not interfere with ∆F508-CFTR stability or corrector action. High-throughput screening and structure-activity analysis identified several classes of potentiators that do not impair corrector action, including tetrahydrobenzothiophenes, thiooxoaminothiazoles, and pyrazole-pyrrole-isoxazoles. The most potent compounds have an EC(50) for ∆F508-CFTR potentiation down to 18 nM and do not reduce corrector efficacy in heterologous ∆F508-CFTR-expressing cells or primary cultures of ∆F508/∆F508 human bronchial epithelia. The ΔF508-CFTR potentiators also activated wild-type and G551D CFTR, albeit weakly. The efficacy of combination therapy for cystic fibrosis caused by the ∆F508 mutation may be improved by replacement of Ivacaftor with a potentiator that does not interfere with corrector action. PMID:26245207

  13. EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1.

    Science.gov (United States)

    Lobo, Miguel J; Amaral, Margarida D; Zaccolo, Manuela; Farinha, Carlos M

    2016-07-01

    Cyclic AMP (cAMP) activates protein kinase A (PKA) but also the guanine nucleotide exchange factor 'exchange protein directly activated by cAMP' (EPAC1; also known as RAPGEF3). Although phosphorylation by PKA is known to regulate CFTR channel gating - the protein defective in cystic fibrosis - the contribution of EPAC1 to CFTR regulation remains largely undefined. Here, we demonstrate that in human airway epithelial cells, cAMP signaling through EPAC1 promotes CFTR stabilization at the plasma membrane by attenuating its endocytosis, independently of PKA activation. EPAC1 and CFTR colocalize and interact through protein adaptor NHERF1 (also known as SLC9A3R1). This interaction is promoted by EPAC1 activation, triggering its translocation to the plasma membrane and binding to NHERF1. Our findings identify a new CFTR-interacting protein and demonstrate that cAMP activates CFTR through two different but complementary pathways - the well-known PKA-dependent channel gating pathway and a new mechanism regulating endocytosis that involves EPAC1. The latter might constitute a novel therapeutic target for treatment of cystic fibrosis.

  14. EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1.

    Science.gov (United States)

    Lobo, Miguel J; Amaral, Margarida D; Zaccolo, Manuela; Farinha, Carlos M

    2016-07-01

    Cyclic AMP (cAMP) activates protein kinase A (PKA) but also the guanine nucleotide exchange factor 'exchange protein directly activated by cAMP' (EPAC1; also known as RAPGEF3). Although phosphorylation by PKA is known to regulate CFTR channel gating - the protein defective in cystic fibrosis - the contribution of EPAC1 to CFTR regulation remains largely undefined. Here, we demonstrate that in human airway epithelial cells, cAMP signaling through EPAC1 promotes CFTR stabilization at the plasma membrane by attenuating its endocytosis, independently of PKA activation. EPAC1 and CFTR colocalize and interact through protein adaptor NHERF1 (also known as SLC9A3R1). This interaction is promoted by EPAC1 activation, triggering its translocation to the plasma membrane and binding to NHERF1. Our findings identify a new CFTR-interacting protein and demonstrate that cAMP activates CFTR through two different but complementary pathways - the well-known PKA-dependent channel gating pathway and a new mechanism regulating endocytosis that involves EPAC1. The latter might constitute a novel therapeutic target for treatment of cystic fibrosis. PMID:27206858

  15. Lumacaftor/ivacaftor combination for cystic fibrosis patients homozygous for Phe508del-CFTR.

    Science.gov (United States)

    Zhang, W; Zhang, X; Zhang, Y H; Strokes, D C; Naren, A P

    2016-04-01

    Cystic fibrosis (CF) is a life-shortening inherited disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel activity resulting from mutations in the CFTR gene. Phe508del is the most prevalent mutation, with approximately 90% of all CF patients carrying it on at least one allele. Over the past two or three decades, significant progress has been made in understanding the pathogenesis of CF, and in the development of effective CF therapies. The approval of Orkambi® (lumacaftor/ivacaftor) marks another milestone in CF therapeutics development, which, with the advent of personalized medicine, could potentially revolutionize CF care and management. This article reviews the rationale, progress and future direction in the development of lumacaftor/ivacaftor combination to treat CF patients homozygous for the Phe508del-CFTR mutation.

  16. Lumacaftor/ivacaftor combination for cystic fibrosis patients homozygous for Phe508del-CFTR.

    Science.gov (United States)

    Zhang, W; Zhang, X; Zhang, Y H; Strokes, D C; Naren, A P

    2016-04-01

    Cystic fibrosis (CF) is a life-shortening inherited disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel activity resulting from mutations in the CFTR gene. Phe508del is the most prevalent mutation, with approximately 90% of all CF patients carrying it on at least one allele. Over the past two or three decades, significant progress has been made in understanding the pathogenesis of CF, and in the development of effective CF therapies. The approval of Orkambi® (lumacaftor/ivacaftor) marks another milestone in CF therapeutics development, which, with the advent of personalized medicine, could potentially revolutionize CF care and management. This article reviews the rationale, progress and future direction in the development of lumacaftor/ivacaftor combination to treat CF patients homozygous for the Phe508del-CFTR mutation. PMID:27252987

  17. Ouabain mimics low temperature rescue of F508del-CFTR in cystic fibrosis epithelial cells

    Directory of Open Access Journals (Sweden)

    Donglei eZhang

    2012-10-01

    Full Text Available ABSTRACT Most cases of cystic fibrosis (CF are caused by the deletion of a single phenylalanine residue at position 508 of the cystic fibrosis transmembrane conductance regulator (CFTR. The mutant F508del-CFTR is retained in the endoplasmic reticulum and degraded, but can be induced by low temperature incubation (29°C to traffic to the plasma membrane where it functions as a chloride channel. Here we show that, cardiac glycosides, at nanomolar concentrations, can partially correct the trafficking of F508del-CFTR in human CF bronchial epithelial cells (CFBE41o- and in an F508del-CFTR mouse model. Comparison of the transcriptional profiles obtained with polarized CFBE41o- cells after treatment with ouabain and by low temperature has revealed a striking similarity between the two corrector treatments that is not shared with other correctors. In summary, our study shows a novel function of ouabain and its analogues in the regulation of F508del-CFTR trafficking and suggests that compounds that mimic this low temperature correction of trafficking will provide new avenues for the development of therapeutics for CF.

  18. Activation of CFTR-mediated CI-Transport by Capsaicinoids in Cell Culture Model

    Institute of Scientific and Technical Information of China (English)

    ZHAO Xue-liang; HOU Ting-ting; GE Hong; SUN Juan-juan; YANG Hong; MA Tong-hui

    2009-01-01

    Previous studies reported that capsaicin potentiates ΔF508 mutant cystic fibrosis transmembrane conductance regulator(CFTR) channel gating defect by transfected cell-based assays.It has been postulated that orally ingested capsaicin may conceptually be used to develop a therapeutic strategy to treat gastrointestinal disorders in CF patients.We tried to reproduce and extend those pre-clinical data of previous studies.Cell-based fluorescence functional measurements in Fischer thyroid epithelial cells(FRT) expressing CFTR showed no effect of capsaicin on potentiating ΔF508-CFTR.while genistein showed a strongly positive activity.Studies show that capsaicin and dihydrocapsaicin activated cAMP-prestimulated wild-type CFTR in a dose-dependent manner with a maximal response of 70% of that activated by genistein,thus gave an apparent EC50 of (40.4±6.8)μmol/L and (150.2±7.4) μmol/L respectively.Preliminary study shows that the binding sites for capsaicin and dihydrocapsaicin may be probably partially overlapped with that for genistein because the maximal activation of wild-type CFTR with genistein is partially blocked by capsaicin and dihydrocapsaicin.

  19. FK506 binding protein 8 peptidylprolyl isomerase activity manages a late stage of cystic fibrosis transmembrane conductance regulator (CFTR) folding and stability.

    Science.gov (United States)

    Hutt, Darren M; Roth, Daniela Martino; Chalfant, Monica A; Youker, Robert T; Matteson, Jeanne; Brodsky, Jeffrey L; Balch, William E

    2012-06-22

    Cystic fibrosis (CF) is caused by mutations in the apical chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) with 90% of patients carrying at least one deletion of the F508 (ΔF508) allele. This mutant form of CFTR is characterized by a folding and trafficking defect that prevents exit from the endoplasmic reticulum. We previously reported that ΔF508 CFTR can be recovered in a complex with Hsp90 and its co-chaperones as an on-pathway folding intermediate, suggesting that Δ508 CF disease arises due to a failure of the proteostasis network (PN), which manages protein folding and degradation in the cell. We have now examined the role of FK506-binding protein 8 (FKBP8), a component of the CFTR interactome, during the biogenesis of wild-type and ΔF508 CFTR. FKBP8 is a member of the peptidylprolyl isomerase family that mediates the cis/trans interconversion of peptidyl prolyl bonds. Our results suggest that FKBP8 is a key PN factor required at a post-Hsp90 step in CFTR biogenesis. In addition, changes in its expression level or alteration of its activity by a peptidylprolyl isomerase inhibitor alter CFTR stability and transport. We propose that CF is caused by the sequential failure of the prevailing PN pathway to stabilize ΔF508-CFTR for endoplasmic reticulum export, a pathway that can be therapeutically managed.

  20. Pathogen and autoantigen homologous regions within the cystic fibrosis transmembrane conductance regulator (CFTR) protein suggest an autoimmune treatable component of cystic fibrosis.

    Science.gov (United States)

    Carter, Chris J

    2011-07-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel provides the glutathione and hypochlorous acid necessary for bactericidal/viricidal actions. CFTR mutations block these effects, diminishing pathogen defence and allowing extracellular pathogen accumulation, where antibody encounter is likely. KEGG pathway analysis of the CFTR interactome shows that CFTR is involved in pathogen entry pathways and immune defence as well as in pathways relevant to comorbid conditions (diabetes, cardiomyopathies and sexual organ development). Pseudomonas aeruginosa and Staphylococcus aureus infections decrease the lifespan of cystic fibrosis patients and Stenotrophomonas maltophilia colonization is increased. Autoantibodies, targeting myeloperoxidase, the bactericidal/permeability-increasing protein and calgranulin may further compromise pathogen defence. Short consensus sequences, within immunogenic extracellular regions of the CFTR protein, are homologous to proteins expressed by P. aeruginosa, S. aureus and S. maltophilia, and to several autoantigens, with a universal overlap between autoantigen/pathogen/CFTR consensi. Antibodies to pathogens are thus likely responsible for the creation of these autoantibodies, which, with pathogen antibodies, may target the CFTR protein acting as antagonists, further compromising its function. This creates a feedforward cycle, diminishing the function of the CFTR protein and increasing the probability of pathogen accumulation and antibody production at every turn. Interruption of this cycle by antibody adsorption or immunosuppressant therapy may be beneficial in cystic fibrosis.

  1. Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90.

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    Luiz Felipe M Prota

    Full Text Available BACKGROUND: Dexamethasone is widely used for pulmonary exacerbation in patients with cystic fibrosis, however, not much is known about the effects of glucocorticoids on the wild-type cystic fibrosis channel transmembrane regulator (CFTR. Our aim was to determine the effects of dexamethasone treatment on wild-type CFTR expression. METHODS AND RESULTS: Dose-response (1 nM to 10 µM and time course (3 to 48 h curves were generated for dexamethasone for mRNA expression in Calu-3 cells using a real-time PCR. Within 24 h, dexamethasone (10 nM showed a 0.3-fold decrease in CFTR mRNA expression, and a 3.2-fold increase in αENaC mRNA expression compared with control groups. Dexamethasone (10 nM induced a 1.97-fold increase in the total protein of wild-type CFTR, confirmed by inhibition by mifepristone. To access surface protein expression, biotinylation followed by Western blotting showed that dexamethasone treatment led to a 2.35-fold increase in the amount of CFTR in the cell surface compared with the untreated control groups. Once protein translation was inhibited with cycloheximide, dexamethasone could not increase the amount of CFTR protein. Protein stability was assessed by inhibition of protein synthesis with cycloheximide (50 µg/ml at different times in cells treated with dexamethasone and in untreated cells. Dexamethasone did not alter the degradation of wild-type CFTR. Assessment of the B band of CFTR within 15 min of metabolic pulse labeling showed a 1.5-fold increase in CFTR protein after treatment with dexamethasone for 24 h. Chaperone 90 (HSP90 binding to CFTR increased 1.55-fold after treatment with dexamethasone for 24 h, whereas chaperone 70 (HSP70 binding decreased 0.30 fold in an immunoprecipitation assay. CONCLUSION: Mature wild-type CFTR protein is regulated by dexamethasone post transcription, involving cotranslational mechanisms with HSP90 and HSP70, which enhances maturation and expression of wild-type CFTR.

  2. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

    Science.gov (United States)

    Veit, Guido; Oliver, Kathryn; Apaja, Pirjo M; Perdomo, Doranda; Bidaud-Meynard, Aurélien; Lin, Sheng-Ting; Guo, Jingyu; Icyuz, Mert; Sorscher, Eric J; Hartman Iv, John L; Lukacs, Gergely L

    2016-05-01

    The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect.

  3. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

    Directory of Open Access Journals (Sweden)

    Guido Veit

    2016-05-01

    Full Text Available The most common cystic fibrosis (CF causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del, results in functional expression defect of the CF transmembrane conductance regulator (CFTR at the apical plasma membrane (PM of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER. Deletion of phenylalanine 670 (ΔF670 in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect.

  4. Small-molecule CFTR activators increase tear secretion and prevent experimental dry eye disease.

    Science.gov (United States)

    Flores, Alyssa M; Casey, Scott D; Felix, Christian M; Phuan, Puay W; Verkman, A S; Levin, Marc H

    2016-05-01

    Dry eye disorders, including Sjögren's syndrome, constitute a common problem in the aging population, with limited effective therapeutic options available. The cAMP-activated Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR) is a major prosecretory channel at the ocular surface. We investigated whether compounds that target CFTR can correct the abnormal tear film in dry eye. Small-molecule activators of human wild-type CFTR identified by high-throughput screening were evaluated in cell culture and in vivo assays, to select compounds that stimulate Cl(-)-driven fluid secretion across the ocular surface in mice. An aminophenyl-1,3,5-triazine, CFTRact-K089, fully activated CFTR in cell cultures with EC50 ∼250 nM and produced an ∼8.5 mV hyperpolarization in ocular surface potential difference. When delivered topically, CFTRact-K089 doubled basal tear volume for 4 h and had no effect in CF mice. CFTRact-K089 showed sustained tear film bioavailability without detectable systemic absorption. In a mouse model of aqueous-deficient dry eye produced by lacrimal ablation, topical administration of 0.1 nmol CFTRact-K089 3 times daily restored tear volume to basal levels, preventing corneal epithelial disruption when initiated at the time of surgery and reversing it when started after development of dry eye. Our results support the potential utility of CFTR-targeted activators as a novel prosecretory treatment for dry eye.-Flores, A. M., Casey, S. D., Felix, C. M., Phuan, P. W., Verkman, A. S., Levin, M. H. Small-molecule CFTR activators increase tear secretion and prevent experimental dry eye disease.

  5. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

    Science.gov (United States)

    Veit, Guido; Oliver, Kathryn; Apaja, Pirjo M; Perdomo, Doranda; Bidaud-Meynard, Aurélien; Lin, Sheng-Ting; Guo, Jingyu; Icyuz, Mert; Sorscher, Eric J; Hartman Iv, John L; Lukacs, Gergely L

    2016-05-01

    The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect. PMID:27168400

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

    Institute of Scientific and Technical Information of China (English)

    Paul; Linsdell

    2014-01-01

    Dysfunction of the cystic fibrosis transmembrane con-ductance regulator(CFTR) chloride channel causes cys-tic fibrosis, while inappropriate activity of this channeloccurs in secretory diarrhea and polycystic kidney dis-ease. Drugs that interact directly with CFTR are there-fore of interest in the treatment of a number of diseasestates. This review focuses on one class of small mol-ecules that interacts directly with CFTR, namely inhibi-tors that act by directly blocking chloride movementthrough the open channel pore. In theory such com-pounds could be of use in the treatment of diarrheaand polycystic kidney disease, however in practice allknown substances acting by this mechanism to inhibitCFTR function lack either the potency or specificity forin vivo use. Nevertheless, this theoretical pharmaco-logical usefulness set the scene for the developmentof more potent, specific CFTR inhibitors. Biophysically,open channel blockers have proven most useful as ex-perimental probes of the structure and function of theCFTR chloride channel pore. Most importantly, the useof these blockers has been fundamental in developing afunctional model of the pore that includes a wide innervestibule that uses positively charged amino acid sidechains to attract both permeant and blocking anionsfrom the cell cytoplasm. CFTR channels are also subjectto this kind of blocking action by endogenous anionspresent in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physio-logical 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 states, of

  7. Targeting ion channels in cystic fibrosis.

    Science.gov (United States)

    Mall, Marcus A; Galietta, Luis J V

    2015-09-01

    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause a characteristic defect in epithelial ion transport that plays a central role in the pathogenesis of cystic fibrosis (CF). Hence, pharmacological correction of this ion transport defect by targeting of mutant CFTR, or alternative ion channels that may compensate for CFTR dysfunction, has long been considered as an attractive approach to a causal therapy of this life-limiting disease. The recent introduction of the CFTR potentiator ivacaftor into the therapy of a subgroup of patients with specific CFTR mutations was a major milestone and enormous stimulus for seeking effective ion transport modulators for all patients with CF. In this review, we discuss recent breakthroughs and setbacks with CFTR modulators designed to rescue mutant CFTR including the common mutation F508del. Further, we examine the alternative chloride channels TMEM16A and SLC26A9, as well as the epithelial sodium channel ENaC as alternative targets in CF lung disease, which remains the major cause of morbidity and mortality in patients with CF. Finally, we will focus on the hurdles that still need to be overcome to make effective ion transport modulation therapies available for all patients with CF irrespective of their CFTR genotype. PMID:26115565

  8. A little CFTR goes a long way: CFTR-dependent sweat secretion from G551D and R117H-5T cystic fibrosis subjects taking ivacaftor.

    Directory of Open Access Journals (Sweden)

    Jessica E Char

    Full Text Available To determine if oral dosing with the CFTR-potentiator ivacaftor (VX-770, Kalydeco improves CFTR-dependent sweating in CF subjects carrying G551D or R117H-5T mutations, we optically measured sweat secretion from 32-143 individually identified glands in each of 8 CF subjects; 6 F508del/G551D, one G551D/R117H-5T, and one I507del/R117H-5T. Two subjects were tested only (- ivacaftor, 3 only (+ ivacaftor and 3 (+/- ivacaftor (1-5 tests per condition. The total number of gland measurements was 852 (- ivacaftor and 906 (+ ivacaftor. A healthy control was tested 4 times (51 glands. For each gland we measured both CFTR-independent (M-sweat and CFTR-dependent (C-sweat; C-sweat was stimulated with a β-adrenergic cocktail that elevated [cAMP]i while blocking muscarinic receptors. Absent ivacaftor, almost all CF glands produced M-sweat on all tests, but only 1/593 glands produced C-sweat (10 tests, 5 subjects. By contrast, 6/6 subjects (113/342 glands produced C-sweat in the (+ ivacaftor condition, but with large inter-subject differences; 3-74% of glands responded with C/M sweat ratios 0.04%-2.57% of the average WT ratio of 0.265. Sweat volume losses cause proportionally larger underestimates of CFTR function at lower sweat rates. The losses were reduced by measuring C/M ratios in 12 glands from each subject that had the highest M-sweat rates. Remaining losses were estimated from single channel data and used to correct the C/M ratios, giving estimates of CFTR function (+ ivacaftor  = 1.6%-7.7% of the WT average. These estimates are in accord with single channel data and transcript analysis, and suggest that significant clinical benefit can be produced by low levels of CFTR function.

  9. Evaluation of CFTR gene mutations in Adana

    OpenAIRE

    Ozlem Goruroglu Ozturk; Filiz Kibar; Esin Damla Ziyanoglu Karacor; Salih Cetiner; Gulhan Sahin; Akgun Yaman

    2013-01-01

    ABSTRACT Objective: Cystic fibrosis is the most common autosomal recessive inherited disorder seen in the white populations. It develops in result of mutations of cystic fibrosis transmembrane regulator (CFTR) gene. Rate of these mutations vary in different geographical regions. In this study, we aimed to determine the frequency of CFTR gene mutations in Adana. Methods: DNA samples of 63 subjects (21 women, 42 men) who were diagnosed as cystic fibrosis at Balcali Hospital of Cukurova Universi...

  10. Evaluation of CFTR gene mutations in Adana

    OpenAIRE

    Öztürk, Özlem Görüroğlu; Filiz KIBAR; Karaçor, Esin Damla Ziyanoğlu; Çetiner, Salih; Şahin, Gülhan; Yaman, Akgün

    2013-01-01

    ABSTRACT Objective: Cystic fibrosis is the most common autosomal recessive inherited disorder seen in the white populations. It develops in result of mutations of cystic fibrosis transmembrane regulator (CFTR) gene. Rate of these mutations vary in different geographical regions. In this study, we aimed to determine the frequency of CFTR gene mutations in Adana. Methods: DNA samples of 63 subjects (21 women, 42 men) who were diagnosed as cystic fibrosis at Balcalı Hospital of Çukurova Unive...

  11. CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction.

    Science.gov (United States)

    Tabeling, Christoph; Yu, Hanpo; Wang, Liming; Ranke, Hannes; Goldenberg, Neil M; Zabini, Diana; Noe, Elena; Krauszman, Adrienn; Gutbier, Birgitt; Yin, Jun; Schaefer, Michael; Arenz, Christoph; Hocke, Andreas C; Suttorp, Norbert; Proia, Richard L; Witzenrath, Martin; Kuebler, Wolfgang M

    2015-03-31

    Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca(2+) mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca(2+) mobilization. Ca(2+) mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction. PMID:25829545

  12. Voltage-Dependent Anion Channel 2 of Arabidopsis thaliana (AtVDAC2 Is Involved in ABA-Mediated Early Seedling Development

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    Xufeng Li

    2009-05-01

    Full Text Available The voltage-dependent anion channel (VDAC is the major transport protein in the outer membrane of mitochondria and plays crucial roles in energy metabolism, apoptosis, and metabolites transport. In plants, the expression of VDACs can be affected by different stresses, including drought, salinity and pathogen defense. In this study, we investigated the expression pattern of AtVDAC2 in A. thaliana and found ABA suppressed the accumulation of AtVDAC2 transcripts. Further, phenotype analysis of this VDAC deregulated-expression transgenic Arabidopsis plants indicated that AtVDAC2 anti-sense line showed an ABA-insensitivity phenotype during the early seedling development under ABA treatment. The results suggested that AtVDAC2 might be involved in ABA signaling in A. thaliana.

  13. Relationship of sperm small heat-shock protein 10 and voltage-dependent anion channel 2 with semen freezability in boars.

    Science.gov (United States)

    Vilagran, Ingrid; Yeste, Marc; Sancho, Sílvia; Casas, Isabel; Rivera del Álamo, Maria M; Bonet, Sergi

    2014-08-01

    Freezability differences between boar ejaculates exist, but there is no useful method to predict the ejaculate freezability before sperm cryopreservation takes place. In this context, the present study sought to determine whether the amounts of small heat-shock protein 10 (also known as outer dense fiber protein 1) (ODF1/HSPB10) and voltage-dependent anion channel 2 (VDAC2) may be used as boar sperm freezability markers. With this aim, 26 boar ejaculates were split into two fractions: one for protein extraction and the other for cryopreservation purposes. Ejaculates were subsequently classified into two groups (good freezability ejaculates [GFE] and poor freezability ejaculates [PFE]) based on viability and sperm motility assessments after 30 and 240 minutes of after thawing. Although the VDAC2 amounts, analyzed through Western blot, were significantly higher (P cryopreservation procedures. PMID:24933094

  14. Measurements of CFTR-Mediated Cl− Secretion in Human Rectal Biopsies Constitute a Robust Biomarker for Cystic Fibrosis Diagnosis and Prognosis

    OpenAIRE

    Sousa, Marisa; Servidoni, Maria F; Vinagre, Adriana M.; Ramalho, Anabela S; Bonadia, Luciana C.; Felício, Verónica; Ribeiro, Maria A..; Uliyakina, Inna; Marson A, Fernando; Kmit, Arthur; Cardoso, Silvia R.; Ribeiro, José D; Carmen S. Bertuzzo; Sousa, Lisete; Kunzelmann, Karl

    2012-01-01

    BACKGROUND: Cystic Fibrosis (CF) is caused by ∼1,900 mutations in the CF transmembrane conductance regulator (CFTR) gene encoding for a cAMP-regulated chloride (Cl(-)) channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases. METHODOLOGY/PRINCIPAL FINDINGS: To further establish measurement of CFTR function as a sensitive and robust biomarker fo...

  15. Intein-mediated Trans-splicing of Chloride Ion Channel CFTR%内含肽介导的氯离子通道蛋白CFTR的反式剪接

    Institute of Scientific and Technical Information of China (English)

    朱甫祥; 刘泽隆; 屈慧鸽; 迟晓艳

    2009-01-01

    研究利用内含肽(intein)的蛋白质反式剪接功能在大肠杆菌中对囊性纤维化跨膜传导调节因子(cystic fibrosis tmnsmembrane regulator,CFTR)的反式剪接作用.CFTR基因突变导致一种常染色体隐性遗传疾病囊性纤维化(cystic fibrosis,CF).将CVTR的cDNA于剪接反应所需的保守性氨基酸残基Ser660前断裂为N端和C端,分别与split mini Ssp DnaB内含肽的106个氨基酸残基的N端和48个氨基酸残基的C端编码序列融合,构建到原核表达载体pBV220.诱导表达后SDS-PAGE可见预期大小剪接形成的CVTR蛋白条带,Western印迹用CFTR特异性抗体进一步证明为剪接所产生的CFTIR蛋白,表明内舍肽可有效催化CFTR的反式剪接.

  16. S-palmitoylation regulates biogenesis of core glycosylated wild-type and F508del CFTR in a post-ER compartment.

    Science.gov (United States)

    McClure, Michelle L; Wen, Hui; Fortenberry, James; Hong, Jeong S; Sorscher, Eric J

    2014-04-15

    Defects in CFTR (cystic fibrosis transmembrane conductance regulator) maturation are central to the pathogenesis of CF (cystic fibrosis). Palmitoylation serves as a key regulator of maturational processing in other integral membrane proteins, but has not been tested previously for functional effects on CFTR. In the present study, we used metabolic labelling to confirm that wild-type and F508del CFTR are palmitoylated, and show that blocking palmitoylation with the pharmacologic inhibitor 2-BP (2-bromopalmitate) decreases steady-state levels of both wild-type and low temperature-corrected F508del CFTR, disrupts post-ER (endoplasmic reticulum) maturation and reduces ion channel function at the cell surface. PATs (protein acyl transferases) comprise a family of 23 gene products that contain a DHHC motif and mediate palmitoylation. Recombinant expression of specific PATs led to increased levels of CFTR protein and enhanced palmitoylation as judged by Western blot and metabolic labelling. Specifically, we show that DHHC-7 (i) increases steady-state levels of wild-type and F508del CFTR band B, (ii) interacts preferentially with the band B glycoform, and (iii) augments radiolabelling by [3H]palmitic acid. Interestingly, immunofluorescence revealed that DHHC-7 also sequesters the F508del protein to a post-ER (Golgi) compartment. Our findings point to the importance of palmitoylation during wild-type and F508del CFTR trafficking.

  17. Abnormal spatial diffusion of Ca2+ in F508del-CFTR airway epithelial cells

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    Becq Frédéric

    2008-10-01

    Full Text Available Abstract Background In airway epithelial cells, calcium mobilization can be elicited by selective autocrine and/or paracrine activation of apical or basolateral membrane heterotrimeric G protein-coupled receptors linked to phospholipase C (PLC stimulation, which generates inositol 1,4,5-trisphosphate (IP3 and 1,2-diacylglycerol (DAG and induces Ca2+ release from endoplasmic reticulum (ER stores. Methods In the present study, we monitored the cytosolic Ca2+ transients using the UV light photolysis technique to uncage caged Ca2+ or caged IP3 into the cytosol of loaded airway epithelial cells of cystic fibrosis (CF and non-CF origin. We compared in these cells the types of Ca2+ receptors present in the ER, and measured their Ca2+ dependent activity before and after correction of F508del-CFTR abnormal trafficking either by low temperature or by the pharmacological corrector miglustat (N-butyldeoxynojirimycin. Results We showed reduction of the inositol 1,4,5-trisphosphate receptors (IP3R dependent-Ca2+ response following both correcting treatments compared to uncorrected cells in such a way that Ca2+ responses (CF+treatment vs wild-type cells were normalized. This normalization of the Ca2+ rate does not affect the activity of Ca2+-dependent chloride channel in miglustat-treated CF cells. Using two inhibitors of IP3R1, we observed a decrease of the implication of IP3R1 in the Ca2+ response in CF corrected cells. We observed a similar Ca2+ mobilization between CF-KM4 cells and CFTR-cDNA transfected CF cells (CF-KM4-reverted. When we restored the F508del-CFTR trafficking in CFTR-reverted cells, the specific IP3R activity was also reduced to a similar level as in non CF cells. At the structural level, the ER morphology of CF cells was highly condensed around the nucleus while in non CF cells or corrected CF cells the ER was extended at the totality of cell. Conclusion These results suggest reversal of the IP3R dysfunction in F508del-CFTR epithelial

  18. The volume-regulated anion channel is formed by LRRC8 heteromers – molecular identification and roles in membrane transport and physiology.

    Science.gov (United States)

    Stauber, Tobias

    2015-09-01

    Cellular volume regulation is fundamental for numerous physiological processes. The volume-regulated anion channel, VRAC, plays a crucial role in regulatory volume decrease. This channel, which is ubiquitously expressed in vertebrates, has been vastly characterized by electrophysiological means. It opens upon cell swelling and conducts chloride and arguably organic osmolytes. VRAC has been proposed to be critically involved in various cellular and organismal functions, including cell proliferation and migration, apoptosis, transepithelial transport, swelling-induced exocytosis and intercellular communication. It may also play a role in pathological states like cancer and ischemia. Despite many efforts, the molecular identity of VRAC had remained elusive for decades, until the recent discovery of heteromers of LRRC8A with other LRRC8 family members as an essential VRAC component. This identification marks a starting point for studies on the structure-function relation, for molecular biological investigations of its cell biology and for re-evaluating the physiological roles of VRAC. This review recapitulates the identification of LRRC8 heteromers as VRAC components, depicts the similarities between LRRC8 proteins and pannexins, and discussed whether VRAC conducts larger osmolytes. Furthermore, proposed physiological functions of VRAC and the present knowledge about the physiological significance of LRRC8 proteins are summarized and collated. PMID:25868000

  19. Effects of curcumin on ion channels and transporters

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    Xuemei eZhang

    2014-03-01

    Full Text Available Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl-1,6-heptadiene-3,5-dione], a polyphenolic compound isolated from the rhizomes of Curcuma longa (turmeric, has been shown to exhibit a wide range of pharmacological activities including anti-inflammatory, anti-cancer, anti-oxidant, anti-atherosclerotic, anti-microbial and wound healing effects. These activities of curcumin are based on its complex molecular structure and chemical features, as well as its ability to interact with multiple signaling molecules. The ability of curcumin to regulate ion channels and transporters was recognized a decade ago. The cystic fibrosis transmembrane conductance regulator (CFTR is a well-studied ion channel target of curcumin. During the process of studying its anti-cancer properties, curcumin was found to inhibit ATP-binding cassette (ABC family members including ABCA1, ABCB1, ABCC1 and ABCG2. Recent studies have revealed that many channels and transporters are modulated by curcumin, such as voltage-gated potassium (Kv channels, high-voltage-gated Ca2+ channels (HVGCC, volume-regulated anion channel (VRAC, Ca2+ release-activated Ca2+ channel (CRAC, aquaporin-4 (AQP-4, glucose transporters, etc. In this review, we aim to provide an overview of the interactions of curcumin with different types of ion channels and transporters and to help better understand and integrate the underlying molecular mechanisms of the multiple pharmacological activities of curcumin.

  20. Measurements of CFTR-Mediated Cl− Secretion in Human Rectal Biopsies Constitute a Robust Biomarker for Cystic Fibrosis Diagnosis and Prognosis

    Science.gov (United States)

    Vinagre, Adriana M.; Ramalho, Anabela S.; Bonadia, Luciana C.; Felício, Verónica; Ribeiro, Maria A.; Uliyakina, Inna; Marson, Fernando A.; Kmit, Arthur; Cardoso, Silvia R.; Ribeiro, José D.; Bertuzzo, Carmen S.; Sousa, Lisete; Kunzelmann, Karl; Ribeiro, Antônio F.; Amaral, Margarida D.

    2012-01-01

    Background Cystic Fibrosis (CF) is caused by ∼1,900 mutations in the CF transmembrane conductance regulator (CFTR) gene encoding for a cAMP-regulated chloride (Cl−) channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases. Methodology/Principal Findings To further establish measurement of CFTR function as a sensitive and robust biomarker for diagnosis and prognosis of CF, we herein assessed cholinergic and cAMP-CFTR-mediated Cl− secretion in 524 freshly excised rectal biopsies from 118 individuals, including patients with confirmed CF clinical diagnosis (n = 51), individuals with clinical CF suspicion (n = 49) and age-matched non-CF controls (n = 18). Conclusive measurements were obtained for 96% of cases. Patients with “Classic CF”, presenting earlier onset of symptoms, pancreatic insufficiency, severe lung disease and low Shwachman-Kulczycki scores were found to lack CFTR-mediated Cl− secretion (<5%). Individuals with milder CF disease presented residual CFTR-mediated Cl− secretion (10–57%) and non-CF controls show CFTR-mediated Cl− secretion ≥30–35% and data evidenced good correlations with various clinical parameters. Finally, comparison of these values with those in “CF suspicion” individuals allowed to confirm CF in 16/49 individuals (33%) and exclude it in 28/49 (57%). Statistical discriminant analyses showed that colonic measurements of CFTR-mediated Cl− secretion are the best discriminator among Classic/Non-Classic CF and non-CF groups. Conclusions/Significance Determination of CFTR-mediated Cl− secretion in rectal biopsies is demonstrated here to be a sensitive, reproducible and robust predictive biomarker for the diagnosis and prognosis of CF. The method also has very high potential for (pre-)clinical trials of CFTR-modulator therapies. PMID:23082198

  1. Measurements of CFTR-mediated Cl- secretion in human rectal biopsies constitute a robust biomarker for Cystic Fibrosis diagnosis and prognosis.

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    Marisa Sousa

    Full Text Available BACKGROUND: Cystic Fibrosis (CF is caused by ∼1,900 mutations in the CF transmembrane conductance regulator (CFTR gene encoding for a cAMP-regulated chloride (Cl(- channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases. METHODOLOGY/PRINCIPAL FINDINGS: To further establish measurement of CFTR function as a sensitive and robust biomarker for diagnosis and prognosis of CF, we herein assessed cholinergic and cAMP-CFTR-mediated Cl(- secretion in 524 freshly excised rectal biopsies from 118 individuals, including patients with confirmed CF clinical diagnosis (n=51, individuals with clinical CF suspicion (n=49 and age-matched non-CF controls (n=18. Conclusive measurements were obtained for 96% of cases. Patients with "Classic CF", presenting earlier onset of symptoms, pancreatic insufficiency, severe lung disease and low Shwachman-Kulczycki scores were found to lack CFTR-mediated Cl(- secretion (<5%. Individuals with milder CF disease presented residual CFTR-mediated Cl(- secretion (10-57% and non-CF controls show CFTR-mediated Cl(- secretion ≥ 30-35% and data evidenced good correlations with various clinical parameters. Finally, comparison of these values with those in "CF suspicion" individuals allowed to confirm CF in 16/49 individuals (33% and exclude it in 28/49 (57%. Statistical discriminant analyses showed that colonic measurements of CFTR-mediated Cl(- secretion are the best discriminator among Classic/Non-Classic CF and non-CF groups. CONCLUSIONS/SIGNIFICANCE: Determination of CFTR-mediated Cl(- secretion in rectal biopsies is demonstrated here to be a sensitive, reproducible and robust predictive biomarker for the diagnosis and prognosis of CF. The method also has very high potential for (pre-clinical trials of CFTR-modulator therapies.

  2. The CF-modifying gene EHF promotes p.Phe508del-CFTR residual function by altering protein glycosylation and trafficking in epithelial cells.

    Science.gov (United States)

    Stanke, Frauke; van Barneveld, Andrea; Hedtfeld, Silke; Wölfl, Stefan; Becker, Tim; Tümmler, Burkhard

    2014-05-01

    The three-base-pair deletion c.1521_1523delCTT (p.Phe508del, F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) is the most frequent disease-causing lesion in cystic fibrosis (CF). The CFTR gene encodes a chloride and bicarbonate channel at the apical membrane of epithelial cells. Altered ion transport of CFTR-expressing epithelia can be used to differentiate manifestations of the so-called CF basic defect. Recently, an 11p13 region has been described as a CF modifier by the North American CF Genetic Modifier Study Consortium. Selecting the epithelial-specific transcription factor EHF (ets homologous factor) as the likely candidate gene on 11p13, we have genotyped two intragenic microsatellites in EHF to replicate the 11p13 finding in the patient cohort of the European CF Twin and Sibling Study. We could observe an association of rare EHF haplotypes among homozygotes for c.1521_1523delCTT in CFTR, which exhibit a CF-untypical manifestation of the CF basic defect such as CFTR-mediated residual chloride secretion and low response to amiloride. We have reviewed transcriptome data obtained from intestinal epithelial samples of homozygotes for c.1521_1523delCTT in CFTR, which were stratified for their EHF genetic background. Transcripts that were upregulated among homozygotes for c.1521_1523delCTT in CFTR, who carry two rare EHF alleles, were enriched for genes that alter protein glycosylation and trafficking, both mechanisms being pivotal for the effective targeting of fully functional p.Phe508del-CFTR to the apical membrane of epithelial cells. We conclude that EHF modifies the CF phenotype by altering capabilities of the epithelial cell to correctly process the folding and trafficking of mutant p.Phe508del-CFTR.

  3. Targeted Integration of a Super-Exon into the CFTR Locus Leads to Functional Correction of a Cystic Fibrosis Cell Line Model.

    Science.gov (United States)

    Bednarski, Christien; Tomczak, Katja; Vom Hövel, Beate; Weber, Wolf-Michael; Cathomen, Toni

    2016-01-01

    In vitro disease models have enabled insights into the pathophysiology of human disease as well as the functional evaluation of new therapies, such as novel genome engineering strategies. In the context of cystic fibrosis (CF), various cellular disease models have been established in recent years, including organoids based on induced pluripotent stem cell technology that allowed for functional readouts of CFTR activity. Yet, many of these in vitro CF models require complex and expensive culturing protocols that are difficult to implement and may not be amenable for high throughput screens. Here, we show that a simple cellular CF disease model based on the bronchial epithelial ΔF508 cell line CFBE41o- can be used to validate functional CFTR correction. We used an engineered nuclease to target the integration of a super-exon, encompassing the sequences of CFTR exons 11 to 27, into exon 11 and re-activated endogenous CFTR expression by treating CFBE41o- cells with a demethylating agent. We demonstrate that the integration of this super-exon resulted in expression of a corrected mRNA from the endogenous CFTR promoter and used short-circuit current measurements in Ussing chambers to corroborate restored ion transport of the repaired CFTR channels. In conclusion, this study proves that the targeted integration of a large super-exon in CFTR exon 11 leads to functional correction of CFTR, suggesting that this strategy can be used to functionally correct all CFTR mutations located downstream of the 5' end of exon 11. PMID:27526025

  4. Rescue of Murine F508del CFTR Activity in Native Intestine by Low Temperature and Proteasome Inhibitors

    NARCIS (Netherlands)

    M. Wilke (Martina); A.G. Bot (Alice); H. Jorna (Huub); B.J. Scholte (Bob); H.R. de Jonge (Hugo)

    2012-01-01

    textabstractMost patients with Cystic Fibrosis (CF) carry at least one allele with the F508del mutation, resulting in a CFTR chloride channel protein with a processing, gating and stability defect, but with substantial residual activity when correctly sorted to the apical membranes of epithelial cel

  5. Evaluation of CFTR gene mutations in Adana

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    Ozlem Goruroglu Ozturk

    2013-04-01

    Full Text Available ABSTRACT Objective: Cystic fibrosis is the most common autosomal recessive inherited disorder seen in the white populations. It develops in result of mutations of cystic fibrosis transmembrane regulator (CFTR gene. Rate of these mutations vary in different geographical regions. In this study, we aimed to determine the frequency of CFTR gene mutations in Adana. Methods: DNA samples of 63 subjects (21 women, 42 men who were diagnosed as cystic fibrosis at Balcali Hospital of Cukurova University, were studied for 19 different CFTR mutations by the strip assay method which is based on reverse hybridization. Results: In cystic fibrosis diagnosed patients, 19 mutations were observed of which 9 were homozygous and 10 were heterozygous. ∆F508 frequency was found as 11.9%, and rate of homozygous was found as 66.7%. Mutation frequencies of W1282X and N1303K were found as 2.40% and 4.80% respectively and rate of homozygous mutations were 50% for both. I148T mutation frequency was found as 3.20% and all were heterozygous. For the whole 19 mutations, frequency of mutation in 63 subjects was 22.3%. Conclusion: Detection of CFTR gene mutations by the strip assay method by reverse hybridization is an easy, fast and informative method. However, due to improvability of the common mutations in probable cystic fibrosis patients because of heterogenity in this region, it is still a major problem and does not exclude cystic fibrosis diagnosis. But this problematic issue can be overcome by evaluating the whole exons of CFTR mutations by advanced molecular tecniques. Key words: CFTR, cystic fibrosis, molecular diagnosis, reverse hibridisation [Cukurova Med J 2013; 38(2.000: 202-208

  6. Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR

    OpenAIRE

    Cui, Guiying; Rahman, Kazi S.; Infield, Daniel T.; Kuang, Christopher; Prince, Chengyu Z.; McCarty, Nael A.

    2014-01-01

    The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1–6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oo...

  7. Detection of CFTR protein in human leukocytes by flow cytometry.

    Science.gov (United States)

    Johansson, Jan; Vezzalini, Marzia; Verzè, Genny; Caldrer, Sara; Bolognin, Silvia; Buffelli, Mario; Bellisola, Giuseppe; Tridello, Gloria; Assael, Baroukh Maurice; Melotti, Paola; Sorio, Claudio

    2014-07-01

    Leukocytes have previously been shown to express detectable levels of the protein cystic fibrosis transmembrane conductance regulator (CFTR). This study aims to evaluate the application of flow cytometric (FC) analysis to detect CFTR expression, and changes thereof, in these cells. Aliquots (200 μL) of peripheral whole blood from 12 healthy control volunteers (CTRLs), 12 carriers of a CFTR mutation (CFC), and 40 patients with cystic fibrosis (CF) carrying various combinations of CFTR mutations were incubated with specific fluorescent probes recognizing CFTR protein expressed on the plasma membrane of leukocytes. FC was applied to analyze CFTR expression in monocytes, lymphocytes, and polymorphonuclear (PMN) cells. CFTR protein was detected in monocytes and lymphocytes, whereas inconclusive results were obtained from the analysis of PMN cells. Mean fluorescence intensity (MFI) ratio value and %CFTR-positive cells above a selected threshold were the two parameters selected to quantify CFTR expression in cells. Lowest variability and the highest reproducibility were obtained when analyzing monocytes. ANOVA results indicated that both parameters were able to discriminate monocytes of healthy controls and CF individuals according to CFTR mutation classes with high accuracy. Significantly increased MFI ratio values were recorded in CFTR-defective cells that were also able to improve CFTR function after ex vivo treatment with PTC124 (Ataluren), an investigative drug designed to permit the ribosome to read through nonsense CFTR mutations. The method described is minimally invasive and may be used in the monitoring of responses to drugs whose efficacy can depend on increased CFTR protein expression levels. © 2014 International Society for Advancement of Cytometry.

  8. The Chloride Anion Acts as a Second Messenger in Mammalian Cells - Modifying the Expression of Specific Genes

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    Ángel G. Valdivieso

    2016-01-01

    Full Text Available Background/Aims: Cystic Fibrosis (CF is caused by mutations in the CFTR gene, encoding a cAMP-activated chloride (Cl- channel. We have previously demonstrated that the expression of several genes can be modulated by the CFTR activity; among them, SRC, MTND4, CISD1, and IL1B. However, the CFTR signalling mechanism involved in the expression of CFTR-dependent genes is unknown. The aim of this work was to determine if intracellular chloride (Cl-i might function as a second messenger modulating the expression of specific genes. Methods: Differential display (DD was applied to IB3-1 cells (CF cells, cultured under conditions that produce different intracellular Cl- concentrations ([Cl-]i, to analyse their expression profile. Results: Several differentially expressed gene products were observed by using DD, suggesting the presence of chloride-dependent gene expression. Two cDNA fragments, derived from differentially expressed mRNAs and showing opposed response to Cl-‚ were isolated, cloned, sequenced and its Cl- dependency validated by reverse transcription quantitative-PCR (RT-qPCR. We identified the gene RPS27, which encodes the multifunctional ribosomal protein RPS27, also known as metallopanstimulin-1 (MPS-1, and the gene GLRX5, encoding glutaredoxin-related protein 5, as chloride-dependent genes. RPS27 was negatively regulated with increased [Cl-]i, approximately from 25-75 mM Cl- (EC50 = 46 ± 7 mM, and positively regulated from 75-125 mM Cl- (EC50 = 110 ± 11 mM (biphasic response. In contrast, GLRX5 was positively modulated by [Cl-]i, showing a typical sigmoidal dose-response curve from 0-50 mM Cl-, reaching a plateau after 50 mM Cl- (EC50 ∼ 34 mM. Conclusion: The results suggest the existence of chloride-dependent genes. The Cl- anion, therefore, might act as a second messenger for channels or receptors able to modulate the intracellular Cl- concentration, regulating in turn the expression of specific genes.

  9. 线粒体电压依赖性阴离子通道与心血管疾病%Voltage-dependent Anion Channel and Cardiovascular Diseases

    Institute of Scientific and Technical Information of China (English)

    夏晶

    2013-01-01

    电压依赖性阴离子通道(VDAC)是位于线粒体外膜的通道蛋白,是线粒体与细胞质之间转运ATP以及其他代谢产物的主要通道,在线粒体代谢和细胞生长中发挥重要调控作用.近期研究发现,在心肌缺血再灌、糖尿病、心衰、高血压和动脉粥样硬化时,VDAC表达明显增加,引起细胞内钙离子循环紊乱、氧化应激,进而导致细胞凋亡,已成为心血管疾病研究的新热点.本文就VDAC的分子功能,调控及其在心血管疾病中的作用和相关机制进行综述.%The voltage-dependent anion channel (VDAC),a mitochondrial membrane channel protein located in the outer of mitochondrial membrane,is the main pathway between mitochondria and cytoplasm exchanging ADP,ATP,and other metabolites,and plays an important role in mitochondrial metabolism and cell growth.A growing evidence showed that VDAC was increased in cardiovascular diseases including myocardial ischemia and reperfusion,diabetes,heart failure,hypertension and atherosclerosis.The abnormal state of VDAC will result in cell death by inducing calcium cycling dysfunction and oxidative stress.And VDAC has become a hot topic in the field of cardiovascular diseases research.In this article,we will introduce the molecular function and regulation of VDAC and its role in cardiovascular diseases.

  10. Volume regulated anion channel currents of rat hippocampal neurons and their contribution to oxygen-and-glucose deprivation induced neuronal death.

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    Huaqiu Zhang

    Full Text Available Volume-regulated anion channels (VRAC are widely expressed chloride channels that are critical for the cell volume regulation. In the mammalian central nervous system, the physiological expression of neuronal VRAC and its role in cerebral ischemia are issues largely unknown. We show that hypoosmotic medium induce an outwardly rectifying chloride conductance in CA1 pyramidal neurons in rat hippocampal slices. The induced chloride conductance was sensitive to some of the VRAC inhibitors, namely, IAA-94 (300 µM and NPPB (100 µM, but not to tamoxifen (10 µM. Using oxygen-and-glucose deprivation (OGD to simulate ischemic conditions in slices, VRAC activation appeared after OGD induced anoxic depolarization (AD that showed a progressive increase in current amplitude over the period of post-OGD reperfusion. The OGD induced VRAC currents were significantly inhibited by inhibitors for glutamate AMPA (30 µM NBQX and NMDA (40 µM AP-5 receptors in the OGD solution, supporting the view that induction of AD requires an excessive Na(+-loading via these receptors that in turn to activate neuronal VRAC. In the presence of NPPB and DCPIB in the post-OGD reperfusion solution, the OGD induced CA1 pyramidal neuron death, as measured by TO-PRO-3-I staining, was significantly reduced, although DCPIB did not appear to be an effective neuronal VRAC blocker. Altogether, we show that rat hippocampal pyramidal neurons express functional VRAC, and ischemic conditions can initial neuronal VRAC activation that may contribute to ischemic neuronal damage.

  11. Stimulation of airway and intestinal mucosal secretion by natural coumarin CFTR activators

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    Hong eYang

    2011-09-01

    Full Text Available Mutations of cystic fibrosis transmembrane conductance regulator (CFTR cause lethal hereditary disease cystic fibrosis (CF that involves extensive destruction and dysfunction of serous epithelium. Possible pharmacological therapy includes correction of defective intracellular processing and abnormal channel gating. In a previous study, we identified five natural coumarin potentiators of Δ508-CFTR including osthole, imperatorin, isopsoralen, praeruptorin A and scoparone. The present study was designed to determine the activity of these coumarine compounds on CFTR activity in animal tissues as a primary evaluation of their therapeutic potential. In the present study, we analyzed the affinity of these coumarin potentiators in activating wild-type CFTR and found that they are all potent activators. Osthole showed the highest affinity with Kd values <50 nmol/L as determined by Ussing chamber short-circuit current assay. Stimulation of rat colonic mucosal secretion by osthole was tested by the Ussing chamber short-circuit current assay. Osthole reached maximal activation of colonic Cl- secretion at 5 mol/L. Stimulation of mouse tracheal mucosal secretion was analyzed by optical measurement of single gland secretion. Fluid secretion rate of tracheal single submucosal gland stimulated by osthole at 10mol/L was 3-fold more rapid than that in negative control. In both cases the stimulated secretions were fully abolished by CFTRinh-172. In conclusion, the effective stimulation of Cl– and fluid secretion in colonic and tracheal mucosa by osthole suggested the therapeutic potential of natural coumarine compounds for the treatment of cystic fibrosis and other CFTR-related diseases.

  12. The amine-containing cutaneous irritant heptylamine inhibits the volume-regulated anion channel and mobilizes intracellular calcium in normal human epidermal keratinocytes.

    Science.gov (United States)

    Raoux, Matthieu; Colomban, Cécile; Delmas, Patrick; Crest, Marcel

    2007-06-01

    Many amines are skin irritants and cause contact dermatitis. However, little is known about their mechanisms of action in keratinocytes except that they induce the release of the inflammatory mediators cytokines and ATP. Here, we tested whether volume-regulated anion channels (VRACs) in primary cultures of normal human epidermal keratinocytes are modulated by the referenced amine-containing cutaneous irritant heptylamine. Under isotonic conditions, we isolated the VRAC current (I(VRAC)) from other conductances using a high Ca(2+)-buffering internal solution. I(VRAC) ran up after patch rupturing and reached a plateau within 15 min. It was reversibly and dose-dependently inhibited by heptylamine with an IC(50) value of 260 microM. Cell-swelling caused by the application of a hypotonic solution increased 2.7-fold I(VRAC) and reduced the inhibition of VRAC by heptylamine with a dose-response curve shifted approximately 10-fold to the right. In addition, we showed, using cell-attached patch recordings, that adding heptylamine to the bath inhibited VRAC activity. This suggests that heptylamine diffuses into the membrane to inhibit VRAC. Finally, we demonstrated that heptylamine induced Ca(2+)-store depletion and that VRAC inhibition was not caused by the increase in cytosolic Ca(2+). Taken together, these results identify heptylamine as a blocker of VRAC and suggest that Ca(2+)-store depletion may be involved in mechanisms of irritant contact dermatitis caused by heptylamine. PMID:17384225

  13. SLAH1, a homologue of the slow type anion channel SLAC1, modulates shoot Cl- accumulation and salt tolerance in Arabidopsis thaliana.

    Science.gov (United States)

    Qiu, Jiaen; Henderson, Sam W; Tester, Mark; Roy, Stuart J; Gilliham, Mathew

    2016-08-01

    Salinity tolerance is correlated with shoot chloride (Cl(-)) exclusion in multiple crops, but the molecular mechanisms of long-distance Cl(-) transport are poorly defined. Here, we characterize the in planta role of AtSLAH1 (a homologue of the slow type anion channel-associated 1 (SLAC1)). This protein, localized to the plasma membrane of root stelar cells, has its expression reduced by salt or ABA, which are key predictions for a protein involved with loading Cl(-) into the root xylem. Artificial microRNA knockdown mutants of AtSLAH1 had significantly reduced shoot Cl(-) accumulation when grown under low Cl(-), whereas shoot Cl(-) increased and the shoot nitrate/chloride ratio decreased following AtSLAH1 constitutive or stelar-specific overexpression when grown in high Cl(-) In both sets of overexpression lines a significant reduction in shoot biomass over the null segregants was observed under high Cl(-) supply, but not low Cl(-) supply. Further in planta data showed AtSLAH3 overexpression increased the shoot nitrate/chloride ratio, consistent with AtSLAH3 favouring nitrate transport. Heterologous expression of AtSLAH1 in Xenopus laevis oocytes led to no detectible transport, suggesting the need for post-translational modifications for AtSLAH1 to be active. Our in planta data are consistent with AtSLAH1 having a role in controlling root-to-shoot Cl(-) transport. PMID:27340232

  14. Restricted ADP movement in cardiomyocytes: Cytosolic diffusion obstacles are complemented with a small number of open mitochondrial voltage-dependent anion channels.

    Science.gov (United States)

    Simson, Päivo; Jepihhina, Natalja; Laasmaa, Martin; Peterson, Pearu; Birkedal, Rikke; Vendelin, Marko

    2016-08-01

    Adequate intracellular energy transfer is crucial for proper cardiac function. In energy starved failing hearts, partial restoration of energy transfer can rescue mechanical performance. There are two types of diffusion obstacles that interfere with energy transfer from mitochondria to ATPases: mitochondrial outer membrane (MOM) with voltage-dependent anion channel (VDAC) permeable to small hydrophilic molecules and cytoplasmatic diffusion barriers grouping ATP-producers and -consumers. So far, there is no method developed to clearly distinguish the contributions of cytoplasmatic barriers and MOM to the overall diffusion restriction. Furthermore, the number of open VDACs in vivo remains unknown. The aim of this work was to establish the partitioning of intracellular diffusion obstacles in cardiomyocytes. We studied the response of mitochondrial oxidative phosphorylation of permeabilized rat cardiomyocytes to changes in extracellular ADP by recording 3D image stacks of NADH autofluorescence. Using cell-specific mathematical models, we determined the permeability of MOM and cytoplasmatic barriers. We found that only ~2% of VDACs are accessible to cytosolic ADP and cytoplasmatic diffusion barriers reduce the apparent diffusion coefficient by 6-10×. In cardiomyocytes, diffusion barriers in the cytoplasm and by the MOM restrict ADP/ATP diffusion to similar extents suggesting a major role of both barriers in energy transfer and other intracellular processes. PMID:27261153

  15. Structure and orientation of two voltage-dependent anion-selective channel isoforms. An attenuated total reflection fourier-transform infrared spectroscopy study.

    Science.gov (United States)

    Abrecht, H; Goormaghtigh, E; Ruysschaert, J M; Homble, F

    2000-12-29

    Two VDAC (voltage-dependent anion-selective channel) isoforms were purified from seed cotyledons of Phaseolus vulgaris by chromatofocusing chromatography. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to study the structural properties of the two isoforms reconstituted in a mixture of asolectin and 5% stigmasterol. The IR spectra of the two VDAC isoforms were highly similar indicating 50 to 53% anti-parallel beta-sheet. The orientation of the beta-strands relative to the barrel axis was calculated from the experimentally obtained dichroic ratios of the amide I beta-sheet component and the amide II band. Comparing the IR spectra of the reconstituted VDAC isoforms with the IR spectra of the bacterial porin OmpF, for which a high resolution structure is available, provided evidence for a general structural organization of the VDAC isoforms similar to that of bacterial porins. Hydrogen-deuterium exchange measurements indicated that the exchange of the amide protons occurs to a higher extent in the two VDAC isoforms than in the OmpF porin.

  16. The role of glutamate release on voltage-dependent anion channels (VDAC-mediated apoptosis in an eleven vessel occlusion model in rats.

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    Eunkuk Park

    Full Text Available Voltage-dependent anion channel (VDAC is the main protein in mitochondria-mediated apoptosis, and the modulation of VDAC may be induced by the excessive release of extracellular glutamate. This study examined the role of glutamate release on VDAC-mediated apoptosis in an eleven vessel occlusion model in rats. Male Sprague-Dawley rats (250-350 g were used for the 11 vessel occlusion ischemic model, which were induced for a 10-min transient occlusion. During the ischemic and initial reperfusion episode, the real-time monitoring of the extracellular glutamate concentration was measured using an amperometric microdialysis biosensor and the cerebral blood flow (CBF was monitored by laser-Doppler flowmetry. To confirm neuronal apoptosis, the brains were removed 72 h after ischemia to detect the neuron-specific nuclear protein and pro-apoptotic proteins (cleaved caspase-3, VDAC, p53 and BAX. The changes in the mitochondrial morphology were measured by atomic force microscopy. A decrease in the % of CBF was observed, and an increase in glutamate release was detected after the onset of ischemia, which continued to increase during the ischemic period. A significantly higher level of glutamate release was observed in the ischemia group. The increased glutamate levels in the ischemia group resulted in the activation of VDAC and pro-apoptotic proteins in the hippocampus with morphological alterations to the mitochondria. This study suggests that an increase in glutamate release promotes VDAC-mediated apoptosis in an 11 vessel occlusion ischemic model.

  17. SLAH1, a homologue of the slow type anion channel SLAC1, modulates shoot Cl − accumulation and salt tolerance in Arabidopsis thaliana

    KAUST Repository

    Qiu, Jiaen

    2016-06-23

    Salinity tolerance is correlated with shoot chloride (Cl–) exclusion in multiple crops, but the molecular mechanisms of long-distance Cl– transport are poorly defined. Here, we characterize the in planta role of AtSLAH1 (a homologue of the slow type anion channel-associated 1 (SLAC1)). This protein, localized to the plasma membrane of root stelar cells, has its expression reduced by salt or ABA, which are key predictions for a protein involved with loading Cl– into the root xylem. Artificial microRNA knockdown mutants of AtSLAH1 had significantly reduced shoot Cl− accumulation when grown under low Cl–, whereas shoot Cl– increased and the shoot nitrate/chloride ratio decreased following AtSLAH1 constitutive or stelar-specific overexpression when grown in high Cl–. In both sets of overexpression lines a significant reduction in shoot biomass over the null segregants was observed under high Cl– supply, but not low Cl– supply. Further in planta data showed AtSLAH3 overexpression increased the shoot nitrate/chloride ratio, consistent with AtSLAH3 favouring nitrate transport. Heterologous expression of AtSLAH1 in Xenopus laevis oocytes led to no detectible transport, suggesting the need for post-translational modifications for AtSLAH1 to be active. Our in planta data are consistent with AtSLAH1 having a role in controlling root-to-shoot Cl– transport.

  18. Multi-physiopathological consequences of the c.1392G>T CFTR mutation revealed by clinical and cellular investigations.

    Science.gov (United States)

    Farhat, Raed; El-Seedy, Ayman; El-Moussaoui, Kamal; Pasquet, Marie-Claude; Adolphe, Catherine; Bieth, Eric; Languepin, Jeanne; Sermet-Gaudelus, Isabelle; Kitzis, Alain; Ladevèze, Véronique

    2015-02-01

    This study combines a clinical approach and multiple level cellular analyses to determine the physiopathological consequences of the c.1392G>T (p.Lys464Asn) CFTR exon 10 mutation, detected in a CF patient with a frameshift deletion in trans and a TG(11)T(5) in cis. Minigene experiment, with different TG(m)T(n) alleles, and nasal cell mRNA extracts were used to study the impact of c.1392G>T on splicing in both in cellulo and in vivo studies. The processing and localization of p.Lys464Asn protein were evaluated, in cellulo, by western blotting analyses and confocal microscopy. Clinical and channel exploration tests were performed on the patient to determine the exact CF phenotype profile and the CFTR chloride transport activity. c.1392G>T affects exon 10 splicing by inducing its complete deletion and encoding a frameshift transcript. The polymorphism TG(11)T(5) aggravates the effects of this mutation on aberrant splicing. Analysis of mRNA obtained from parental airway epithelial cells confirmed these in cellulo results. At the protein level the p.Lys464Asn protein showed neither maturated form nor membrane localization. Furthermore, the in vivo channel tests confirmed the absence of CFTR activity. Thus, the c.1392G>T mutation alone or in association with the TG repeats and the poly T tract revealed obvious impacts on splicing and CFTR protein processing and functionality. The c.[T(5); 1392G>T] complex allele contributes to the CF phenotype by affecting splicing and inducing a severe misprocessing defect. These results demonstrate that the classical CFTR mutations classification is not sufficient: in vivo and in cellulo studies of a possible complex allele in a patient are required to provide correct CFTR mutation classification, adequate medical counseling, and adapted therapeutic strategies.

  19. CFTR protein repair therapy in cystic fibrosis.

    Science.gov (United States)

    Quintana-Gallego, Esther; Delgado-Pecellín, Isabel; Calero Acuña, Carmen

    2014-04-01

    Cystic fibrosis is a single gene, autosomal recessive disorder, in which more than 1,900 mutations grouped into 6 classes have been described. It is an example a disease that could be well placed to benefit from personalised medicine. There are currently 2 very different approaches that aim to correct the basic defect: gene therapy, aimed at correcting the genetic alteration, and therapy aimed at correcting the defect in the CFTR protein. The latter is beginning to show promising results, with several molecules under development. Ataluren (PTC124) is a molecule designed to make the ribosomes become less sensitive to the premature stop codons responsible for class i mutations. Lumacaftor (VX-809) is a CFTR corrector directed at class ii mutations, among which Phe508del is the most frequent, with encouraging results. Ivacaftor (VX-770) is a potentiator, the only one marketed to date, which has shown good efficacy for the class iii mutation Gly551Asp in children over the age of 6 and adults. These drugs, or a combination of them, are currently undergoing various clinical trials for other less common genetic mutations. In the last 5 years, CFTR has been designated as a therapeutic target. Ivacaftor is the first drug to treat the basic defect in cystic fibrosis, but only provides a response in a small number of patients. New drugs capable of restoring the CFTR protein damaged by the most common mutations are required.

  20. Endocytic Sorting of CFTR variants Monitored by Single Cell Fluorescence Ratio Image Analysis (FRIA) in Living Cells

    Science.gov (United States)

    Barriere, H.; Apaja, P.; Okiyoneda, T.; Lukacs, G. L.

    2016-01-01

    Summary The wild-type CFTR channel undergoes constitutive internalization and recycling at the plasma membrane. This process is initiated by the recognition of the Tyr- and di-Leu-based endocytic motifs of CFTR by the AP-2 adaptor complex, leading to the formation of clathrin-coated vesicles and the channel delivery to sorting/recycling endosomes. Accumulating evidence suggests that conformationally defective mutant CFTRs (e.g. rescued ΔF508 and glycosylation-deficient channel) are unstable at the plasma membrane and undergo augmented ubiquitination in post-Golgi compartments. Ubiquitination conceivably accounts for the metabolic instability at cell surface by provoking accelerated internalization, as well as rerouting the channel from recycling towards lysosomal degradation. We developed an in vivo fluorescence ratio imaging assay (FRIA) that in concert with genetic manipulation can be utilized to establish the post-endocytic fate and sorting determinants of mutant CFTRs. PMID:21594793

  1. CFTR and Wnt/beta-catenin signaling in lung development

    Directory of Open Access Journals (Sweden)

    Love Damon

    2008-07-01

    Full Text Available Abstract Background Cystic fibrosis transmembrane conductance regulator (CFTR was shown previously to modify stretch induced differentiation in the lung. The mechanism for CFTR modulation of lung development was examined by in utero gene transfer of either a sense or antisense construct to alter CFTR expression levels. The BAT-gal transgenic reporter mouse line, expressing β-galactosidase under a canonical Wnt/β-catenin-responsive promoter, was used to assess the relative roles of CFTR, Wnt, and parathyroid hormone-related peptide (PTHrP in lung organogenesis. Adenoviruses containing full-length CFTR, a short anti-sense CFTR gene fragment, or a reporter gene as control were used in an intra-amniotic gene therapy procedure to transiently modify CFTR expression in the fetal lung. Results A direct correlation between CFTR expression levels and PTHrP levels was found. An inverse correlation between CFTR and Wnt signaling activities was demonstrated. Conclusion These data are consistent with CFTR participating in the mechanicosensory process essential to regulate Wnt/β-Catenin signaling required for lung organogenesis.

  2. N-Alpha-Acetyltransferases and Regulation of CFTR Expression.

    Directory of Open Access Journals (Sweden)

    Ali J Vetter

    Full Text Available The majority of cystic fibrosis (CF-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR. The mutant preferentially interacts with a subunit of an N-alpha-acetyltransferase A. Based on recent reports that acetylation of the N-termini of some N-end rule substrates control their ubiquitination and subsequent degradation, a potential role for this modification in regulation of CFTR expression was assessed. Knockdown experiments identified two complexes, which affect G85E CFTR proteins levels, NatA and NatB. Effects of the knockdowns on mRNA levels, translation rates, and degradation rates established that the two complexes regulate G85E CFTR through two separate mechanisms. NatA acts indirectly by regulating transcription levels and NatB acts through a previously identified, but incompletely understood posttranslational mechanism. This regulation did not effect trafficking of G85E CFTR, which remains retained in the ER, nor did it alter the degradation rate of CFTR. A mutation predicted to inhibit N-terminal acetylation of CFTR, Q2P, was without effect, suggesting neither system acts directly on CFTR. These results contradict the prediction that N-terminal acetylation of CFTR determines its fitness as a proteasome substrate, but rather NatB plays a role in the conformational maturation of CFTR in the ER through actions on an unidentified protein.

  3. N-Alpha-Acetyltransferases and Regulation of CFTR Expression.

    Science.gov (United States)

    Vetter, Ali J; Karamyshev, Andrey L; Patrick, Anna E; Hudson, Henry; Thomas, Philip J

    2016-01-01

    The majority of cystic fibrosis (CF)-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR. The mutant preferentially interacts with a subunit of an N-alpha-acetyltransferase A. Based on recent reports that acetylation of the N-termini of some N-end rule substrates control their ubiquitination and subsequent degradation, a potential role for this modification in regulation of CFTR expression was assessed. Knockdown experiments identified two complexes, which affect G85E CFTR proteins levels, NatA and NatB. Effects of the knockdowns on mRNA levels, translation rates, and degradation rates established that the two complexes regulate G85E CFTR through two separate mechanisms. NatA acts indirectly by regulating transcription levels and NatB acts through a previously identified, but incompletely understood posttranslational mechanism. This regulation did not effect trafficking of G85E CFTR, which remains retained in the ER, nor did it alter the degradation rate of CFTR. A mutation predicted to inhibit N-terminal acetylation of CFTR, Q2P, was without effect, suggesting neither system acts directly on CFTR. These results contradict the prediction that N-terminal acetylation of CFTR determines its fitness as a proteasome substrate, but rather NatB plays a role in the conformational maturation of CFTR in the ER through actions on an unidentified protein.

  4. N-Alpha-Acetyltransferases and Regulation of CFTR Expression.

    Science.gov (United States)

    Vetter, Ali J; Karamyshev, Andrey L; Patrick, Anna E; Hudson, Henry; Thomas, Philip J

    2016-01-01

    The majority of cystic fibrosis (CF)-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR. The mutant preferentially interacts with a subunit of an N-alpha-acetyltransferase A. Based on recent reports that acetylation of the N-termini of some N-end rule substrates control their ubiquitination and subsequent degradation, a potential role for this modification in regulation of CFTR expression was assessed. Knockdown experiments identified two complexes, which affect G85E CFTR proteins levels, NatA and NatB. Effects of the knockdowns on mRNA levels, translation rates, and degradation rates established that the two complexes regulate G85E CFTR through two separate mechanisms. NatA acts indirectly by regulating transcription levels and NatB acts through a previously identified, but incompletely understood posttranslational mechanism. This regulation did not effect trafficking of G85E CFTR, which remains retained in the ER, nor did it alter the degradation rate of CFTR. A mutation predicted to inhibit N-terminal acetylation of CFTR, Q2P, was without effect, suggesting neither system acts directly on CFTR. These results contradict the prediction that N-terminal acetylation of CFTR determines its fitness as a proteasome substrate, but rather NatB plays a role in the conformational maturation of CFTR in the ER through actions on an unidentified protein. PMID:27182737

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

    OpenAIRE

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

    2016-01-01

    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...

  6. Resonant spectra of quadrupolar anions

    CERN Document Server

    Fossez, K; Nazarewicz, W; Michel, N; Garrett, W R; Płoszajczak, M

    2016-01-01

    In quadrupole-bound anions, an extra electron is attached at a sufficiently large quadrupole moment of a neutral molecule, which is lacking a permanent dipole moment. The nature of the bound states and low-lying resonances of such anions is of interest for understanding the threshold behavior of open quantum systems in general. In this work, we investigate the properties of quadrupolar anions as extreme halo systems, the formation of rotational bands, and the transition from a subcritical to supercritical electric quadrupole moment. We solve the electron-plus-molecule problem using a non-adiabatic coupled-channel formalism by employing the Berggren ensemble, which explicitly contains bound states, narrow resonances, and the scattering continuum. We demonstrate that binding energies and radii of quadrupolar anions strictly follow the scaling laws for two-body halo systems. Contrary to the case of dipolar anions, ground-state band of quadrupolar anions smoothly extend into the continuum, and many rotational ban...

  7. The power stroke driven by ATP binding in CFTR as studied by molecular dynamics simulations.

    Science.gov (United States)

    Furukawa-Hagiya, Tomoka; Furuta, Tadaomi; Chiba, Shuntaro; Sohma, Yoshiro; Sakurai, Minoru

    2013-01-10

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP binding cassette (ABC) protein superfamily. Currently, it remains unclear how ATP binding causes the opening of the channel gate at the molecular level. To clarify this mechanism, we first constructed an atomic model of the inward-facing CFTR using the X-ray structures of other ABC proteins. Molecular dynamics (MD) simulations were then performed to explore the structure and dynamics of the inward-facing CFTR in a membrane environment. In the MgATP-bound state, two nucleotide-binding domains (NBDs) formed a head-to-tail type of dimer, in which the ATP molecules were sandwiched between the Walker A and signature motifs. Alternatively, one of the final MD structures in the apo state was similar to that of a "closed-apo" conformation found in the X-ray analysis of ATP-free MsbA. Principal component analysis for the MD trajectory indicated that NBD dimerization causes significant structural and dynamical changes in the transmembrane domains (TMDs), which is likely indicative of the formation of a chloride ion access path. This study suggests that the free energy gain from ATP binding acts as a driving force not only for NBD dimerization but also for NBD-TMD concerted motions. PMID:23214920

  8. Restoration of R117H CFTR folding and function in human airway cells through combination treatment with VX-809 and VX-770.

    Science.gov (United States)

    Gentzsch, Martina; Ren, Hong Y; Houck, Scott A; Quinney, Nancy L; Cholon, Deborah M; Sopha, Pattarawut; Chaudhry, Imron G; Das, Jhuma; Dokholyan, Nikolay V; Randell, Scott H; Cyr, Douglas M

    2016-09-01

    Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients. PMID:27402691

  9. An unexpected effect of TNF-α on F508del-CFTR maturation and function [v2; ref status: indexed, http://f1000r.es/5tv

    Directory of Open Access Journals (Sweden)

    Sara Bitam

    2015-09-01

    Full Text Available Cystic fibrosis (CF is a multifactorial disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR, which encodes a cAMP-dependent Cl- channel. The most frequent mutation, F508del, leads to the synthesis of a prematurely degraded, otherwise partially functional protein. CFTR is expressed in many epithelia, with major consequences in the airways of patients with CF, characterized by both fluid transport abnormalities and persistent inflammatory responses. The relationship between the acute phase of inflammation and the expression of wild type (WT CFTR or F508del-CFTR is poorly understood. The aim of the present study was to investigate this effect. The results show that 10 min exposure to TNF-alpha (0.5-50ng/ml of F508del-CFTR-transfected HeLa cells and human bronchial cells expressing F508del-CFTR in primary culture (HBE leads to the maturation of F508del-CFTR and induces CFTR chloride currents. The enhanced CFTR expression and function upon TNFα is sustained, in HBE cells, for at least 24 h. The underlying mechanism of action involves a protein kinase C (PKC signaling pathway, and occurs through insertion of vesicles containing F508del-CFTR to the plasma membrane, with TNFα behaving as a corrector molecule. In conclusion, a novel and unexpected action of TNFα has been discovered and points to the importance of systematic studies on the roles of inflammatory mediators in the maturation of abnormally folded proteins in general and in the context of CF in particular.

  10. An unexpected effect of TNF-α on F508del-CFTR maturation and function [v1; ref status: indexed, http://f1000r.es/5jf

    Directory of Open Access Journals (Sweden)

    Sara Bitam

    2015-07-01

    Full Text Available Cystic fibrosis (CF is a multifactorial disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR, which encodes a cAMP-dependent Cl- channel. The most frequent mutation, F508del, leads to the synthesis of a prematurely degraded, otherwise partially functional protein. CFTR is expressed in many epithelia, with major consequences in the airways of patients with CF, characterized by both fluid transport abnormalities and persistent inflammatory responses. The relationship between the acute phase of inflammation and the expression of wild type (WT CFTR or F508del-CFTR is poorly understood. The aim of the present study was to investigate this effect. The results show that 10 min exposure to TNF-alpha (0.5-50ng/ml of F508del-CFTR-transfected HeLa cells and human bronchial cells expressing F508del-CFTR in primary culture (HBE leads to the maturation of F508del-CFTR and induces CFTR chloride currents. The enhanced CFTR expression and function upon TNFα is sustained, in HBE cells, for at least 24 h. The underlying mechanism of action involves a protein kinase C (PKC signaling pathway, and occurs through insertion of vesicles containing F508del-CFTR to the plasma membrane, with TNFα behaving as a corrector molecule. In conclusion, a novel and unexpected action of TNFα has been discovered and points to the importance of systematic studies on the roles of inflammatory mediators in the maturation of abnormally folded proteins in general and in the context of CF in particular.

  11. ABSOLUTE CONFIGURATION AND BIOLOGICAL PROPERTIES OF ENANTIOMERS OF CFTR INHIBITOR BPO-27.

    Science.gov (United States)

    Snyder, David S; Tradtrantip, Lukmanee; Battula, Sailaja; Yao, Chenjuan; Phuan, Puay-Wah; Fettinger, James C; Kurth, Mark J; Verkman, A S

    2013-05-01

    We previously reported benzopyrimido-pyrrolo-oxazinedione (BPO) inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and showed their efficacy in a model of polycystic kidney disease. Here, we separated the enantiomers of lead compound BPO-27, (1), which contains a single chiral center, and determined their absolute configuration, activity and metabolic stability. Following separation by chiral supercritical fluid chromatography, the R enantiomer, as determined by x-ray crystallography, inhibited CFTR chloride conductance with IC50 ~ 4 nM, while S enantiomer was inactive. In vitro metabolic stability in hepatic microsomes showed both enantiomers as stable, with <5 % metabolism in 4 h. Following bolus interperitoneal administration in mice, serum (R)-1 decayed with t1/2 ~ 1.6 h and gave sustained therapeutic concentrations in kidney.

  12. Steady-state interactions of glibenclamide with CFTR: evidence for multiple sites in the pore.

    Science.gov (United States)

    Zhang, Z R; Zeltwanger, S; McCarty, N A

    2004-05-01

    The objective of the present study was to clarify the mechanism by which the sulfonylurea drug, glibenclamide, inhibits single CFTR channels in excised patches from Xenopus oocytes. Glibenclamide blocks the open pore of the channel via binding at multiple sites with varying kinetics. In the absence of glibenclamide, open-channel bursts exhibited a flickery intraburst closed state (C1); this is due to block of the pore by the pH buffer, TES. Application of 25 microM glibenclamide to the cytoplasmic solution resulted in the appearance of two drug-induced intraburst closed states (C2, C3) of widely different duration, which differed in pH-dependence. The kinetics of interaction with the C3 state, but not the C2 state, were strongly voltage-dependent. The durations of both the C2 and C3 states were concentration-dependent, indicating a non-linear reaction scheme. Application of drug also increased the burst duration, which is consistent with an open-channel blocking mechanism. A kinetic model is proposed. These results indicate that glibenclamide interacts with open CFTR channels in a complex manner, involving interactions with multiple binding sites in the channel pore.

  13. The glutamate aspartate transporter (GLAST) mediates L-glutamate-stimulated ascorbate-release via swelling-activated anion channels in cultured neonatal rodent astrocytes.

    Science.gov (United States)

    Lane, Darius J R; Lawen, Alfons

    2013-03-01

    Vitamin C (ascorbate) plays important neuroprotective and neuromodulatory roles in the mammalian brain. Astrocytes are crucially involved in brain ascorbate homeostasis and may assist in regenerating extracellular ascorbate from its oxidised forms. Ascorbate accumulated by astrocytes can be released rapidly by a process that is stimulated by the excitatory amino acid, L-glutamate. This process is thought to be neuroprotective against excitotoxicity. Although of potential clinical interest, the mechanism of this stimulated ascorbate-release remains unknown. Here, we report that primary cultures of mouse and rat astrocytes release ascorbate following initial uptake of dehydroascorbate and accumulation of intracellular ascorbate. Ascorbate-release was not due to cellular lysis, as assessed by cellular release of the cytosolic enzyme lactate dehydrogenase, and was stimulated by L-glutamate and L-aspartate, but not the non-excitatory amino acid L-glutamine. This stimulation was due to glutamate-induced cellular swelling, as it was both attenuated by hypertonic and emulated by hypotonic media. Glutamate-stimulated ascorbate-release was also sensitive to inhibitors of volume-sensitive anion channels, suggesting that the latter may provide the conduit for ascorbate efflux. Glutamate-stimulated ascorbate-release was not recapitulated by selective agonists of either ionotropic or group I metabotropic glutamate receptors, but was completely blocked by either of two compounds, TFB-TBOA and UCPH-101, which non-selectively and selectively inhibit the glial Na(+)-dependent excitatory amino acid transporter, GLAST, respectively. These results suggest that an impairment of astrocytic ascorbate-release may exacerbate neuronal dysfunction in neurodegenerative disorders and acute brain injury in which excitotoxicity and/or GLAST deregulation have been implicated. PMID:22886112

  14. Regulated recycling of mutant CFTR is partially restored by pharmacological treatment.

    Science.gov (United States)

    Holleran, John P; Zeng, Jianxin; Frizzell, Raymond A; Watkins, Simon C

    2013-06-15

    Efficient trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) to and from the cell surface is essential for maintaining channel density at the plasma membrane (PM) and ensuring proper physiological activity. The most common mutation, F508del, exhibits reduced surface expression and impaired function despite treatment with currently available pharmacological small molecules, called correctors. To gain more detailed insight into whether CFTR enters compartments that allow corrector stabilization in the cell periphery, we investigated the peripheral trafficking itineraries and kinetics of wild type (WT) and F508del in living cells using high-speed fluorescence microscopy together with fluorogen activating protein detection. We directly visualized internalization and accumulation of CFTR WT from the PM to a perinuclear compartment that colocalized with the endosomal recycling compartment (ERC) markers Rab11 and EHD1, reaching steady-state distribution by 25 minutes. Stimulation by protein kinase A (PKA) depleted this intracellular pool and redistributed CFTR channels to the cell surface, elicited by reduced endocytosis and active translocation to the PM. Corrector or temperature rescue of F508del also resulted in targeting to the ERC and exhibited subsequent PKA-stimulated trafficking to the PM. Corrector treatment (24 hours) led to persistent residence of F508del in the ERC, while thermally destabilized F508del was targeted to lysosomal compartments by 3 hours. Acute addition of individual correctors, C4 or C18, acted on peripheral trafficking steps to partially block lysosomal targeting of thermally destabilized F508del. Taken together, corrector treatment redirects F508del trafficking from a degradative pathway to a regulated recycling route, and proteins that mediate this process become potential targets for improving the efficacy of current and future correctors.

  15. Correction of chloride transport and mislocalization of CFTR protein by vardenafil in the gastrointestinal tract of cystic fibrosis mice.

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    Barbara Dhooghe

    Full Text Available Although lung disease is the major cause of mortality in cystic fibrosis (CF, gastrointestinal (GI manifestations are the first hallmarks in 15-20% of affected newborns presenting with meconium ileus, and remain major causes of morbidity throughout life. We have previously shown that cGMP-dependent phosphodiesterase type 5 (PDE5 inhibitors rescue defective CF Transmembrane conductance Regulator (CFTR-dependent chloride transport across the mouse CF nasal mucosa. Using F508del-CF mice, we examined the transrectal potential difference 1 hour after intraperitoneal injection of the PDE5 inhibitor vardenafil or saline to assess the amiloride-sensitive sodium transport and the chloride gradient and forskolin-dependent chloride transport across the GI tract. In the same conditions, we performed immunohistostaining studies in distal colon to investigate CFTR expression and localization. F508del-CF mice displayed increased sodium transport and reduced chloride transport compared to their wild-type littermates. Vardenafil, applied at a human therapeutic dose (0.14 mg/kg used to treat erectile dysfunction, increased chloride transport in F508del-CF mice. No effect on sodium transport was detected. In crypt colonocytes of wild-type mice, the immunofluorescence CFTR signal was mostly detected in the apical cell compartment. In F508del-CF mice, a 25% reduced signal was observed, located mostly in the subapical region. Vardenafil increased the peak of intensity of the fluorescence CFTR signal in F508del-CF mice and displaced it towards the apical cell compartment. Our findings point out the intestinal mucosa as a valuable tissue to study CFTR transport function and localization and to evaluate efficacy of therapeutic strategies in CF. From our data we conclude that vardenafil mediates potentiation of the CFTR chloride channel and corrects mislocalization of the mutant protein. The study provides compelling support for targeting the cGMP signaling pathway in CF

  16. In vivo readout of CFTR function: ratiometric measurement of CFTR-dependent secretion by individual, identifiable human sweat glands.

    Directory of Open Access Journals (Sweden)

    Jeffrey J Wine

    Full Text Available To assess CFTR function in vivo, we developed a bioassay that monitors and compares CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (~50 individual, identified glands in each subject. Sweating was stimulated by intradermally injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-layer that contained dispersed, water soluble dye particles that partitioned into the sweat bubbles, making them highly visible. CFTR-independent secretion (M-sweat was stimulated with methacholine, which binds to muscarinic receptors and elevates cytosolic calcium. CFTR-dependent secretion (C-sweat was stimulated with a β-adrenergic cocktail that elevates cytosolic cAMP while blocking muscarinic receptors. A C-sweat/M-sweat ratio was determined on a gland-by-gland basis to compensate for differences unrelated to CFTR function, such as gland size. The average ratio provides an approximately linear readout of CFTR function: the heterozygote ratio is ~0.5 the control ratio and for CF subjects the ratio is zero. During assay development, we measured C/M ratios in 6 healthy controls, 4 CF heterozygotes, 18 CF subjects and 4 subjects with 'CFTR-related' conditions. The assay discriminated all groups clearly. It also revealed consistent differences in the C/M ratio among subjects within groups. We hypothesize that these differences reflect, at least in part, levels of CFTR expression, which are known to vary widely. When C-sweat rates become very low the C/M ratio also tended to decrease; we hypothesize that this nonlinearity reflects ductal fluid absorption. We also discovered that M-sweating potentiates the subsequent C-sweat response. We then used potentiation as a surrogate for drugs that can increase CFTR-dependent secretion. This bioassay provides an additional method for assessing CFTR function in vivo, and is well suited for within-subject tests of systemic, CFTR-directed therapeutics.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  19. Potent, metabolically stable benzopyrimido-pyrrolo-oxazine-dione (BPO) CFTR inhibitors for polycystic kidney disease.

    Science.gov (United States)

    Snyder, David S; Tradtrantip, Lukmanee; Yao, Chenjuan; Kurth, Mark J; Verkman, A S

    2011-08-11

    We previously reported the discovery of pyrimido-pyrrolo-quinoxalinedione (PPQ) inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and showed their efficacy in an organ culture model of polycystic kidney disease (PKD) (J. Med. Chem. 2009, 52, 6447-6455). Here, we report related benzopyrimido-pyrrolo-oxazinedione (BPO) CFTR inhibitors. To establish structure-activity relationships and select lead compound(s) with improved potency, metabolic stability, and aqueous solubility compared to the most potent prior compound 8 (PPQ-102, IC(50) ∼ 90 nM), we synthesized 16 PPQ analogues and 11 BPO analogues. The analogues were efficiently synthesized in 5-6 steps and 11-61% overall yield. Modification of 8 by bromine substitution at the 5-position of the furan ring, replacement of the secondary amine with an ether bridge, and carboxylation, gave 6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [4',5':3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid 42 (BPO-27), which fully inhibited CFTR with IC(50) ∼ 8 nM and, compared to 8, had >10-fold greater metabolic stability and much greater polarity/aqueous solubility. In an embryonic kidney culture model of PKD, 42 prevented cyst growth with IC(50) ∼ 100 nM. Benzopyrimido-pyrrolo-oxazinediones such as 42 are potential development candidates for antisecretory therapy of PKD.

  20. Research Progress in Voltage-dependent Anion Channel%电压依赖性阴离子通道的研究进展

    Institute of Scientific and Technical Information of China (English)

    戴琼艳(综述); 段满林(审校)

    2015-01-01

    电压依赖性阴离子通道( VDAC)位于线粒体外膜,形成了线粒体和代谢产物之间的分界,作为“守门员”控制着代谢产物的进出以及线粒体与其他细胞器的对话,同时,VDAC 也是线粒体介导凋亡的关键成员。除了调节线粒体的代谢和产能功能,VDAC 通过与不同配体和蛋白相互作用,作为细胞生存和死亡信号的汇聚点,这些功能可能使VDAC成为合理的新治疗发展的靶点。现就哺乳动物VDAC蛋白(尤其是VDAC1)的结构、功能、与相关蛋白的关系以及 VDAC 参与的疾病作一综述。%Found at the outer mitochondrial membrane , the voltage-dependent anion channel ( VDAC ) forms the main interface between the mitochondria and cellular metabolisms,thus functions as a gatekeeper, controlling cross-talk between mitochondria and the rest of the cell.Meanwhile,VDAC has also been recog-nized as a key protein in mitochondria-mediated apoptosis.In addition to regulating the metabolic and ener-getic functions of mitochondria,VDAC appears to be a convergence point for cell survival and cell death sig-nals via its association with various ligands and proteins ,making it to be a rational target for new therapeu-tics.Here is to make a review of mammalian VDAC ,especially VDAC1,addressing its structure,functions, the relationship between associated proteins and its involvement in several diseases .

  1. The CFTR frameshift mutation 3905insT and its effect at transcript and protein level.

    Science.gov (United States)

    Sanz, Javier; von Känel, Thomas; Schneider, Mircea; Steiner, Bernhard; Schaller, André; Gallati, Sabina

    2010-02-01

    Cystic fibrosis (CF) is one of the most common genetic diseases in the Caucasian population and is characterized by chronic obstructive pulmonary disease, exocrine pancreatic insufficiency, and elevation of sodium and chloride concentrations in the sweat and infertility in men. The disease is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a protein that functions as chloride channel at the apical membrane of different epithelia. Owing to the high genotypic and phenotypic disease heterogeneity, effects and consequences of the majority of the CFTR mutations have not yet been studied. Recently, the frameshift mutation 3905insT was identified as the second most frequent mutation in the Swiss population and found to be associated with a severe phenotype. The frameshift mutation produces a premature termination codon (PTC) in exon 20, and transcripts bearing this PTC are potential targets for degradation through nonsense-mediated mRNA decay (NMD) and/or for exon skipping through nonsense-associated alternative splicing (NAS). Using RT-PCR analysis in lymphocytes and different tissue types from patients carrying the mutation, we showed that the PTC introduced by the mutation does neither elicit a degradation of the mRNA through NMD nor an alternative splicing through NAS. Moreover, immunocytochemical analysis in nasal epithelial cells revealed a significantly reduced amount of CFTR at the apical membrane providing a possible molecular explanation for the more severe phenotype observed in F508del/3905insT compound heterozygotes compared with F508del homozygotes. However, further experiments are needed to elucidate the fate of the 3905insT CFTR in the cell after its biosynthesis.

  2. An image analysis method to quantify CFTR subcellular localization.

    Science.gov (United States)

    Pizzo, Lucilla; Fariello, María Inés; Lepanto, Paola; Aguilar, Pablo S; Kierbel, Arlinet

    2014-08-01

    Aberrant protein subcellular localization caused by mutation is a prominent feature of many human diseases. In Cystic Fibrosis (CF), a recessive lethal disorder that results from dysfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the most common mutation is a deletion of phenylalanine-508 (pF508del). Such mutation produces a misfolded protein that fails to reach the cell surface. To date, over 1900 mutations have been identified in CFTR gene, but only a minority has been analyzed at the protein level. To establish if a particular CFTR variant alters its subcellular distribution, it is necessary to quantitatively determine protein localization in the appropriate cellular context. To date, most quantitative studies on CFTR localization have been based on immunoprecipitation and western blot. In this work, we developed and validated a confocal microscopy-image analysis method to quantitatively examine CFTR at the apical membrane of epithelial cells. Polarized MDCK cells transiently transfected with EGFP-CFTR constructs and stained for an apical marker were used. EGFP-CFTR fluorescence intensity in a region defined by the apical marker was normalized to EGFP-CFTR whole cell fluorescence intensity, rendering "apical CFTR ratio". We obtained an apical CFTR ratio of 0.67 ± 0.05 for wtCFTR and 0.11 ± 0.02 for pF508del. In addition, this image analysis method was able to discriminate intermediate phenotypes: partial rescue of the pF508del by incubation at 27 °C rendered an apical CFTR ratio value of 0.23 ± 0.01. We concluded the method has a good sensitivity and accurately detects milder phenotypes. Improving axial resolution through deconvolution further increased the sensitivity of the system as rendered an apical CFTR ratio of 0.76 ± 0.03 for wild type and 0.05 ± 0.02 for pF508del. The presented procedure is faster and simpler when compared with other available methods and it is therefore suitable as a screening method to identify

  3. Reduced expression of Tis7/IFRD1 protein in murine and human cystic fibrosis airway epithelial cell models homozygous for the F508del-CFTR mutation.

    Science.gov (United States)

    Blanchard, Elise; Marie, Solenne; Riffault, Laure; Bonora, Monique; Tabary, Olivier; Clement, Annick; Jacquot, Jacky

    2011-08-01

    12-O-tetradecanoyl phorbol-13-acetate-induced sequence 7/interferon related development regulator 1 (Tis7/IFRD1) has been recently identified as a modifier gene in lung inflammatory disease severity in patients with cystic fibrosis (CF), based upon its capacity to regulate inflammatory activities in neutrophils. In CF patients, the F508del mutation in the Cftr gene encoding a chloride channel, the CF transmembrane conductance regulator (CFTR) in airway epithelial cells results in an exaggerated inflammatory response of these cells. At present, it is unknown whether the Tis7/IFRD1 gene product is expressed in airway epithelial cells. We therefore investigated the possibility there is an intrinsic alteration in Tis7/IFRD1 protein level in cells lacking CFTR function in tracheal homogenates of F508del-CFTR mice and in a F508del-CFTR human bronchial epithelial cell line (CFBE41o(-) cells). When Tis7/IFRD1 protein was detectable, trachea from F508del-CFTR mice showed a reduction in the level of Tis7/IFRD1 protein compared to wild-type control littermates. A significant reduction of IFRD1 protein level was found in CFBE41o(-) cells compared to normal bronchial epithelial cells 16HBE14o(-). Surprisingly, messenger RNA level of IFRD1 in CFBE41o(-) cells was found elevated. Treating CFBE41o(-) cells with the antioxidant glutathione rescued the IFRD1 protein level closer to control level and also reduced the pro-inflammatory cytokine IL-8 release. This work provides evidence for the first time of reduced level of IFRD1 protein in murine and human F508del-CFTR airway epithelial cell models, possibly mediated in response to oxidative stress which might contribute to the exaggerated inflammatory airway response observed in CF patients homozygous for the F508del mutation.

  4. Resonant spectra of quadrupolar anions

    Science.gov (United States)

    Fossez, K.; Mao, Xingze; Nazarewicz, W.; Michel, N.; Garrett, W. R.; Płoszajczak, M.

    2016-09-01

    In quadrupole-bound anions, an extra electron is attached at a sufficiently large quadrupole moment of a neutral molecule, which is lacking a permanent dipole moment. The nature of the bound states and low-lying resonances of such anions is of interest for understanding the threshold behavior of open quantum systems in general. In this work, we investigate the properties of quadrupolar anions as halo systems, the formation of rotational bands, and the transition from a subcritical to supercritical electric quadrupole moment. We solve the electron-plus-rotor problem using a nonadiabatic coupled-channel formalism by employing the Berggren ensemble, which explicitly contains bound states, narrow resonances, and the scattering continuum. The rotor is treated as a linear triad of point charges with zero monopole and dipole moments and nonzero quadrupole moment. We demonstrate that binding energies and radii of quadrupolar anions strictly follow the scaling laws for two-body halo systems. Contrary to the case of dipolar anions, ground-state band of quadrupolar anions smoothly extend into the continuum, and many rotational bands could be identified above the detachment threshold. We study the evolution of a bound state of an anion as it dives into the continuum at a critical quadrupole moment and we show that the associated critical exponent is α =2 . Everything considered, quadrupolar anions represent a perfect laboratory for the studies of marginally bound open quantum systems.

  5. Nasal Potential Difference in Cystic Fibrosis considering Severe CFTR Mutations

    OpenAIRE

    2015-01-01

    The gold standard for diagnosing cystic fibrosis (CF) is a sweat chloride value above 60 mEq/L. However, this historical and important tool has limitations; other techniques should be studied, including the nasal potential difference (NPD) test. CFTR gene sequencing can identify CFTR mutations, but this method is time-consuming and too expensive to be used in all CF centers. The present study compared CF patients with two classes I-III CFTR mutations (10 patients) (G1), CF patients with class...

  6. Loop diuretics are open-channel blockers of the cystic fibrosis transmembrane conductance regulator with distinct kinetics

    Science.gov (United States)

    Ju, Min; Scott-Ward, Toby S; Liu, Jia; Khuituan, Pissared; Li, Hongyu; Cai, Zhiwei; Husbands, Stephen M; Sheppard, David N

    2014-01-01

    BACKGROUND AND PURPOSE Loop diuretics are widely used to inhibit the Na+, K+, 2Cl− co-transporter, but they also inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Here, we investigated the mechanism of CFTR inhibition by loop diuretics and explored the effects of chemical structure on channel blockade. EXPERIMENTAL APPROACH Using the patch-clamp technique, we tested the effects of bumetanide, furosemide, piretanide and xipamide on recombinant wild-type human CFTR. KEY RESULTS When added to the intracellular solution, loop diuretics inhibited CFTR Cl− currents with potency approaching that of glibenclamide, a widely used CFTR blocker with some structural similarity to loop diuretics. To begin to study the kinetics of channel blockade, we examined the time dependence of macroscopic current inhibition following a hyperpolarizing voltage step. Like glibenclamide, piretanide blockade of CFTR was time and voltage dependent. By contrast, furosemide blockade was voltage dependent, but time independent. Consistent with these data, furosemide blocked individual CFTR Cl− channels with ‘very fast’ speed and drug-induced blocking events overlapped brief channel closures, whereas piretanide inhibited individual channels with ‘intermediate’ speed and drug-induced blocking events were distinct from channel closures. CONCLUSIONS AND IMPLICATIONS Structure–activity analysis of the loop diuretics suggests that the phenoxy group present in bumetanide and piretanide, but absent in furosemide and xipamide, might account for the different kinetics of channel block by locking loop diuretics within the intracellular vestibule of the CFTR pore. We conclude that loop diuretics are open-channel blockers of CFTR with distinct kinetics, affected by molecular dimensions and lipophilicity. PMID:24117047

  7. CHD6 regulates the topological arrangement of the CFTR locus.

    Science.gov (United States)

    Sancho, Ana; Li, SiDe; Paul, Thankam; Zhang, Fan; Aguilo, Francesca; Vashisht, Ajay; Balasubramaniyan, Natarajan; Leleiko, Neal S; Suchy, Frederick J; Wohlschlegel, James A; Zhang, Weijia; Walsh, Martin J

    2015-05-15

    The control of transcription is regulated through the well-coordinated spatial and temporal interactions between distal genomic regulatory elements required for specialized cell-type and developmental gene expression programs. With recent findings CFTR has served as a model to understand the principles that govern genome-wide and topological organization of distal intra-chromosomal contacts as it relates to transcriptional control. This is due to the extensive characterization of the DNase hypersensitivity sites, modification of chromatin, transcription factor binding sites and the arrangement of these sites in CFTR consistent with the restrictive expression in epithelial cell types. Here, we identified CHD6 from a screen among several chromatin-remodeling proteins as a putative epigenetic modulator of CFTR expression. Moreover, our findings of CTCF interactions with CHD6 are consistent with the role described previously for CTCF in CFTR regulation. Our results now reveal that the CHD6 protein lies within the infrastructure of multiple transcriptional complexes, such as the FACT, PBAF, PAF1C, Mediator, SMC/Cohesion and MLL complexes. This model underlies the fundamental role CHD6 facilitates by tethering cis-acting regulatory elements of CFTR in proximity to these multi-subunit transcriptional protein complexes. Finally, we indicate that CHD6 structurally coordinates a three-dimensional stricture between intragenic elements of CFTR bound by several cell-type specific transcription factors, such as CDX2, SOX18, HNF4α and HNF1α. Therefore, our results reveal new insights into the epigenetic regulation of CFTR expression, whereas the manipulation of CFTR gene topology could be considered for treating specific indications of cystic fibrosis and/or pancreatitis.

  8. Assessing the Disease-Liability of Mutations in CFTR

    OpenAIRE

    Ferec, Claude; Cutting, Garry R

    2012-01-01

    Over 1900 mutations have been reported in the cystic fibrosis transmembrane conductance regulator (CFTR), the gene defective in patients with cystic fibrosis. These mutations have been discovered primarily in individuals who have features consistent with the diagnosis of CF. In some cases, it has been recognized that the mutations are not causative of cystic fibrosis but are responsible for disorders with features similar to CF, and these conditions have been termed CFTR-related disorders or ...

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

    Science.gov (United States)

    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

  10. Characterizing responses to CFTR-modulating drugs using rectal organoids derived from subjects with cystic fibrosis.

    Science.gov (United States)

    Dekkers, Johanna F; Berkers, Gitte; Kruisselbrink, Evelien; Vonk, Annelotte; de Jonge, Hugo R; Janssens, Hettie M; Bronsveld, Inez; van de Graaf, Eduard A; Nieuwenhuis, Edward E S; Houwen, Roderick H J; Vleggaar, Frank P; Escher, Johanna C; de Rijke, Yolanda B; Majoor, Christof J; Heijerman, Harry G M; de Winter-de Groot, Karin M; Clevers, Hans; van der Ent, Cornelis K; Beekman, Jeffrey M

    2016-06-22

    Identifying subjects with cystic fibrosis (CF) who may benefit from cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs is time-consuming, costly, and especially challenging for individuals with rare uncharacterized CFTR mutations. We studied CFTR function and responses to two drugs-the prototypical CFTR potentiator VX-770 (ivacaftor/KALYDECO) and the CFTR corrector VX-809 (lumacaftor)-in organoid cultures derived from the rectal epithelia of subjects with CF, who expressed a broad range of CFTR mutations. We observed that CFTR residual function and responses to drug therapy depended on both the CFTR mutation and the genetic background of the subjects. In vitro drug responses in rectal organoids positively correlated with published outcome data from clinical trials with VX-809 and VX-770, allowing us to predict from preclinical data the potential for CF patients carrying rare CFTR mutations to respond to drug therapy. We demonstrated proof of principle by selecting two subjects expressing an uncharacterized rare CFTR genotype (G1249R/F508del) who showed clinical responses to treatment with ivacaftor and one subject (F508del/R347P) who showed a limited response to drug therapy both in vitro and in vivo. These data suggest that in vitro measurements of CFTR function in patient-derived rectal organoids may be useful for identifying subjects who would benefit from CFTR-correcting treatment, independent of their CFTR mutation. PMID:27334259

  11. Assessment of cystic fibrosis transmembrane conductance regulator (CFTR) activity in CFTR-null mice after bone marrow transplantation

    OpenAIRE

    Bruscia, Emanuela M.; Price, Joanna E.; Cheng, Ee-chun; Weiner, Scott; Caputo, Christina; Ferreira, Elisa C.; Egan, Marie E.; Krause, Diane S.

    2006-01-01

    Several studies have demonstrated that bone marrow (BM)-derived cells give rise to rare epithelial cells in the gastrointestinal (GI) and respiratory tracts after BM transplantation into myeloablated recipients. We investigate whether, after transplantation of cystic fibrosis transmembrane conductance regulator (CFTR)-positive BM-derived cells, BM-derived GI and airway epithelial cells can provide CFTR activity in the GI tract and nasal epithelium of recipient cystic fibrosis mice. CFTR−/− mi...

  12. Physiological Adaptation of the Bacterium Lactococcus lactis in Response to the Production of Human CFTR

    NARCIS (Netherlands)

    Steen, Anton; Wiederhold, Elena; Gandhi, Tejas; Breitling, Rainer; Slotboom, Dirk Jan

    2011-01-01

    Biochemical and biophysical characterization of CFTR (the cystic fibrosis transmembrane conductance regulator) is thwarted by difficulties to obtain sufficient quantities of correctly folded and functional protein. Here we have produced human CFTR in the prokaryotic expression host Lactococcus lacti

  13. 囊性纤维化跨膜转运调节体氯离子通道——跨上皮离子转运的多功能引擎%The cystic fibrosis transmembrane conductance regulator Cl-channel: a versatile engine for transepithelial ion transport

    Institute of Scientific and Technical Information of China (English)

    李红宇; 蔡志伟; 陈正豪; 鞠敏; 徐喆; David N.Sheppard

    2007-01-01

    -binding domains (NBDs) and a unique regulatory domain (RD). The MSDs assemble to form a low conductance (6-10 pS) anion-selective pore with deep intracellular and shallow extracellular vestibules separated by a selectivity filter.The NBDs form a head-to-tail dimer with two ATP-binding sites (termed sites 1 and 2) located at the dimer interface. Anion flow through CFTR is gated by the interaction of ATP with sites 1 and 2 powering cycles of NBD dimer association and dissociation and hence, conformational changes in the MSDs that open and close the channel pore. The RD is an unstructured domain with multiple consensus phosphorylation sites, phosphorylation of which stimulates CFTR function by enhancing the interaction of ATP with the NBDs. Tight spatial and temporal control of CFTR activity is achieved by macromolecular signalling complexes in which scaffolding proteins colocalise CFTR and plasma membrane receptors with protein kinases and phosphatases. Moreover, a macromolecular complex composed of CFTR and metabolic enzymes (a CFTR metabolon) permits CFTR activity to be coupled tightly to metabolic pathways within cells so that CFTR inhibition conserves vital energy stores. CFTR is expressed in epithelial tissues throughout the body, lining ducts and tubes. It functions to control the quantity and composition of epithelial secretions by driving either the absorption or secretion of salt and water. Of note, in the respiratory airways CFTR plays an additional important role in host defence.Malfunction of CFTR disrupts transepithelial ion transport leading to a wide spectrum of human disease.

  14. Characterizing responses to CFTR-modulating drugs using rectal organoids derived from subjects with cystic fibrosis

    NARCIS (Netherlands)

    Dekkers, Johanna F; Berkers, Gitte; Kruisselbrink, Evelien; Vonk, Annelotte; de Jonge, Hugo R; Janssens, Hettie M; Bronsveld, Inez; van de Graaf, Eduard A; Nieuwenhuis, Edward E S; Houwen, Roderick H J; Vleggaar, Frank P; Escher, Johanna C; de Rijke, Yolanda B; Majoor, Christof J; Heijerman, Harry G M; de Winter-de Groot, Karin M; Clevers, Hans; van der Ent, Cornelis K; Beekman, Jeffrey M

    2016-01-01

    Identifying subjects with cystic fibrosis (CF) who may benefit from cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs is time-consuming, costly, and especially challenging for individuals with rare uncharacterized CFTR mutations. We studied CFTR function and responses to tw

  15. Analysis of cystic fibrosis–associated P67L CFTR illustrates barriers to personalized therapeutics for orphan diseases

    Science.gov (United States)

    Sabusap, Carleen M.; Wang, Wei; McNicholas, Carmel M.; Chung, W. Joon; Fu, Lianwu; Wen, Hui; Mazur, Marina; Kirk, Kevin L.; Collawn, James F.; Hong, Jeong S.; Sorscher, Eric J.

    2016-01-01

    Emerging knowledge indicates the difficulty in categorizing unusual cystic fibrosis (CF) mutations, with regard to both pathogenic mechanism and theratype. As case in point, we present data concerning P67L mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a defect carried by a small number of individuals with CF and sometimes attributed to a channel conductance abnormality. Findings from our laboratory and others establish that P67L causes protein misfolding, disrupts maturation, confers gating defects, is thermally stable, and exhibits near normal conductance. These results provide one framework by which rare CF alleles such as P67L can be more comprehensively profiled vis-à-vis molecular pathogenesis. We also demonstrate that emerging CF treatments — ivacaftor and lumacaftor — can mediate pronounced pharmacologic activation of P67L CFTR. Infrequent CF alleles are often improperly characterized, in part, due to the small numbers of patients involved. Moreover, access to new personalized treatments among patients with ultra-orphan genotypes has been limited by difficulty arranging phase III clinical trials, and off-label prescribing has been impaired by high drug cost and difficulty arranging third party reimbursement. Rare CFTR mutations such as P67L are emblematic of the challenges to “precision” medicine, including use of the best available mechanistic knowledge to treat patients with unusual forms of disease. PMID:27660821

  16. Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation.

    Science.gov (United States)

    De Stefano, Daniela; Villella, Valeria R; Esposito, Speranza; Tosco, Antonella; Sepe, Angela; De Gregorio, Fabiola; Salvadori, Laura; Grassia, Rosa; Leone, Carlo A; De Rosa, Giuseppe; Maiuri, Maria C; Pettoello-Mantovani, Massimo; Guido, Stefano; Bossi, Anna; Zolin, Anna; Venerando, Andrea; Pinna, Lorenzo A; Mehta, Anil; Bona, Gianni; Kroemer, Guido; Maiuri, Luigi; Raia, Valeria

    2014-01-01

    Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in Cftr(F508del) homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.

  17. The H-loop in the Second Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator is Required for Efficient Chloride Channel Closing

    OpenAIRE

    Kloch, Monika; Milewski, Michał; Nurowska, Ewa; Dworakowska, Beata; Cutting, Garry R; Dołowy, Krzysztof

    2010-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine res...

  18. Transcriptional networks driving enhancer function in the CFTR gene.

    Science.gov (United States)

    Kerschner, Jenny L; Harris, Ann

    2012-09-01

    A critical cis-regulatory element for the CFTR (cystic fibrosis transmembrane conductance regulator) gene is located in intron 11, 100 kb distal to the promoter, with which it interacts. This sequence contains an intestine-selective enhancer and associates with enhancer signature proteins, such as p300, in addition to tissue-specific TFs (transcription factors). In the present study we identify critical TFs that are recruited to this element and demonstrate their importance in regulating CFTR expression. In vitro DNase I footprinting and EMSAs (electrophoretic mobility-shift assays) identified four cell-type-selective regions that bound TFs in vitro. ChIP (chromatin immunoprecipitation) identified FOXA1/A2 (forkhead box A1/A2), HNF1 (hepatocyte nuclear factor 1) and CDX2 (caudal-type homeobox 2) as in vivo trans-interacting factors. Mutation of their binding sites in the intron 11 core compromised its enhancer activity when measured by reporter gene assay. Moreover, siRNA (small interfering RNA)-mediated knockdown of CDX2 caused a significant reduction in endogenous CFTR transcription in intestinal cells, suggesting that this factor is critical for the maintenance of high levels of CFTR expression in these cells. The ChIP data also demonstrate that these TFs interact with multiple cis-regulatory elements across the CFTR locus, implicating a more global role in intestinal expression of the gene.

  19. CFTR and Ca2+ signaling in cystic fibrosis

    Directory of Open Access Journals (Sweden)

    Fabrice eAntigny

    2011-10-01

    Full Text Available Among the diverse physiological functions exerted by calcium signaling in living cells, its role in the regulation of protein biogenesis and trafficking remains incompletely understood. In cystic fibrosis (CF disease the most common CFTR (Cystic Fibrosis Transmembrane conductance Regulator mutation, F508del-CFTR generates a misprocessed protein that is abnormally retained in the endoplasmic reticulum (ER compartment, rapidly degraded by the ubiquitine/proteasome pathway and hence absent at the plasma membrane of CF epithelial cells. Recent studies have demonstrated that intracellular calcium signals consequent to activation of apical G protein-coupled receptors (GPCRs by different agonists are increased in CF airway epithelia. Moreover, the regulation of various intracellular calcium storage compartments, such as ER is also abnormal in CF cells. Although the molecular mechanism to explain this increase remains puzzling in epithelial cells, the F508del-CFTR mutation is proposed to be the origin of abnormal Ca2+ influx linking the calcium signaling to CFTR pathobiology. This article reviews the relationships between CFTR and calcium signaling in the context of the genetic disease cystic fibrosis.

  20. CFTR Modulators: Shedding Light on Precision Medicine for Cystic Fibrosis

    Science.gov (United States)

    Lopes-Pacheco, Miquéias

    2016-01-01

    Cystic fibrosis (CF) is the most common life-threatening monogenic disease afflicting Caucasian people. It affects the respiratory, gastrointestinal, glandular and reproductive systems. The major cause of morbidity and mortality in CF is the respiratory disorder caused by a vicious cycle of obstruction of the airways, inflammation and infection that leads to epithelial damage, tissue remodeling and end-stage lung disease. Over the past decades, life expectancy of CF patients has increased due to early diagnosis and improved treatments; however, these patients still present limited quality of life. Many attempts have been made to rescue CF transmembrane conductance regulator (CFTR) expression, function and stability, thereby overcoming the molecular basis of CF. Gene and protein variances caused by CFTR mutants lead to different CF phenotypes, which then require different treatments to quell the patients’ debilitating symptoms. In order to seek better approaches to treat CF patients and maximize therapeutic effects, CFTR mutants have been stratified into six groups (although several of these mutations present pleiotropic defects). The research with CFTR modulators (read-through agents, correctors, potentiators, stabilizers and amplifiers) has achieved remarkable progress, and these drugs are translating into pharmaceuticals and personalized treatments for CF patients. This review summarizes the main molecular and clinical features of CF, emphasizes the latest clinical trials using CFTR modulators, sheds light on the molecular mechanisms underlying these new and emerging treatments, and discusses the major breakthroughs and challenges to treating all CF patients. PMID:27656143

  1. Nasal Potential Difference in Cystic Fibrosis considering Severe CFTR Mutations

    Directory of Open Access Journals (Sweden)

    Ronny Tah Yen Ng

    2015-01-01

    Full Text Available The gold standard for diagnosing cystic fibrosis (CF is a sweat chloride value above 60 mEq/L. However, this historical and important tool has limitations; other techniques should be studied, including the nasal potential difference (NPD test. CFTR gene sequencing can identify CFTR mutations, but this method is time-consuming and too expensive to be used in all CF centers. The present study compared CF patients with two classes I-III CFTR mutations (10 patients (G1, CF patients with classes IV-VI CFTR mutations (five patients (G2, and 21 healthy subjects (G3. The CF patients and healthy subjects also underwent the NPD test. A statistical analysis was performed using the Mann-Whitney, Kruskal-Wallis, χ2, and Fisher’s exact tests, α=0.05. No differences were observed between the CF patients and healthy controls for the PDMax, Δamiloride, and Δchloride + free + amiloride markers from the NPD test. For the finger value, a difference between G2 and G3 was described. The Wilschanski index values were different between G1 and G3. In conclusion, our data showed that NPD is useful for CF diagnosis when classes I-III CFTR mutations are screened. However, if classes IV-VI are considered, the NPD test showed an overlap in values with healthy subjects.

  2. The lactoperoxidase system links anion transport to host defense in cystic fibrosis.

    Science.gov (United States)

    Conner, Gregory E; Wijkstrom-Frei, Corinne; Randell, Scott H; Fernandez, Vania E; Salathe, Matthias

    2007-01-23

    Chronic respiratory infections in cystic fibrosis result from CFTR channel mutations but how these impair antibacterial defense is less clear. Airway host defense depends on lactoperoxidase (LPO) that requires thiocyanate (SCN-) to function and epithelia use CFTR to concentrate SCN- at the apical surface. To test whether CFTR mutations result in impaired LPO-mediated host defense, CF epithelial SCN- transport was measured. CF epithelia had significantly lower transport rates and did not accumulate SCN- in the apical compartment. The lower CF [SCN-] did not support LPO antibacterial activity. Modeling of airway LPO activity suggested that reduced transport impairs LPO-mediated defense and cannot be compensated by LPO or H2O2 upregulation.

  3. Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant

    Energy Technology Data Exchange (ETDEWEB)

    Atwell, Shane; Brouillette, Christie G.; Conners, Kris; Emtage, Spencer; Gheyi, Tarun; Guggino, William B.; Hendle, Jorg; Hunt, John F.; Lewis, Hal A.; Lu, Frances; Protasevich, Irina I.; Rodgers, Logan A.; Romero, Rich; Wasserman, Stephen R.; Weber, Patricia C.; Wetmore, Diana; Zhang, Feiyu F.; Zhao, Xun (Cystic); (UAB); (JHU); (Columbia); (Lilly)

    2010-04-26

    Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646({Delta}405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-{angstrom} resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The {Delta}F508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

  4. Unraveling the toxicity mechanisms of the herbicide diclofop-methyl in rice: modulation of the activity of key enzymes involved in citrate metabolism and induction of cell membrane anion channels.

    Science.gov (United States)

    Ding, Haiyan; Lu, Haiping; Lavoie, Michel; Xie, Jun; Li, Yali; Lv, Xiaolu; Fu, Zhengwei; Qian, Haifeng

    2014-11-01

    Residual soil concentrations of the herbicide diclofop-methyl (DM) can be toxic to other nontarget plant species, but the toxicity mechanisms at play are not fully understood. In the present study, we analyzed the toxic effect of DM on root growth and metabolism in the rice species Oryza sativa. The results show that a 48-h exposure to a trace level (5 μg/L) of DM inhibits rice root growth by almost 70%. A 48-h exposure to 5 μg/L DM also leads to an ≈2.5-fold increase in citrate synthase (CS) activity (and CS gene transcription) and an ≈2-fold decrease in the citrate lyase gene transcripts, which lead to an increase in the intracellular concentration of citrate and in citrate exudation rate. Addition of a specific inhibitor of cell membrane anion channel, anthracene-9-carboxylic acid, decreased citrate release in the culture, suggesting that DM-induced citrate loss from the cells is mediated by a specific membrane-bound channel protein. This study brings new insights into the key biochemical mechanisms leading to DM toxicity in rice.

  5. A molecular mechanism for aberrantCFTR-dependent HCO3– transport in cystic fibrosis

    OpenAIRE

    Ko, Shigeru B. H.; Shcheynikov, Nikolay; Choi, Joo Young; Luo, Xiang; Ishibashi, Kenichi; Thomas, Philip J.; Kim, Joo Young; Kim, Kyung Hwan; Lee, Min Goo; Naruse, Satoru; Muallem, Shmuel

    2002-01-01

    Aberrant HCO3– transport is a hallmark of cystic fibrosis (CF) and is associated with aberrant Cl–-dependent HCO3– transport by the cystic fibrosis transmembrane conductance regulator (CFTR). We show here that HCO3– current by CFTR cannot account for CFTR-activated HCO3– transport and that CFTR does not activate AE1–AE4. In contrast, CFTR markedly activates Cl– and OH–/HCO3– transport by members of the SLC26 family DRA, SLC26A6 and pendrin. Most notably, the SLC26s are electrogenic transporte...

  6. Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

    Energy Technology Data Exchange (ETDEWEB)

    Marasini, Carlotta, E-mail: marasini@ge.ibf.cnr.it [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy); Galeno, Lauretta; Moran, Oscar [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer CFTR mutations produce cystic fibrosis. Black-Right-Pointing-Pointer Chloride transport depends on the regulatory domain phosphorylation. Black-Right-Pointing-Pointer Regulatory domain is intrinsically disordered. Black-Right-Pointing-Pointer Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and {beta}-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of {alpha}-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two

  7. Partial correction of the CFTR-dependent ABPA mouse model with recombinant adeno-associated virus gene transfer of truncated CFTR gene.

    Science.gov (United States)

    Mueller, Christian; Torrez, Daniel; Braag, Sofia; Martino, Ashley; Clarke, Tracy; Campbell-Thompson, Martha; Flotte, Terence R

    2008-01-01

    Recently, we have developed a model of airway inflammation in a CFTR knockout mouse utilizing Aspergillus fumigatus crude protein extract (Af-cpe) to mimic allergic bronchopulmonary aspergillosis (ABPA) 1, an unusual IgE-mediated hypersensitivity syndrome seen in up to 15% of cystic fibrosis (CF) patients and rarely elsewhere. We hypothesized that replacement of CFTR via targeted gene delivery to airway epithelium would correct aberrant epithelial cytokine signaling and ameliorate the ABPA phenotype in CFTR-deficient (CFTR 489X - /-, FABP-hCFTR + / +) mice. CFTR knockout mice underwent intra-tracheal (IT) delivery of recombinant adeno-associated virus serotype 5 (rAAV5Delta-264CFTR) or rAAV5-GFP at 2.58 x 10(12) viral genomes/mouse. All mice were then sensitized with two serial injections (200 microg) of crude Af antigen via the intra-peritoneal (IP) route. Untreated mice were sensitized without virus exposure. Challenges were performed 2 weeks after final sensitization, using a 0.25% solution containing Aspergillus fumigatus crude protein extract delivered by inhalation on three consecutive days. The rAAV5Delta-264CFTR-treated mice had lower total serum IgE levels (172513 ng/ml +/- 1312) than rAAV5-GFP controls (26 892 ng/ml +/- 3715) (p = 0.037) and non-treated, sensitized controls (24 816 +/- 4219 ng/ml). Serum IgG1 levels also were lower in mice receiving the CFTR vector. Interestingly, splenocytes from rAAV5Delta-264CFTR-treated mice secreted less IL-13, INFg, TNFa, RANTES and GM-CSF after ConA stimulation. Gene therapy with rAAV5Delta-264CFTR attenuated the hyper-IgE response in this reproducible CF mouse model of ABPA, with systemic effects also evident in the cytokine response of stimulated splenocytes. PMID:18023072

  8. CFTR Mutations in Congenital Absence of Vas Deferens

    Directory of Open Access Journals (Sweden)

    Ramin Radpour

    2007-01-01

    Full Text Available A qualitative diagnosis of infertility requires attention to female and male physical abnormalities,endocrine anomalies and genetic conditions that interfere with reproduction. Many genes arelikely to be involved in the complex process of reproduction. Cystic fibrosis (CF incidence variesin different White people populations (a higher incidence of CF is observed in northern–westernEuropean populations than in southern European populations, and therefore the incidence ofcongenital bilateral absence of the vas deferens (CBAVD may also vary in different Whitepeople populations. As CF is mainly observed in White people, hardly any data are available ofCBAVD in non-White people, but frequent polymorphisms such as 5T are observed in mostpopulations. The spectrum and distribution of cystic fibrosis transmembrane conductanceregulator gene (CFTR mutations differs between CBAVD and CF patients, and even comparedwith control individuals. Combinations of particular alleles at several polymorphic loci yieldinsufficient functional CFTR. The combination of the 5T allele in one copy of the CFTR genewith a cystic fibrosis mutation in the other copy is the most common cause of CBAVD in Iran.Because of techniques such as intracytoplasmic sperm injection (ICSI, CBAVD patients are nowable to father children, however such couples have an increased risk of having a child with cysticfibrosis, and therefore genetic testing and counseling should be provided. Around 10% ofobstructive azoospermia is congenital and is due to mutations the CF gene. This paper reviews therelationship of mutations in the CFTR gene with CBAVD.

  9. Insulin-like growth factor 1 (IGF-1 enhances the protein expression of CFTR.

    Directory of Open Access Journals (Sweden)

    Ha Won Lee

    Full Text Available Low levels of insulin-like growth factor 1 (IGF-1 have been observed in the serum of cystic fibrosis (CF patients. However, the effects of low serum IGF-1 on the cystic fibrosis transmembrane conductance regulator (CFTR, whose defective function is the primary cause of cystic fibrosis, have not been studied. Here, we show in human cells that IGF-1 increases the steady-state levels of mature wildtype CFTR in a CFTR-associated ligand (CAL- and TC10-dependent manner; moreover, IGF-1 increases CFTR-mediated chloride transport. Using an acceptor photobleaching fluorescence resonance energy transfer (FRET assay, we have confirmed the binding of CAL and CFTR in the Golgi. We also show that CAL overexpression inhibits forskolin-induced increases in the cell-surface expression of CFTR. We found that IGF-1 activates TC10, and active TC10 alters the functional association between CAL and CFTR. Furthermore, IGF-1 and active TC10 can reverse the CAL-mediated reduction in the cell-surface expression of CFTR. IGF-1 does not increase the expression of ΔF508 CFTR, whose processing is arrested in the ER. This finding is consistent with our observation that IGF-1 alters the functional interaction of CAL and CFTR in the Golgi. However, when ΔF508 CFTR is rescued with low temperature or the corrector VRT-325 and proceeds to the Golgi, IGF-1 can increase the expression of the rescued ΔF508 CFTR. Our data support a model indicating that CAL-CFTR binding in the Golgi inhibits CFTR trafficking to the cell surface, leading CFTR to the degradation pathway instead. IGF-1-activated TC10 changes the interaction of CFTR and CAL, allowing CFTR to progress to the plasma membrane. These findings offer a potential strategy using a combinational treatment of IGF-1 and correctors to increase the post-Golgi expression of CFTR in cystic fibrosis patients bearing the ΔF508 mutation.

  10. Insulin-like growth factor 1 (IGF-1) enhances the protein expression of CFTR.

    Science.gov (United States)

    Lee, Ha Won; Cheng, Jie; Kovbasnjuk, Olga; Donowitz, Mark; Guggino, William B

    2013-01-01

    Low levels of insulin-like growth factor 1 (IGF-1) have been observed in the serum of cystic fibrosis (CF) patients. However, the effects of low serum IGF-1 on the cystic fibrosis transmembrane conductance regulator (CFTR), whose defective function is the primary cause of cystic fibrosis, have not been studied. Here, we show in human cells that IGF-1 increases the steady-state levels of mature wildtype CFTR in a CFTR-associated ligand (CAL)- and TC10-dependent manner; moreover, IGF-1 increases CFTR-mediated chloride transport. Using an acceptor photobleaching fluorescence resonance energy transfer (FRET) assay, we have confirmed the binding of CAL and CFTR in the Golgi. We also show that CAL overexpression inhibits forskolin-induced increases in the cell-surface expression of CFTR. We found that IGF-1 activates TC10, and active TC10 alters the functional association between CAL and CFTR. Furthermore, IGF-1 and active TC10 can reverse the CAL-mediated reduction in the cell-surface expression of CFTR. IGF-1 does not increase the expression of ΔF508 CFTR, whose processing is arrested in the ER. This finding is consistent with our observation that IGF-1 alters the functional interaction of CAL and CFTR in the Golgi. However, when ΔF508 CFTR is rescued with low temperature or the corrector VRT-325 and proceeds to the Golgi, IGF-1 can increase the expression of the rescued ΔF508 CFTR. Our data support a model indicating that CAL-CFTR binding in the Golgi inhibits CFTR trafficking to the cell surface, leading CFTR to the degradation pathway instead. IGF-1-activated TC10 changes the interaction of CFTR and CAL, allowing CFTR to progress to the plasma membrane. These findings offer a potential strategy using a combinational treatment of IGF-1 and correctors to increase the post-Golgi expression of CFTR in cystic fibrosis patients bearing the ΔF508 mutation.

  11. In vivo pharmacology and antidiarrheal efficacy of a thiazolidinone CFTR inhibitor in rodents.

    Science.gov (United States)

    Sonawane, N D; Muanprasat, Chatchai; Nagatani, Ray; Song, Yuanlin; Verkman, A S

    2005-01-01

    A small-molecule inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTR(inh)-172), reduces enterotoxin-induced intestinal fluid secretion in rodents. Here, we study CFTR(inh)-172 pharmacology and antidiarrheal efficacy in rodents using (14)C-labeled CFTR(inh)-172, liquid chromatography/mass spectrometry, and a closed intestinal loop model of fluid secretion. CFTR(inh)-172 was cleared primarily by renal glomerular filtration without chemical modification. CFTR(inh)-172 accumulated in liver within 5 min after intravenous infusion in mice, and was concentrated fivefold in bile over blood. At 30-240 min, CFTR(inh)-172 was found mainly in liver, intestine, and kidney, with little detectable in the brain, heart, skeletal muscle, or lung. Pharmacokinetic analysis in rats following intravenous bolus infusion showed a distribution volume of 770 mL with redistribution and elimination half-times of 0.14 h and 10.3 h, respectively. CFTR(inh)-172 was stable in hepatic microsomes. Closed-loop studies in mice indicated that a single intraperitoneal injection of 20 microg CFTR(inh)-172 inhibited fluid accumulation at 6 h after cholera toxin by >90% in duodenum and jejunum, approximately 60% in ileum and accumulation of CFTR(inh)-172 account for its efficacy as an antidiarrheal. PMID:15761937

  12. Optimal correction of distinct CFTR folding mutants in rectal cystic fibrosis organoids.

    Science.gov (United States)

    Dekkers, Johanna F; Gogorza Gondra, Ricardo A; Kruisselbrink, Evelien; Vonk, Annelotte M; Janssens, Hettie M; de Winter-de Groot, Karin M; van der Ent, Cornelis K; Beekman, Jeffrey M

    2016-08-01

    Small-molecule therapies that restore defects in cystic fibrosis transmembrane conductance regulator (CFTR) gating (potentiators) or trafficking (correctors) are being developed for cystic fibrosis (CF) in a mutation-specific fashion. Options for pharmacological correction of CFTR-p.Phe508del (F508del) are being extensively studied but correction of other trafficking mutants that may also benefit from corrector treatment remains largely unknown.We studied correction of the folding mutants CFTR-p.Phe508del, -p.Ala455Glu (A455E) and -p.Asn1303Lys (N1303K) by VX-809 and 18 other correctors (C1-C18) using a functional CFTR assay in human intestinal CF organoids.Function of both CFTR-p.Phe508del and -p.Ala455Glu was enhanced by a variety of correctors but no residual or corrector-induced activity was associated with CFTR-p.Asn1303Lys. Importantly, VX-809-induced correction was most dominant for CFTR-p.Phe508del, while correction of CFTR-p.Ala455Glu was highest by a subgroup of compounds called bithiazoles (C4, C13, C14 and C17) and C5.These data support the development of mutation-specific correctors for optimal treatment of different CFTR trafficking mutants, and identify C5 and bithiazoles as the most promising compounds for correction of CFTR-p.Ala455Glu. PMID:27103391

  13. A novel fluorescent sensor for measurement of CFTR function by flow cytometry.

    Science.gov (United States)

    Vijftigschild, Lodewijk A W; van der Ent, Cornelis K; Beekman, Jeffrey M

    2013-06-01

    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis. CFTR-dependent iodide transport measured by fluorescent quenching of ectopically expressed halide-sensitive yellow fluorescent protein (YFP) is widely being used to study CFTR function by microscopy or plate readers. Since YFP fluorescence in these systems is dependent on YFP expression levels and iodide concentration, differences in sensor expression level between experimental units are normalized at the start of each experiment. To allow accurate measurement of CFTR function by flow cytometry, we reasoned that co-expression of an iodide insensitive fluorescent protein would allow for normalization of sensor expression levels and more accurate quantification of CFTR function. Our data indicated that dsRed and mKate fluorescence are iodide insensitive, and we determined an optimal format for co-expression of these fluorescent proteins with halide-sensitive YFP. We showed using microscopy that ratiometric measurement (YFP/mKate) corrects for differences in sensor expression levels. Ratiometric measurements were essential to accurately measure CFTR function by flow cytometry that we here describe for the first time. Mixing of wild type or mutant CFTR expressing cells indicated that addition of approximately 10% of wild type CFTR expressing cells could be distinguished by ratiometric YFP quenching. Flow cytometric ratiometric YFP quenching also allowed us to study CFTR mutants associated with differential residual function upon ectopic expression. Compared with conventional plate-bound CFTR function assays, the flow cytometric approach described here can be used to study CFTR function in suspension cells. It may be further adapted to study CFTR function in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function by cell sorting.

  14. Chloride channels in stroke

    Institute of Scientific and Technical Information of China (English)

    Ya-ping ZHANG; Hao ZHANG; Dayue Darrel DUAN

    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 (Clˉ) channels in vascular remodeling and stroke.At least three Clˉ channel genes are expressed in VSMCs:1) the TMEM16A (or Ano1),which may encode the calcium-activated Clˉ channels (CACCs); 2) the CLC-3 Clˉ channel and Clˉ/H+ antiporter,which is closely related to the volume-regulated Clˉ channels (VRCCs); and 3) the cystic fibrosis transmembrane conductance regulator (CFTR),which encodes the PKA-and PKC-activated Clˉ 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,Clˉ current mediated by gammaaminobutyric acid (GABA) receptor has also been implicated a role in ischemic neuron death.This review focuses on the functional roles of Clˉ channels in the development of stroke and provides a perspective on the future directions for research and the potential to develop Clˉ channels as new targets for the prevention and treatment of stroke.

  15. Anion Channel Inhibitor NPPB-Inhibited Fluoride Accumulation in Tea Plant (Camellia sinensis Is Related to the Regulation of Ca2+, CaM and Depolarization of Plasma Membrane Potential

    Directory of Open Access Journals (Sweden)

    Xian-Chen Zhang

    2016-01-01

    Full Text Available Tea plant is known to be a hyper-accumulator of fluoride (F. Over-intake of F has been shown to have adverse effects on human health, e.g., dental fluorosis. Thus, understanding the mechanisms fluoride accumulation and developing potential approaches to decrease F uptake in tea plants might be beneficial for human health. In the present study, we found that pretreatment with the anion channel inhibitor NPPB reduced F accumulation in tea plants. Simultaneously, we observed that NPPB triggered Ca2+ efflux from mature zone of tea root and significantly increased relative CaM in tea roots. Besides, pretreatment with the Ca2+ chelator (EGTA and CaM antagonists (CPZ and TFP suppressed NPPB-elevated cytosolic Ca2+ fluorescence intensity and CaM concentration in tea roots, respectively. Interestingly, NPPB-inhibited F accumulation was found to be significantly alleviated in tea plants pretreated with either Ca2+ chelator (EGTA or CaM antagonists (CPZ and TFP. In addition, NPPB significantly depolarized membrane potential transiently and we argue that the net Ca2+ and H+ efflux across the plasma membrane contributed to the restoration of membrane potential. Overall, our results suggest that regulation of Ca2+-CaM and plasma membrane potential depolarization are involved in NPPB-inhibited F accumulation in tea plants.

  16. Anion Channel Inhibitor NPPB-Inhibited Fluoride Accumulation in Tea Plant (Camellia sinensis) Is Related to the Regulation of Ca²⁺, CaM and Depolarization of Plasma Membrane Potential.

    Science.gov (United States)

    Zhang, Xian-Chen; Gao, Hong-Jian; Yang, Tian-Yuan; Wu, Hong-Hong; Wang, Yu-Mei; Zhang, Zheng-Zhu; Wan, Xiao-Chun

    2016-01-01

    Tea plant is known to be a hyper-accumulator of fluoride (F). Over-intake of F has been shown to have adverse effects on human health, e.g., dental fluorosis. Thus, understanding the mechanisms fluoride accumulation and developing potential approaches to decrease F uptake in tea plants might be beneficial for human health. In the present study, we found that pretreatment with the anion channel inhibitor NPPB reduced F accumulation in tea plants. Simultaneously, we observed that NPPB triggered Ca(2+) efflux from mature zone of tea root and significantly increased relative CaM in tea roots. Besides, pretreatment with the Ca(2+) chelator (EGTA) and CaM antagonists (CPZ and TFP) suppressed NPPB-elevated cytosolic Ca(2+) fluorescence intensity and CaM concentration in tea roots, respectively. Interestingly, NPPB-inhibited F accumulation was found to be significantly alleviated in tea plants pretreated with either Ca(2+) chelator (EGTA) or CaM antagonists (CPZ and TFP). In addition, NPPB significantly depolarized membrane potential transiently and we argue that the net Ca(2+) and H⁺ efflux across the plasma membrane contributed to the restoration of membrane potential. Overall, our results suggest that regulation of Ca(2+)-CaM and plasma membrane potential depolarization are involved in NPPB-inhibited F accumulation in tea plants. PMID:26742036

  17. Anion Channel Inhibitor NPPB-Inhibited Fluoride Accumulation in Tea Plant (Camellia sinensis) Is Related to the Regulation of Ca2+, CaM and Depolarization of Plasma Membrane Potential

    Science.gov (United States)

    Zhang, Xian-Chen; Gao, Hong-Jian; Yang, Tian-Yuan; Wu, Hong-Hong; Wang, Yu-Mei; Zhang, Zheng-Zhu; Wan, Xiao-Chun

    2016-01-01

    Tea plant is known to be a hyper-accumulator of fluoride (F). Over-intake of F has been shown to have adverse effects on human health, e.g., dental fluorosis. Thus, understanding the mechanisms fluoride accumulation and developing potential approaches to decrease F uptake in tea plants might be beneficial for human health. In the present study, we found that pretreatment with the anion channel inhibitor NPPB reduced F accumulation in tea plants. Simultaneously, we observed that NPPB triggered Ca2+ efflux from mature zone of tea root and significantly increased relative CaM in tea roots. Besides, pretreatment with the Ca2+ chelator (EGTA) and CaM antagonists (CPZ and TFP) suppressed NPPB-elevated cytosolic Ca2+ fluorescence intensity and CaM concentration in tea roots, respectively. Interestingly, NPPB-inhibited F accumulation was found to be significantly alleviated in tea plants pretreated with either Ca2+ chelator (EGTA) or CaM antagonists (CPZ and TFP). In addition, NPPB significantly depolarized membrane potential transiently and we argue that the net Ca2+ and H+ efflux across the plasma membrane contributed to the restoration of membrane potential. Overall, our results suggest that regulation of Ca2+-CaM and plasma membrane potential depolarization are involved in NPPB-inhibited F accumulation in tea plants. PMID:26742036

  18. Anion Channel Inhibitor NPPB-Inhibited Fluoride Accumulation in Tea Plant (Camellia sinensis) Is Related to the Regulation of Ca²⁺, CaM and Depolarization of Plasma Membrane Potential.

    Science.gov (United States)

    Zhang, Xian-Chen; Gao, Hong-Jian; Yang, Tian-Yuan; Wu, Hong-Hong; Wang, Yu-Mei; Zhang, Zheng-Zhu; Wan, Xiao-Chun

    2016-01-05

    Tea plant is known to be a hyper-accumulator of fluoride (F). Over-intake of F has been shown to have adverse effects on human health, e.g., dental fluorosis. Thus, understanding the mechanisms fluoride accumulation and developing potential approaches to decrease F uptake in tea plants might be beneficial for human health. In the present study, we found that pretreatment with the anion channel inhibitor NPPB reduced F accumulation in tea plants. Simultaneously, we observed that NPPB triggered Ca(2+) efflux from mature zone of tea root and significantly increased relative CaM in tea roots. Besides, pretreatment with the Ca(2+) chelator (EGTA) and CaM antagonists (CPZ and TFP) suppressed NPPB-elevated cytosolic Ca(2+) fluorescence intensity and CaM concentration in tea roots, respectively. Interestingly, NPPB-inhibited F accumulation was found to be significantly alleviated in tea plants pretreated with either Ca(2+) chelator (EGTA) or CaM antagonists (CPZ and TFP). In addition, NPPB significantly depolarized membrane potential transiently and we argue that the net Ca(2+) and H⁺ efflux across the plasma membrane contributed to the restoration of membrane potential. Overall, our results suggest that regulation of Ca(2+)-CaM and plasma membrane potential depolarization are involved in NPPB-inhibited F accumulation in tea plants.

  19. Aza-Bambusurils En Route to Anion Transporters.

    Science.gov (United States)

    Singh, Mandeep; Solel, Ephrath; Keinan, Ehud; Reany, Ofer

    2016-06-20

    Previous calculations of anion binding with various bambusuril analogs predicted that the replacement of oxygen by nitrogen atoms to produce semiaza-bambus[6]urils would award these new cavitands with multiple anion binding properties. This study validates the hypothesis by efficient synthesis, crystallography, thermogravimetric analysis and calorimetry. These unique host molecules are easily accessible from the corresponding semithio-bambusurils in a one-pot reaction, which converts a single anion receptor into a potential anion channel. Solid-state structures exhibit simultaneous accommodation of three anions, linearly positioned within the cavity along the main symmetry axis. The ability to hold anions at a short distance of about 4 Å is reminiscent of natural chloride channels in E. coli, which exhibit similar distances between their adjacent anion binding sites. The calculated transition-state energy for double-anion movement through the channel suggests that although these host-guest complexes are thermodynamically stable they enjoy high kinetic flexibility to render them efficient anion channels. PMID:27225332

  20. Impact of heterozygote CFTR Mutations in COPD patients with Chronic Bronchitis

    OpenAIRE

    Raju, S. Vamsee; Tate, Jody H; Peacock, Sandra KG; Fang, Ping; Oster, Robert A.; Dransfield, Mark T.; Steven M Rowe

    2014-01-01

    Background Cigarette smoking causes Chronic Obstructive Pulmonary Disease (COPD), the 3rd leading cause of death in the U.S. CFTR ion transport dysfunction has been implicated in COPD pathogenesis, and is associated with chronic bronchitis. However, susceptibility to smoke induced lung injury is variable and the underlying genetic contributors remain unclear. We hypothesized that presence of CFTR mutation heterozygosity may alter susceptibility to cigarette smoke induced CFTR dysfunction. Con...

  1. Anion exchange membrane

    Science.gov (United States)

    Verkade, John G; Wadhwa, Kuldeep; Kong, Xueqian; Schmidt-Rohr, Klaus

    2013-05-07

    An anion exchange membrane and fuel cell incorporating the anion exchange membrane are detailed in which proazaphosphatrane and azaphosphatrane cations are covalently bonded to a sulfonated fluoropolymer support along with anionic counterions. A positive charge is dispersed in the aforementioned cations which are buried in the support to reduce the cation-anion interactions and increase the mobility of hydroxide ions, for example, across the membrane. The anion exchange membrane has the ability to operate at high temperatures and in highly alkaline environments with high conductivity and low resistance.

  2. Osteoblast CFTR inactivation reduces differentiation and osteoprotegerin expression in a mouse model of cystic fibrosis-related bone disease.

    Directory of Open Access Journals (Sweden)

    Michael S Stalvey

    Full Text Available Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF. CF-related bone disease (CFBD is characterized by uncoupled bone turnover--impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR, the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr-/- mouse model. In the murine calvarial organ culture assay, Cftr-/- calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+ littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr-/- compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl mRNA was detected, significantly less osteoprotegerin (Opg was expressed in Cftr-/- compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr-/- murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt

  3. Advancing clinical development pathways for new CFTR modulators in cystic fibrosis.

    Science.gov (United States)

    Mayer-Hamblett, Nicole; Boyle, Michael; VanDevanter, Donald

    2016-05-01

    Cystic fibrosis (CF) is a life-shortening genetic disease affecting approximately 70,000 individuals worldwide. Until recently, drug development efforts have emphasised therapies treating downstream signs and symptoms resulting from the underlying CF biological defect: reduced function of the CF transmembrane conductance regulator (CFTR) protein. The current CF drug development landscape has expanded to include therapies that enhance CFTR function by either restoring wild-type CFTR protein expression or increasing (modulating) the function of mutant CFTR proteins in cells. To date, two systemic small-molecule CFTR modulators have been evaluated in pivotal clinical trials in individuals with CF and specific mutant CFTR genotypes that have led to regulatory review and/or approval. Advances in the discovery of CFTR modulators as a promising new class of therapies have been impressive, yet work remains to develop highly effective, disease-modifying modulators for individuals of all CF genotypes. The objectives of this review are to outline the challenges and opportunities in drug development created by systemic genotype-specific CFTR modulators, highlight the advantages of sweat chloride as an established biomarker of CFTR activity to streamline early-phase development and summarise options for later phase clinical trial designs that respond to the adoption of approved genotype-specific modulators into standard of care. An optimal development framework will be needed to move the most promising therapies efficiently through the drug development pipeline and ultimately deliver efficacious and safe therapies to all individuals with CF.

  4. Discovery of novel potent ΔF508-CFTR correctors that target the nucleotide binding domain.

    Science.gov (United States)

    Odolczyk, Norbert; Fritsch, Janine; Norez, Caroline; Servel, Nathalie; da Cunha, Melanie Faria; Bitam, Sara; Kupniewska, Anna; Wiszniewski, Ludovic; Colas, Julien; Tarnowski, Krzysztof; Tondelier, Danielle; Roldan, Ariel; Saussereau, Emilie L; Melin-Heschel, Patricia; Wieczorek, Grzegorz; Lukacs, Gergely L; Dadlez, Michal; Faure, Grazyna; Herrmann, Harald; Ollero, Mario; Becq, Frédéric; Zielenkiewicz, Piotr; Edelman, Aleksander

    2013-10-01

    The deletion of Phe508 (ΔF508) in the first nucleotide binding domain (NBD1) of CFTR is the most common mutation associated with cystic fibrosis. The ΔF508-CFTR mutant is recognized as improperly folded and targeted for proteasomal degradation. Based on molecular dynamics simulation results, we hypothesized that interaction between ΔF508-NBD1 and housekeeping proteins prevents ΔF508-CFTR delivery to the plasma membrane. Based on this assumption we applied structure-based virtual screening to identify new low-molecular-weight compounds that should bind to ΔF508-NBD1 and act as protein-protein interaction inhibitors. Using different functional assays for CFTR activity, we demonstrated that in silico-selected compounds induced functional expression of ΔF508-CFTR in transfected HeLa cells, human bronchial CF cells in primary culture, and in the nasal epithelium of homozygous ΔF508-CFTR mice. The proposed compounds disrupt keratin8-ΔF508-CFTR interaction in ΔF508-CFTR HeLa cells. Structural analysis of ΔF508-NBD1 in the presence of these compounds suggests their binding to NBD1. We conclude that our strategy leads to the discovery of new compounds that are among the most potent correctors of ΔF508-CFTR trafficking defect known to date.

  5. Cholic acid induces a Cftr dependent biliary secretion and liver growth response in mice.

    Directory of Open Access Journals (Sweden)

    Frank A J A Bodewes

    Full Text Available The cause of Cystic fibrosis liver disease (CFLD, is unknown. It is well recognized that hepatic exposure to hydrophobic bile salts is associated with the development of liver disease. For this reason, we hypothesize that, CFTR dependent variations, in the hepatic handling of hydrophobic bile salts, are related to the development CFLD. To test our hypothesis we studied, in Cftr-/- and control mice, bile production, bile composition and liver pathology, in normal feeding condition and during cholate exposure, either acute (intravenous or chronic (three weeks via the diet. In Cftr-/- and control mice the basal bile production was comparable. Intravenous taurocholate increased bile production to the same extent in Cftr-/- and control mice. However, chronic cholate exposure increased the bile flow significantly less in Cftr-/- mice than in controls, together with significantly higher biliary bile salt concentration in Cftr-/- mice. Prolonged cholate exposure, however, did not induce CFLD like pathology in Cftr-/- mice. Chronic cholate exposure did induce a significant increase in liver mass in controls that was absent in Cftr-/- mice. Chronic cholate administration induces a cystic fibrosis-specific hepatobiliary phenotype, including changes in bile composition. These changes could not be associated with CFLD like pathological changes in CF mouse livers. However, chronic cholate administration induces liver growth in controls that is absent in Cftr-/- mice. Our findings point to an impaired adaptive homeotrophic liver response to prolonged hydrophobic bile salt exposure in CF conditions.

  6. β2-Adrenergic receptor agonists activate CFTR in intestinal organoids and subjects with cystic fibrosis.

    Science.gov (United States)

    Vijftigschild, Lodewijk A W; Berkers, Gitte; Dekkers, Johanna F; Zomer-van Ommen, Domenique D; Matthes, Elizabeth; Kruisselbrink, Evelien; Vonk, Annelotte; Hensen, Chantal E; Heida-Michel, Sabine; Geerdink, Margot; Janssens, Hettie M; van de Graaf, Eduard A; Bronsveld, Inez; de Winter-de Groot, Karin M; Majoor, Christof J; Heijerman, Harry G M; de Jonge, Hugo R; Hanrahan, John W; van der Ent, Cornelis K; Beekman, Jeffrey M

    2016-09-01

    We hypothesized that people with cystic fibrosis (CF) who express CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations associated with residual function may benefit from G-protein coupled receptor (GPCR)-targeting drugs that can activate and enhance CFTR function.We used intestinal organoids to screen a GPCR-modulating compound library and identified β2-adrenergic receptor agonists as the most potent inducers of CFTR function.β2-Agonist-induced organoid swelling correlated with the CFTR genotype, and could be induced in homozygous CFTR-F508del organoids and highly differentiated primary CF airway epithelial cells after rescue of CFTR trafficking by small molecules. The in vivo response to treatment with an oral or inhaled β2-agonist (salbutamol) in CF patients with residual CFTR function was evaluated in a pilot study. 10 subjects with a R117H or A455E mutation were included and showed changes in the nasal potential difference measurement after treatment with oral salbutamol, including a significant improvement of the baseline potential difference of the nasal mucosa (+6.35 mV, pCFTR activation when administered ex vivo to organoids.This proof-of-concept study suggests that organoids can be used to identify drugs that activate CFTR function in vivo and to select route of administration. PMID:27471203

  7. SNaPshot Assay for the Detection of the Most Common CFTR Mutations in Infertile Men

    OpenAIRE

    Predrag Noveski; Svetlana Madjunkova; Marija Mircevska; Toso Plaseski; Vanja Filipovski; Dijana Plaseska-Karanfilska

    2014-01-01

    Congenital bilateral absence of vas deferens (CBAVD) is the most common CFTR-related disorder (CFTR-RD) that explains about 1-2% of the male infertility cases. Controversial data have been published regarding the involvement of CFTR mutations in infertile men with non-obstructive azoospermia and oligozoospermia. Here, we describe single base extension (SNaPshot) assay for detection of 11 common CFTR mutations: F508del, G542X, N1303K, 621+1G->T, G551D, R553X, R1162X, W1282X, R117H, 2184insA an...

  8. SNaPshot assay for the detection of the most common CFTR mutations in infertile men.

    Directory of Open Access Journals (Sweden)

    Predrag Noveski

    Full Text Available Congenital bilateral absence of vas deferens (CBAVD is the most common CFTR-related disorder (CFTR-RD that explains about 1-2% of the male infertility cases. Controversial data have been published regarding the involvement of CFTR mutations in infertile men with non-obstructive azoospermia and oligozoospermia. Here, we describe single base extension (SNaPshot assay for detection of 11 common CFTR mutations: F508del, G542X, N1303K, 621+1G->T, G551D, R553X, R1162X, W1282X, R117H, 2184insA and 1717-1G->A and IVS8polyT variants. The assay was validated on 50 previously genotyped samples and was used to screen a total of 369 infertile men with different impairment of spermatogenesis and 136 fertile controls. Our results show that double heterozygosity of cystic fibrosis (CF and CFTR-related disorder (CFTR-RD mutations are found in a high percentage (22.7% of infertile men with obstructive azoospermia, but not in other studied groups of infertile men. The SNaPshot assay described here is an inexpensive, fast and robust method for primary screening of the most common CFTR mutations both in patients with classical CF and CFTR-RD. It can contribute to better understanding of the role of CFTR mutations in impaired spermatogenesis, ultimately leading to improved management of infertile men.

  9. Anions in Cometary Comae

    Science.gov (United States)

    Charnley, Steven B.

    2011-01-01

    The presence of negative ions (anions) in cometary comae is known from Giotto mass spectrometry of IP/Halley. The anions 0-, OH-, C-, CH- and CN- have been detected, as well as unidentified anions with masses 22-65 and 85-110 amu (Chaizy et al. 1991). Organic molecular anions are known to have a significant impact on the charge balance of interstellar clouds and circumstellar envelopes and have been shown to act as catalysts for the gas-phase synthesis of larger hydrocarbon molecules in the ISM, but their importance in cometary comae has not yet been explored. We present details of the first attempt to model the chemistry of anions in cometary comae. Based on the combined chemical and hydro dynamical model of Rodgers & Charnley (2002), we investigate the role of large carbon-chain anions in cometary coma chemistry. We calculate the effects of these anions on coma thermodynamics, charge balance and examine their impact on molecule formation.

  10. Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating.

    Science.gov (United States)

    Szollosi, Andras; Muallem, Daniella R; Csanády, László; Vergani, Paola

    2011-06-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460-1348 and 460-1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains

  11. CFTR, PRSS1 und SPINK1 Varianten bei chronischer Pankreatitis

    OpenAIRE

    Rosendahl, Jonas

    2010-01-01

    Chronic pancreatitis (CP) is defined as a continuing or relapsing inflammatory disease of the pancreas, leading to endocrine and/or exocrine insufficiency and irreversible morphological changes. Currently, it is thought that the imbalance of pancreatic proteases and their inhibitors results in the development of chronic pancreatitis. Until now, genetic variations of four genes have been associated to chronic pancreatitis: PRSS1, PRSS2, SPINK1 and CFTR. Since direct DNA-sequencing has becom...

  12. Mechanisms of CFTR folding at the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Soo Jung Kim

    2012-12-01

    Full Text Available In the past decade much has been learned about how CFTR folds and misfolds as the etiologic cause of cystic fibrosis (CF. CFTR folding is complex and hierarchical, takes place in multiple cellular compartments and physical environments, and involves several large networks of folding machineries. Insertion of transmembrane (TM segments into the endoplasmic reticulum (ER membrane and tertiary folding of cytosolic domains begin cotranslationally as the nascent polypeptide emerges from the ribosome, whereas posttranslational folding establishes critical domain-domain contacts needed to form a physiologically stable structure. Within the membrane, N- and C-terminal TM helices are sorted into bundles that project from the cytosol to form docking sites for nucleotide binding domains, NBD1 and NBD2, which in turn form a sandwich dimer for ATP binding. While tertiary folding is required for domain assembly, proper domain assembly also reciprocally affects folding of individual domains analogous to a jigsaw puzzle wherein the structure of each interlocking piece influences its neighbors. Superimposed on this process is an elaborate proteostatic network of cellular chaperones and folding machineries that facilitate the timing and coordination of specific folding steps in and across the ER membrane. While the details of this process require further refinement, we finally have a useful framework to understand key folding defect(s caused by ∆F508 that provides a molecular target(s for the next generation of CFTR small molecule correctors aimed at the specific defect present in the majority of CF patients.

  13. CFTR gene mutations in isolated chronic obstructive pulmonary disease

    Energy Technology Data Exchange (ETDEWEB)

    Pignatti, P.F.; Bombien, C.; Marigo, C. [and others

    1994-09-01

    In order to identify a possible hereditary predisposition to the development of chronic obstructive pulmonary disease (COPD), we have looked for the presence of cystic fibrosis transmembrane regulator (CFTR) gene DNA sequence modifications in 28 unrelated patients with no signs of cystic fibrosis. The known mutations in Italian CF patients, as well as the most frequent worldwide CF mutations, were investigated. In addition, a denaturing gradient gel electrophoresis analysis of about half of the coding sequence of the gene in 56 chromosomes from the patients and in 102 chromosomes from control individuals affected by other pulmonary diseases and from normal controls was performed. Nine different CFTR gene mutations and polymorphisms were found in seven patients, a highly significant increase over controls. Two of the patients were compound heterozygotes. Two frequent CF mutations were detected: deletion F508 and R117H; two rare CF mutations: R1066C and 3667ins4; and five CF sequence variants: R75Q (which was also described as a disease-causing mutation in male sterility cases due to the absence of the vasa deferentia), G576A, 2736 A{r_arrow}G, L997F, and 3271+18C{r_arrow}T. Seven (78%) of the mutations are localized in transmembrane domains. Six (86%) of the patients with defined mutations and polymorphisms had bronchiectasis. These results indicate that CFTR gene mutations and sequence alterations may be involved in the etiopathogenesis of some cases of COPD.

  14. Effects of irradiation and W11-a12 on anion-selective channel of mouse peritoneal macrophage%电离辐射对小鼠腹腔巨噬细胞阴离子通道活动的影响及康复新对其的作用

    Institute of Scientific and Technical Information of China (English)

    舒崇湘; 叶本兰; 程天民; 萧家思

    2001-01-01

    目的 探讨电离辐射对小鼠腹腔巨噬细胞阴离子通道活动的影响及康复新对其的作用。方法 采用电生理膜片钳离子单通道记录方法。结果 用调理的zymosan刺激腹腔巨噬细胞后,阴离子通道开放的阳性率在受照射组显著减少,且受刺激至通道开放的时间间隔显著延长。受照射组较正常对照组通道开放概率显著降低、开放时间常数缩短而关闭时间常数延长,康复新对正常对照组离子通道活动的影响不显著,但可使照射组的通道开放有一定程度的加强。结论 电离辐射抑制巨噬细胞膜上阴离子通道的开放可能是其抑制巨噬细胞功能的一个重要途径。康复新可减弱电离辐射的这一抑制作用。%Objective To study the effects of irradiation and W11-a12,a kind of repair-promoting drug,on anion-selective channel in membranes of mouse peritoneal macrophage. Methods The activity of anion-selective channel was recorded from cell-attached patches with patch clamp techniques. Results The effects of irradiation on anion-selective channel in membranes of peritoneal macrophage included:①decreasing the mean number of activated channels by the presence of zymosan; ②prolonging the mean time from stimulus to the opening of channels; ③depressing the opening of channels by decreasing open-state probability,shortening open-time and prolonging close-time. The effects of irradiation could partly be depressed by W11-a12. Conclusion Irradiation will depress the anion-selective channel of peritoneal macrophage, which may be an important way to depress the function of macrophage.

  15. Disrupted interaction between CFTR and AF-6/afadin aggravates malignant phenotypes of colon cancer.

    Science.gov (United States)

    Sun, Ting Ting; Wang, Yan; Cheng, Hong; Xiao, Hu Zhang; Xiang, Juan Juan; Zhang, Jie Ting; Yu, Siu Bun Sydney; Martin, Tracey Amanda; Ye, Lin; Tsang, Lai Ling; Jiang, Wen Guo; Xiaohua, Jiang; Chan, Hsiao Chang

    2014-03-01

    How mutations or dysfunction of CFTR may increase the risk of malignancies in various tissues remains an open question. Here we report the interaction between CFTR and an adherens junction molecule, AF-6/afadin, and its involvement in the development of colon cancer. We have found that CFTR and AF-6/afadin are co-localized at the cell-cell contacts and physically interact with each other in colon cancer cell lines. Knockdown of CFTR results in reduced epithelial tightness and enhanced malignancies, with increased degradation and reduced stability of AF-6/afadin protein. The enhanced invasive phenotype of CFTR-knockdown cells can be completely reversed by either AF-6/afadin over-expression or ERK inhibitor, indicating the involvement of AF-6/MAPK pathway. More interestingly, the expression levels of CFTR and AF-6/afadin are significantly downregulated in human colon cancer tissues and lower expression of CFTR and/or AF-6/afadin is correlated with poor prognosis of colon cancer patients. The present study has revealed a previously unrecognized interaction between CFTR and AF-6/afadin that is involved in the pathogenesis of colon cancer and indicated the potential of the two as novel markers of metastasis and prognostic predictors for human colon cancer.

  16. Mechanism-based corrector combination restores Delta F508-CFTR folding and function

    NARCIS (Netherlands)

    Okiyoneda, Tsukasa; Veit, Guido; Dekkers, Johanna F.; Bagdany, Miklos; Soya, Naoto; Xu, Haijin; Roldan, Ariel; Verkman, Alan S.; Kurth, Mark; Simon, Agnes; Hegedus, Tamas; Beekman, Jeffrey M.; Lukacs, Gergely L.

    2013-01-01

    The most common cystic fibrosis mutation, Delta F508 in nucleotide binding domain 1 (NBD1), impairs cystic fibrosis transmembrane conductance regulator (CFTR)-coupled domain folding, plasma membrane expression, function and stability. VX-809, a promising investigational corrector of Delta F508-CFTR

  17. Conformationally rigid histone deacetylase inhibitors correct DF508-CFTR protein function

    DEFF Research Database (Denmark)

    Vickers, Chris J.; Olsen, Christian Adam; Hutt, Darren M.;

    2011-01-01

    Histone deacetylase (HDAC) inhibitors have shown partial efficacy toward correcting cystic fibrosis transmembrane conductance regulator (CFTR) protein function in ΔF508- CFTR models. While current treatment options for CF generally concentrate on disease symptoms such as management of inflammation...

  18. Defective CFTR-dependent CREB activation results in impaired spermatogenesis and azoospermia.

    Directory of Open Access Journals (Sweden)

    Wen Ming Xu

    Full Text Available Cystic fibrosis (CF is the most common life-limiting recessive genetic disease among Caucasians caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR with over 95% male patients infertile. However, whether CFTR mutations could affect spermatogenesis and result in azoospermia remains an open question. Here we report compromised spermatogenesis, with significantly reduced testicular weight and sperm count, and decreased cAMP-responsive element binding protein (CREB expression in the testes of CFTR knockout mice. The involvement of CFTR in HCO(3 (- transport and the expression of the HCO(3 (- sensor, soluble adenylyl cyclase (sAC, are demonstrated for the first time in the primary culture of rat Sertoli cells. Inhibition of CFTR or depletion of HCO(3 (- could reduce FSH-stimulated, sAC-dependent cAMP production and phosphorylation of CREB, the key transcription factor in spermatogenesis. Decreased CFTR and CREB expression are also observed in human testes with azoospermia. The present study reveals a previously undefined role of CFTR and sAC in regulating the cAMP-CREB signaling pathway in Sertoli cells, defect of which may result in impaired spermatogenesis and azoospermia. Altered CFTR-sAC-cAMP-CREB functional loop may also underline the pathogenesis of various CF-related diseases.

  19. Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR

    DEFF Research Database (Denmark)

    Wainwright, Claire E; Elborn, J Stuart; Ramsey, Bonnie W;

    2015-01-01

    BACKGROUND: Cystic fibrosis is a life-limiting disease that is caused by defective or deficient cystic fibrosis transmembrane conductance regulator (CFTR) protein activity. Phe508del is the most common CFTR mutation. METHODS: We conducted two phase 3, randomized, double-blind, placebo......-controlled studies that were designed to assess the effects of lumacaftor (VX-809), a CFTR corrector, in combination with ivacaftor (VX-770), a CFTR potentiator, in patients 12 years of age or older who had cystic fibrosis and were homozygous for the Phe508del CFTR mutation. In both studies, patients were randomly......-ivacaftor and placebo groups. The rate of discontinuation due to an adverse event was 4.2% among patients who received lumacaftor-ivacaftor versus 1.6% among those who received placebo. CONCLUSIONS: These data show that lumacaftor in combination with ivacaftor provided a benefit for patients with cystic fibrosis...

  20. Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the CFTR gene

    DEFF Research Database (Denmark)

    Phylactides, M.; Rowntree, R.; Nuthall, H.;

    2002-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) gene shows a complex pattern of expression, with temporal and spatial regulation that is not accounted for by elements in the promoter. One approach to identifying the regulatory elements for CFTR is the mapping of DNase I...... hypersensitive sites (DHS) within the locus. We previously identified at least 12 clusters of DHS across the CFTR gene and here further evaluate DHS in introns 2,3,10,16,17a, 18, 20 and 21 to assess their functional importance in regulation of CFTR gene expression. Transient transfections of enhancer....../reporter constructs containing the DHS regions showed that those in introns 20 and 21 augmented the activity of the CFTR promoter. Structural analysis of the DNA sequence at the DHS suggested that only the one intron 21 might be caused by inherent DNA structures. Cell specificity of the DHS suggested a role for the...

  1. Ileal mucosal bile acid absorption is increased in Cftr knockout mice

    Directory of Open Access Journals (Sweden)

    Somasundaram Sivagurunathan

    2001-10-01

    Full Text Available Abstract Background Excessive loss of bile acids in stool has been reported in patients with cystic fibrosis. Some data suggest that a defect in mucosal bile acid transport may be the mechanism of bile acid malabsorption in these individuals. However, the molecular basis of this defect is unknown. This study examines the expression of the ileal bile acid transporter protein (IBAT and rates of diffusional (sodium independent and active (sodium dependent uptake of the radiolabeled bile acid taurocholate in mice with targeted disruption of the cftr gene. Methods Wild-type, heterozygous cftr (+/- and homozygous cftr (-/- mice were studied. Five one-cm segments of terminal ileum were excised, everted and mounted onto thin stainless steel rods and incubated in buffer containing tracer 3H-taurocholate. Simultaneously, adjacent segments of terminal ileum were taken and processed for immunohistochemistry and Western blots using an antibody against the IBAT protein. Results In all ileal segments, taurocholate uptake rates were fourfold higher in cftr (-/- and two-fold higher in cftr (+/- mice compared to wild-type mice. Passive uptake was not significantly higher in cftr (-/- mice than in controls. IBAT protein was comparably increased. Immuno-staining revealed that the greatest increases occurred in the crypts of cftr (-/- animals. Conclusions In the ileum, IBAT protein densities and taurocholate uptake rates are elevated in cftr (-/- mice > cftr (+/- > wild-type mice. These findings indicate that bile acid malabsorption in cystic fibrosis is not caused by a decrease in IBAT activity at the brush border. Alternative mechanisms are proposed, such as impaired bile acid uptake caused by the thick mucus barrier in the distal small bowel, coupled with a direct negative regulatory role for cftr in IBAT function.

  2. Cystic fibrosis transmembrane conductance regulator protein (CFTR) expression in the developing human brain: comparative immunohistochemical study between patients with normal and mutated CFTR.

    Science.gov (United States)

    Marcorelles, Pascale; Friocourt, Gaëlle; Uguen, Arnaud; Ledé, Françoise; Férec, Claude; Laquerrière, Annie

    2014-11-01

    Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein has recently been shown to be expressed in the human adult central nervous system (CNS). As CFTR expression has also been documented during embryonic development in several organs, such as the respiratory tract, the intestine and the male reproductive system, suggesting a possible role during development we decided to investigate the expression of CFTR in the human developing CNS. In addition, as some, although rare, neurological symptoms have been reported in patients with CF, we compared the expression of normal and mutated CFTR at several fetal stages. Immunohistochemistry was performed on brain and spinal cord samples of foetuses between 13 and 40 weeks of gestation and compared with five patients with cystic fibrosis (CF) of similar ages. We showed in this study that CFTR is only expressed in neurons and has an early and widespread distribution during development. Although we did not observe any cerebral abnormality in patients with CF, we observed a slight delay in the maturation of several brain structures. We also observed different expression and localization of CFTR depending on the brain structure or the cell maturation stage. Our findings, along with a literature review on the neurological phenotypes of patients with CF, suggest that this gene may play previously unsuspected roles in neuronal maturation or function.

  3. Cholangiocyte anion exchange and biliary bicarbonate excretion

    Institute of Scientific and Technical Information of China (English)

    Jesús M Banales; Jesús Prieto; Juan F Medina

    2006-01-01

    Primary canalicular bile undergoes a process of fluidization and alkalinization along the biliary tract that is influenced by several factors including hormones, innervation/neuropeptides, and biliary constituents. Theexcretion of bicarbonate at both the canaliculi and the bile ducts is an important contributor to the generation of the so-called bile-salt independent flow. Bicarbonate is secreted from hepatocytes and cholangiocytes through parallel mechanisms which involve chloride efflux through activation of Cl- channels, and further bicarbonate secretion via AE2/SLC4A2-mediated Cl-/HCO3-exchange. Glucagon and secretin are two relevant hormones which seem to act very similarly in their target cells (hepatocytes for the former and cholangiocytes for the latter). These hormones interact with their specific G protein-coupled receptors, causing increases in intracellular levels of cAMP and activation of cAMP-dependent Cl- and HCO3- secretory mechanisms. Both hepatocytes and cholangiocytes appear to have cAMP-responsive intracellular vesicles in which AE2/SLC4A2 colocalizes with cell specific Cl- channels (CFTR in cholangiocytes and not yet determined in hepatocytes) and aquaporins (AQP8 in hepatocytes and AQP1 in cholangiocytes). cAMP-induced coordinated trafficking of these vesicles to either canalicular or cholangiocyte lumenal membranes and further exocytosis results in increased osmotic forces and passive movement of water with net bicarbonate-rich hydrocholeresis.

  4. Molecular mechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins

    International Nuclear Information System (INIS)

    The initial step in reduced glutathione (GSH) turnover in all mammalian cells is its transport across the plasma membrane into the extracellular space; however, the mechanisms of GSH transport are not clearly defined. GSH export is required for the delivery of its constituent amino acids to other tissues, detoxification of drugs, metals, and other reactive compounds of both endogenous and exogenous origin, protection against oxidant stress, and secretion of hepatic bile. Recent studies indicate that some members of the multidrug resistance-associated protein (MRP/CFTR or ABCC) family of ATP-binding cassette (ABC) proteins, as well as some members of the organic anion transporting polypeptide (OATP or SLC21A) family of transporters contribute to this process. In particular, five of the 12 members of the MRP/CFTR family appear to mediate GSH export from cells namely, MRP1, MRP2, MRP4, MRP5, and CFTR. Additionally, two members of the OATP family, rat Oatp1 and Oatp2, have been identified as GSH transporters. For the Oatp1 transporter, efflux of GSH may provide the driving force for the uptake of extracellular substrates. In humans, OATP-B and OATP8 do not appear to transport GSH; however, other members of this family have yet to be characterized in regards to GSH transport. In yeast, the ABC proteins Ycf1p and Bpt1p transport GSH from the cytosol into the vacuole, whereas Hgt1p mediates GSH uptake across the plasma membrane. Because transport is a key step in GSH homeostasis and is intimately linked to its biological functions, GSH export proteins are likely to modulate essential cellular functions

  5. Correlation of apical fluid-regulating channel proteins with lung function in human COPD lungs.

    Directory of Open Access Journals (Sweden)

    Runzhen Zhao

    Full Text Available Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC, cystic fibrosis transmembrane conductance regulator (CFTR, and aquaporin 5 (AQP5 proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI and II (ATII-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3 was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD.

  6. Interference with ubiquitination in CFTR modifies stability of core glycosylated and cell surface pools.

    Science.gov (United States)

    Lee, Seakwoo; Henderson, Mark J; Schiffhauer, Eric; Despanie, Jordan; Henry, Katherine; Kang, Po Wei; Walker, Douglas; McClure, Michelle L; Wilson, Landon; Sorscher, Eric J; Zeitlin, Pamela L

    2014-07-01

    It is recognized that both wild-type and mutant CFTR proteins undergo ubiquitination at multiple lysines in the proteins and in one or more subcellular locations. We hypothesized that ubiquitin is added to specific sites in wild-type CFTR to stabilize it and at other sites to signal for proteolysis. Mass spectrometric analysis of wild-type CFTR identified ubiquitinated lysines 68, 710, 716, 1041, and 1080. We demonstrate that the ubiquitinated K710, K716, and K1041 residues stabilize wild-type CFTR, protecting it from proteolysis. The polyubiquitin linkage is predominantly K63. N-tail mutants, K14R and K68R, lead to increased mature band CCFTR, which can be augmented by proteasomal (but not lysosomal) inhibition, allowing trafficking to the surface. The amount of CFTR in the K1041R mutant was drastically reduced and consisted of bands A/B, suggesting that the site in transmembrane 10 (TM10) was critical to further processing beyond the proteasome. The K1218R mutant increases total and cell surface CFTR, which is further accumulated by proteasomal and lysosomal inhibition. Thus, ubiquitination at residue 1218 may destabilize wild-type CFTR in both the endoplasmic reticulum (ER) and recycling pools. Small molecules targeting the region of residue 1218 to block ubiquitination or to preserving structure at residues 710 to 716 might be protein sparing for some forms of cystic fibrosis.

  7. Unravelling druggable signalling networks that control F508del-CFTR proteostasis.

    Science.gov (United States)

    Hegde, Ramanath Narayana; Parashuraman, Seetharaman; Iorio, Francesco; Ciciriello, Fabiana; Capuani, Fabrizio; Carissimo, Annamaria; Carrella, Diego; Belcastro, Vincenzo; Subramanian, Advait; Bounti, Laura; Persico, Maria; Carlile, Graeme; Galietta, Luis; Thomas, David Y; Di Bernardo, Diego; Luini, Alberto

    2015-12-23

    Cystic fibrosis (CF) is caused by mutations in CF transmembrane conductance regulator (CFTR). The most frequent mutation (F508del-CFTR) results in altered proteostasis, that is, in the misfolding and intracellular degradation of the protein. The F508del-CFTR proteostasis machinery and its homeostatic regulation are well studied, while the question whether 'classical' signalling pathways and phosphorylation cascades might control proteostasis remains barely explored. Here, we have unravelled signalling cascades acting selectively on the F508del-CFTR folding-trafficking defects by analysing the mechanisms of action of F508del-CFTR proteostasis regulator drugs through an approach based on transcriptional profiling followed by deconvolution of their gene signatures. Targeting multiple components of these signalling pathways resulted in potent and specific correction of F508del-CFTR proteostasis and in synergy with pharmacochaperones. These results provide new insights into the physiology of cellular proteostasis and a rational basis for developing effective pharmacological correctors of the F508del-CFTR defect.

  8. CFTR Deletion in Mouse Testis Induces VDAC1 Mediated Inflammatory Pathway Critical for Spermatogenesis

    Science.gov (United States)

    Huijuan, Liao; Jiang, Xie; Ming, Yang; Huaqin, Sun; Wenming, Xu

    2016-01-01

    Cystic fibrosis is the most common genetic disease among Caucasians and affects tissues including lung, pancreas and reproductive tracts. It has been shown that Endoplasmic Reticulum (ER) stress and heat shock response are two major deregulated functional modules related to CFTR dysfunction. To identify the impact of CFTR deletion during spermatogenesis, we examined the expression of spermiogenesis-related genes in the testis of CFTR mutant mice (CF mice). We confirmed expression changes of MSY2, a germ cell specific RNA binding protein, resulting from deletion of CFTR in testis. Furthermore, real time PCR and Western blot results showed that an inflammatory response was activated in CF mice testis, as reflected by the altered expression of cytokines. We demonstrate for the first time that expression of MSY2 is decreased in CF mice. Our results suggest that CFTR deletion in testis influences inflammatory responses and these features are likely to be due to the unique environment of the seminiferous tubule during the spermatogenesis process. The current study also suggests avenues to understand the pathophysiology of CFTR during spermatogenesis and provides targets for the possible treatment of CFTR-related infertility. PMID:27483469

  9. CFTR Deletion in Mouse Testis Induces VDAC1 Mediated Inflammatory Pathway Critical for Spermatogenesis.

    Science.gov (United States)

    Yan, Chen; Lang, Qin; Huijuan, Liao; Jiang, Xie; Ming, Yang; Huaqin, Sun; Wenming, Xu

    2016-01-01

    Cystic fibrosis is the most common genetic disease among Caucasians and affects tissues including lung, pancreas and reproductive tracts. It has been shown that Endoplasmic Reticulum (ER) stress and heat shock response are two major deregulated functional modules related to CFTR dysfunction. To identify the impact of CFTR deletion during spermatogenesis, we examined the expression of spermiogenesis-related genes in the testis of CFTR mutant mice (CF mice). We confirmed expression changes of MSY2, a germ cell specific RNA binding protein, resulting from deletion of CFTR in testis. Furthermore, real time PCR and Western blot results showed that an inflammatory response was activated in CF mice testis, as reflected by the altered expression of cytokines. We demonstrate for the first time that expression of MSY2 is decreased in CF mice. Our results suggest that CFTR deletion in testis influences inflammatory responses and these features are likely to be due to the unique environment of the seminiferous tubule during the spermatogenesis process. The current study also suggests avenues to understand the pathophysiology of CFTR during spermatogenesis and provides targets for the possible treatment of CFTR-related infertility. PMID:27483469

  10. Physiological adaptation of the bacterium Lactococcus lactis in response to the production of human CFTR.

    Science.gov (United States)

    Steen, Anton; Wiederhold, Elena; Gandhi, Tejas; Breitling, Rainer; Slotboom, Dirk Jan

    2011-07-01

    Biochemical and biophysical characterization of CFTR (the cystic fibrosis transmembrane conductance regulator) is thwarted by difficulties to obtain sufficient quantities of correctly folded and functional protein. Here we have produced human CFTR in the prokaryotic expression host Lactococcus lactis. The full-length protein was detected in the membrane of the bacterium, but the yields were too low (proteins) for in vitro functional and structural characterization, and induction of the expression of CFTR resulted in growth arrest. We used isobaric tagging for relative and absolute quantitation based quantitative proteomics to find out why production of CFTR in L. lactis was problematic. Protein abundances in membrane and soluble fractions were monitored as a function of induction time, both in CFTR expression cells and in control cells that did not express CFTR. Eight hundred and forty six proteins were identified and quantified (35% of the predicted proteome), including 163 integral membrane proteins. Expression of CFTR resulted in an increase in abundance of stress-related proteins (e.g. heat-shock and cell envelope stress), indicating the presence of misfolded proteins in the membrane. In contrast to the reported consequences of membrane protein overexpression in Escherichia coli, there were no indications that the membrane protein insertion machinery (Sec) became overloaded upon CFTR production in L. lactis. Nutrients and ATP became limiting in the control cells as the culture entered the late exponential and stationary growth phases but this did not happen in the CFTR expressing cells, which had stopped growing upon induction. The different stress responses elicited in E. coli and L. lactis upon membrane protein production indicate that different strategies are needed to overcome low expression yields and toxicity.

  11. Intrinsic anion oxidation potentials.

    Science.gov (United States)

    Johansson, Patrik

    2006-11-01

    Anions of lithium battery salts have been investigated by electronic structure calculations with the objective to find a computational measure to correlate with the observed (in)stability of nonaqueous lithium battery electrolytes vs oxidation often encountered in practice. Accurate prediction of intrinsic anion oxidation potentials is here made possible by computing the vertical free energy difference between anion and neutral radical (Delta Gv) and further strengthened by an empirical correction using only the anion volume as a parameter. The 6-311+G(2df,p) basis set, the VSXC functional, and the C-PCM SCRF algorithm were used. The Delta Gv calculations can be performed using any standard computational chemistry software. PMID:17078600

  12. Cysteine string protein promotes proteasomal degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) by increasing its interaction with the C terminus of Hsp70-interacting protein and promoting CFTR ubiquitylation.

    Science.gov (United States)

    Schmidt, Béla Z; Watts, Rebecca J; Aridor, Meir; Frizzell, Raymond A

    2009-02-13

    Cysteine string protein (Csp) is a J-domain-containing protein whose overexpression blocks the exit of cystic fibrosis transmembrane conductance regulator (CFTR) from the endoplasmic reticulum (ER). Another method of blocking ER exit, the overexpression of Sar1-GTP, however, yielded twice as much immature CFTR compared with Csp overexpression. This finding suggested that Csp not only inhibits CFTR ER exit but also facilitates the degradation of immature CFTR. This was confirmed by treatment with a proteasome inhibitor, which returned the level of immature CFTR to that found in cells expressing Sar1-GTP only. CspH43Q, which does not interact with Hsc70/Hsp70 efficiently, did not promote CFTR degradation, suggesting that the pro-degradative effect of Csp requires Hsc70/Hsp70 binding/activation. In agreement with this, Csp overexpression increased the amount of Hsc70/Hsp70 co-immunoprecipitated with CFTR, whereas overexpression of CspH43Q did not. The Hsc70/Hsp70 binding partner C terminus of Hsp70-interacting protein (CHIP) can target CFTR for proteasome-mediated degradation. Csp overexpression also increased the amount of CHIP co-immunoprecipitated with CFTR. In addition, CHIP interacted directly with Csp, which was confirmed by in vitro binding experiments. Csp overexpression also increased CFTR ubiquitylation and reduced the half-life of immature CFTR. These findings indicate that Csp not only regulates the exit of CFTR from the ER, but that this action is accompanied by Hsc70/Hsp70 and CHIP-mediated CFTR degradation.

  13. Molecular basis of potassium channels in pancreatic duct epithelial cells

    DEFF Research Database (Denmark)

    Hayashi, M.; Novak, Ivana

    2013-01-01

    Potassium channels regulate excitability, epithelial ion transport, proliferation, and apoptosis. In pancreatic ducts, K channels hyperpolarize the membrane potential and provide the driving force for anion secretion. This review focuses on the molecular candidates of functional K channels...

  14. Retinal pigment epithelial function: a role for CFTR?

    Science.gov (United States)

    Blaug, Sasha; Quinn, Richard; Quong, Judy; Jalickee, Stephen; Miller, Sheldon S

    2003-01-01

    In the vertebrate eye, the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE) are separated by a small extracellular (subretinal) space whose volume and chemical composition varies in the light and dark. Light onset triggers relatively fast (ms) retinal responses and much slower voltage and resistance changes (s to min) at the apical and basolateral membranes of the RPE. Two of these slow RPE responses, the fast oscillation (FO) and the light peak, are measured clinically as part of the electrooculogram (EOG). Both EOG responses are mediated in part by apical and basolateral membranes proteins that form a pathway for the movement of salt and osmotically obliged fluid across the RPE, from retina to choroid. This transport pathway serves to control the volume and chemical composition of the subretinal and choroidal extracellular spaces. In human fetal RPE, we have identified one of these proteins, the cystic fibrosis transmembrane conductance regulator (CFTR) by RT-PCR, immunolocalization, and electrophysiological techniques. Evidence is presented to suggest that the FO component of the EOG is mediated directly or indirectly by CFTR. PMID:12675485

  15. The "Goldilocks Effect" in Cystic Fibrosis: identification of a lung phenotype in the cftr knockout and heterozygous mouse

    Directory of Open Access Journals (Sweden)

    Bates Jason HT

    2004-07-01

    Full Text Available Abstract Background Cystic Fibrosis is a pleiotropic disease in humans with primary morbidity and mortality associated with a lung disease phenotype. However, knockout in the mouse of cftr, the gene whose mutant alleles are responsible for cystic fibrosis, has previously failed to produce a readily, quantifiable lung phenotype. Results Using measurements of pulmonary mechanics, a definitive lung phenotype was demonstrated in the cftr-/- mouse. Lungs showed decreased compliance and increased airway resistance in young animals as compared to cftr+/+ littermates. These changes were noted in animals less than 60 days old, prior to any long term inflammatory effects that might occur, and are consistent with structural differences in the cftr-/- lungs. Surprisingly, the cftr+/- animals exhibited a lung phenotype distinct from either the homozygous normal or knockout genotypes. The heterozygous mice showed increased lung compliance and decreased airway resistance when compared to either homozygous phenotype, suggesting a heterozygous advantage that might explain the high frequency of this mutation in certain populations. Conclusions In the mouse the gene dosage of cftr results in distinct differences in pulmonary mechanics of the adult. Distinct phenotypes were demonstrated in each genotype, cftr-/-, cftr +/-, and cftr+/+. These results are consistent with a developmental role for CFTR in the lung.

  16. Side chain and backbone contributions of Phe508 to CFTR folding

    Energy Technology Data Exchange (ETDEWEB)

    Thibodeau, Patrick H.; Brautigam, Chad A.; Machius, Mischa; Thomas, Philip J. (U. of Texas-SMED)

    2010-12-07

    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

  17. Rescue of F508del-CFTR by RXR motif inactivation triggers proteome modulation associated with the unfolded protein response.

    Science.gov (United States)

    Gomes-Alves, Patrícia; Couto, Francisco; Pesquita, Cátia; Coelho, Ana V; Penque, Deborah

    2010-04-01

    F508del-CFTR, the most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, disrupts intracellular trafficking leading to cystic fibrosis (CF). The trafficking defect of F508del-CFTR can be rescued by simultaneous inactivation of its four RXR motifs (4RK). Proteins involved in the F508del-CFTR trafficking defect and/or rescue are therefore potential CF therapeutic targets. We sought to identify these proteins by investigating differential proteome modulation in BHK cells over-expressing wt-CFTR, F508del-CFTR or the revertant F508del/4RK-CFTR. By 2-dimensional electrophoresis-based proteomics and western blot approaches we demonstrated that over-expression of F508del/4RK-CFTR modulates the expression of a large number of proteins, many of which are reported interactors of CFTR and/or 14-3-3 with potential roles in CFTR trafficking. GRP78/BiP, a marker of ER stress and unfolded protein response (UPR), is up-regulated in cells over-expressing either F508del-CFTR or F598del/4RK-CFTR. However, over-expression of F508del/4RK-CFTR induces the up-regulation of many other UPR-associated proteins (e.g. GRP94, PDI, GRP75/mortalin) and, interestingly, the down-regulation of proteasome components associated with CFTR degradation, such as the proteasome activator PA28 (PSME2) and COP9 signalosome (COPS5/CSN5). Moreover, the F508del-CFTR-induced proteostasis imbalance, which involves some heat shock chaperones (e.g. HSP72/Hpa2), ER-EF-hand Ca(2+)-binding proteins (calumenin) and the proteasome activator PA28 (PSME2), tends to be 'restored', i.e., in BHK cells over-expressing F508del/4RK-CFTR those proteins tend to have expression levels similar to the wild-type ones. These findings indicate that a particular cellular environment orchestrated by the UPR contributes to and/or is compatible with F508del/4RK-CFTR rescue.

  18. Rescue of F508del-CFTR by RXR motif inactivation triggers proteome modulation associated with the unfolded protein response.

    Science.gov (United States)

    Gomes-Alves, Patrícia; Couto, Francisco; Pesquita, Cátia; Coelho, Ana V; Penque, Deborah

    2010-04-01

    F508del-CFTR, the most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, disrupts intracellular trafficking leading to cystic fibrosis (CF). The trafficking defect of F508del-CFTR can be rescued by simultaneous inactivation of its four RXR motifs (4RK). Proteins involved in the F508del-CFTR trafficking defect and/or rescue are therefore potential CF therapeutic targets. We sought to identify these proteins by investigating differential proteome modulation in BHK cells over-expressing wt-CFTR, F508del-CFTR or the revertant F508del/4RK-CFTR. By 2-dimensional electrophoresis-based proteomics and western blot approaches we demonstrated that over-expression of F508del/4RK-CFTR modulates the expression of a large number of proteins, many of which are reported interactors of CFTR and/or 14-3-3 with potential roles in CFTR trafficking. GRP78/BiP, a marker of ER stress and unfolded protein response (UPR), is up-regulated in cells over-expressing either F508del-CFTR or F598del/4RK-CFTR. However, over-expression of F508del/4RK-CFTR induces the up-regulation of many other UPR-associated proteins (e.g. GRP94, PDI, GRP75/mortalin) and, interestingly, the down-regulation of proteasome components associated with CFTR degradation, such as the proteasome activator PA28 (PSME2) and COP9 signalosome (COPS5/CSN5). Moreover, the F508del-CFTR-induced proteostasis imbalance, which involves some heat shock chaperones (e.g. HSP72/Hpa2), ER-EF-hand Ca(2+)-binding proteins (calumenin) and the proteasome activator PA28 (PSME2), tends to be 'restored', i.e., in BHK cells over-expressing F508del/4RK-CFTR those proteins tend to have expression levels similar to the wild-type ones. These findings indicate that a particular cellular environment orchestrated by the UPR contributes to and/or is compatible with F508del/4RK-CFTR rescue. PMID:20044041

  19. Anion-π catalysis.

    Science.gov (United States)

    Zhao, Yingjie; Beuchat, César; Domoto, Yuya; Gajewy, Jadwiga; Wilson, Adam; Mareda, Jiri; Sakai, Naomi; Matile, Stefan

    2014-02-01

    The introduction of new noncovalent interactions to build functional systems is of fundamental importance. We here report experimental and theoretical evidence that anion-π interactions can contribute to catalysis. The Kemp elimination is used as a classical tool to discover conceptually innovative catalysts for reactions with anionic transition states. For anion-π catalysis, a carboxylate base and a solubilizer are covalently attached to the π-acidic surface of naphthalenediimides. On these π-acidic surfaces, transition-state stabilizations up to ΔΔGTS = 31.8 ± 0.4 kJ mol(-1) are found. This value corresponds to a transition-state recognition of KTS = 2.7 ± 0.5 μM and a catalytic proficiency of 3.8 × 10(5) M(-1). Significantly increasing transition-state stabilization with increasing π-acidity of the catalyst, observed for two separate series, demonstrates the existence of "anion-π catalysis." In sharp contrast, increasing π-acidity of the best naphthalenediimide catalysts does not influence the more than 12 000-times weaker substrate recognition (KM = 34.5 ± 1.6 μM). Together with the disappearance of Michaelis-Menten kinetics on the expanded π-surfaces of perylenediimides, this finding supports that contributions from π-π interactions are not very important for anion-π catalysis. The linker between the π-acidic surface and the carboxylate base strongly influences activity. Insufficient length and flexibility cause incompatibility with saturation kinetics. Moreover, preorganizing linkers do not improve catalysis much, suggesting that the ideal positioning of the carboxylate base on the π-acidic surface is achieved by intramolecular anion-π interactions rather than by an optimized structure of the linker. Computational simulations are in excellent agreement with experimental results. They confirm, inter alia, that the stabilization of the anionic transition states (but not the neutral ground states) increases with the π-acidity of the

  20. Anion-π catalysis.

    Science.gov (United States)

    Zhao, Yingjie; Beuchat, César; Domoto, Yuya; Gajewy, Jadwiga; Wilson, Adam; Mareda, Jiri; Sakai, Naomi; Matile, Stefan

    2014-02-01

    The introduction of new noncovalent interactions to build functional systems is of fundamental importance. We here report experimental and theoretical evidence that anion-π interactions can contribute to catalysis. The Kemp elimination is used as a classical tool to discover conceptually innovative catalysts for reactions with anionic transition states. For anion-π catalysis, a carboxylate base and a solubilizer are covalently attached to the π-acidic surface of naphthalenediimides. On these π-acidic surfaces, transition-state stabilizations up to ΔΔGTS = 31.8 ± 0.4 kJ mol(-1) are found. This value corresponds to a transition-state recognition of KTS = 2.7 ± 0.5 μM and a catalytic proficiency of 3.8 × 10(5) M(-1). Significantly increasing transition-state stabilization with increasing π-acidity of the catalyst, observed for two separate series, demonstrates the existence of "anion-π catalysis." In sharp contrast, increasing π-acidity of the best naphthalenediimide catalysts does not influence the more than 12 000-times weaker substrate recognition (KM = 34.5 ± 1.6 μM). Together with the disappearance of Michaelis-Menten kinetics on the expanded π-surfaces of perylenediimides, this finding supports that contributions from π-π interactions are not very important for anion-π catalysis. The linker between the π-acidic surface and the carboxylate base strongly influences activity. Insufficient length and flexibility cause incompatibility with saturation kinetics. Moreover, preorganizing linkers do not improve catalysis much, suggesting that the ideal positioning of the carboxylate base on the π-acidic surface is achieved by intramolecular anion-π interactions rather than by an optimized structure of the linker. Computational simulations are in excellent agreement with experimental results. They confirm, inter alia, that the stabilization of the anionic transition states (but not the neutral ground states) increases with the π-acidity of the

  1. Efficient, non-toxic anion transport by synthetic carriers in cells and epithelia

    Science.gov (United States)

    Li, Hongyu; Valkenier, Hennie; Judd, Luke W.; Brotherhood, Peter R.; Hussain, Sabir; Cooper, James A.; Jurček, Ondřej; Sparkes, Hazel A.; Sheppard, David N.; Davis, Anthony P.

    2016-01-01

    Transmembrane anion transporters (anionophores) have potential for new modes of biological activity, including therapeutic applications. In particular they might replace the activity of defective anion channels in conditions such as cystic fibrosis. However, data on the biological effects of anionophores are scarce, and it remains uncertain whether such molecules are fundamentally toxic. Here, we report a biological study of an extensive series of powerful anion carriers. Fifteen anionophores were assayed in single cells by monitoring anion transport in real time through fluorescence emission from halide-sensitive yellow fluorescent protein. A bis-(p-nitrophenyl)ureidodecalin shows especially promising activity, including deliverability, potency and persistence. Electrophysiological tests show strong effects in epithelia, close to those of natural anion channels. Toxicity assays yield negative results in three cell lines, suggesting that promotion of anion transport may not be deleterious to cells. We therefore conclude that synthetic anion carriers are realistic candidates for further investigation as treatments for cystic fibrosis.

  2. CFTR is required for maximal transepithelial liquid transport in pig alveolar epithelia

    OpenAIRE

    Li, Xiaopeng; Comellas, Alejandro P.; Karp, Philip H.; Ernst, Sarah E.; Moninger, Thomas O.; Gansemer, Nicholas D.; Taft, Peter J.; Pezzulo, Alejandro A; Michael V Rector; Rossen, Nathan; Stoltz, David A.; McCray, Paul B.; Welsh, Michael J.; Zabner, Joseph

    2012-01-01

    A balance between alveolar liquid absorption and secretion is critical for maintaining optimal alveolar subphase liquid height and facilitating gas exchange in the alveolar space. However, the role of cystic fibrosis transmembrane regulator protein (CFTR) in this homeostatic process has remained elusive. Using a newly developed porcine model of cystic fibrosis, in which CFTR is absent, we investigated ion transport properties and alveolar liquid transport in isolated type II alveolar epitheli...

  3. Physiological Adaptation of the Bacterium Lactococcus lactis in Response to the Production of Human CFTR*

    OpenAIRE

    A. Steen; Wiederhold, E.; T Gandhi; Breitling, R.; D. J. Slotboom

    2010-01-01

    Biochemical and biophysical characterization of CFTR (the cystic fibrosis transmembrane conductance regulator) is thwarted by difficulties to obtain sufficient quantities of correctly folded and functional protein. Here we have produced human CFTR in the prokaryotic expression host Lactococcus lactis. The full-length protein was detected in the membrane of the bacterium, but the yields were too low (< 0.1% of membrane proteins) for in vitro functional and structural characterization, and indu...

  4. Adeno-associated virus–targeted disruption of the CFTR gene in cloned ferrets

    OpenAIRE

    Sun, Xingshen; Yan, Ziying; Yi, Yaling; Li, Ziyi; Lei, Diana; Rogers, Christopher S.; Chen, Juan; Zhang, Yulong; Welsh, Michael J.; Leno, Gregory H.; Engelhardt, John F.

    2008-01-01

    Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene–deficient model...

  5. Rare large homozygous CFTR gene deletion in an Iranian patient with cystic fibrosis

    OpenAIRE

    Farjadian, Shirin; Moghtaderi, Mozhgan; Zuntini, Roberta; Ferrari, Simona

    2014-01-01

    Cystic fibrosis, a common autosomal recessive genetic disorder among Caucasians, is caused by defects in the transmembrane conductance regulatory (CFTR) gene. The analysis of CFTR gene mutations is useful to better characterize the disease, and for preconceptional screening, prenatal and preimplantation genetic diagnosis. Here we report the results of a genetic analysis in a 16-year-old boy from southwestern Iran diagnosed as having cystic fibrosis in infancy based on gastrointestinal and pul...

  6. Phenotypic variability of R117H-CFTR expression within monozygotic twins.

    Science.gov (United States)

    Waller, Michael D; Simmonds, Nicholas J

    2016-08-01

    Whilst cystic fibrosis is a monogenic condition, variation in phenotype exists for the same CFTR genotype, which is influenced by multiple genetic and non-genetic (environmental) factors. The R117H-CFTR mutation has variability directly relating to in cis poly-thymidine alleles, producing a differing spectrum of disease. This paper provides evidence of extreme phenotype variability - including fertility status - in the context of male monogenetic twins, discussing mechanisms and highlighting the diagnostic and treatment challenges. PMID:27364092

  7. Relating the disease mutation spectrum to the evolution of the cystic fibrosis transmembrane conductance regulator (CFTR.

    Directory of Open Access Journals (Sweden)

    Lavanya Rishishwar

    Full Text Available Cystic fibrosis (CF is the most common genetic disease among Caucasians, and accordingly the cystic fibrosis transmembrane conductance regulator (CFTR protein has perhaps the best characterized disease mutation spectrum with more than 1,500 causative mutations having been identified. In this study, we took advantage of that wealth of mutational information in an effort to relate site-specific evolutionary parameters with the propensity and severity of CFTR disease-causing mutations. To do this, we devised a scoring scheme for known CFTR disease-causing mutations based on the Grantham amino acid chemical difference matrix. CFTR site-specific evolutionary constraint values were then computed for seven different evolutionary metrics across a range of increasing evolutionary depths. The CFTR mutational scores and the various site-specific evolutionary constraint values were compared in order to evaluate which evolutionary measures best reflect the disease-causing mutation spectrum. Site-specific evolutionary constraint values from the widely used comparative method PolyPhen2 show the best correlation with the CFTR mutation score spectrum, whereas more straightforward conservation based measures (ConSurf and ScoreCons show the greatest ability to predict individual CFTR disease-causing mutations. While far greater than could be expected by chance alone, the fraction of the variability in mutation scores explained by the PolyPhen2 metric (3.6%, along with the best set of paired sensitivity (58% and specificity (60% values for the prediction of disease-causing residues, were marginal. These data indicate that evolutionary constraint levels are informative but far from determinant with respect to disease-causing mutations in CFTR. Nevertheless, this work shows that, when combined with additional lines of evidence, information on site-specific evolutionary conservation can and should be used to guide site-directed mutagenesis experiments by more narrowly

  8. Natural Compound Curcumin-a Channel Potentiator Rather Than a Corrector of the Defective Intracellular Processing of △F508 Mutant Cystic Fibrosis Transmembrane Conductance Regulator

    Institute of Scientific and Technical Information of China (English)

    LIU Xin; GUAN Li; HE Cheng-yan; ZHANG Xiao-jing; XU Li-na; SHANG De-jing; MA Tong-hui; YANG Hong

    2008-01-01

    Cystic fibrosis(CF)is a severe genetic disease caused by the gene mutation of the cystic fibrosis transmembrane conductance regulator(CFTR)chloride channel.The most common point mutation △F508,which leads to impaired intracellular processing and channel gating of CFTR, appears in about 90%CF patients.The natural compound curcumin was reported to correct the processing defect of △F508-CFTR and proposed as a potential therapeutic drug to cure CF.In the present study.we analyzed the efrect of curcumin on △F508-CFTR and demonstrated that curcumin can restore the impaired chloride conductance of △F508 mutant CFTR.The activity is rapid,reversible and cAMP-dependent.However,we couldn't reproduce the previously reported correction of the defective membrane trafficking of △F508-CFTR by curcumin.Therefore,curcumin may not be a superior lead compound for developing anti-CF drugs.

  9. Refining the continuum of CFTR-associated disorders in the era of newborn screening.

    Science.gov (United States)

    Levy, H; Nugent, M; Schneck, K; Stachiw-Hietpas, D; Laxova, A; Lakser, O; Rock, M; Dahmer, M K; Biller, J; Nasr, S Z; Baker, M; McColley, S A; Simpson, P; Farrell, P M

    2016-05-01

    Clinical heterogeneity in cystic fibrosis (CF) often causes diagnostic uncertainty in infants without symptoms and in older patients with milder phenotypes. We performed a cross-sectional evaluation of a comprehensive set of clinical and laboratory descriptors in a physician-defined cohort (N = 376; Children's Hospital of Wisconsin and the American Family Children's Hospital CF centers in Milwaukee and Madison, WI, USA) to determine the robustness of categorizing CF (N = 300), cystic fibrosis transmembrane conductance regulator (CFTR)-related disorder (N = 19), and CFTR-related (CRMS) metabolic syndrome (N = 57) according to current consensus guidelines. Outcome measures included patient demographics, clinical measures, sweat chloride levels, CFTR genotype, age at diagnosis, airway microbiology, pancreatic function, infection, and nutritional status. The CF cohort had a significantly higher median sweat chloride level (105 mmol/l) than CFTR-related disorder patients (43 mmol/l) and CFTR-related metabolic syndrome patients (35 mmol/l; p ≤ 0.001). Patient groups significantly differed in pancreatic sufficiency, immunoreactive trypsinogen levels, sweat chloride values, genotype, and positive Pseudomonas aeruginosa cultures (p ≤ 0.001). An automated classification algorithm using recursive partitioning demonstrated concordance between physician diagnoses and consensus guidelines. Our analysis suggests that integrating clinical information with sweat chloride levels, CFTR genotype, and pancreatic sufficiency provides a context for continued longitudinal monitoring of patients for personalized and effective treatment. PMID:26671754

  10. Vanadogermanate cluster anions.

    Science.gov (United States)

    Whitfield, T; Wang, X; Jacobson, A J

    2003-06-16

    Three novel vanadogermanate cluster anions have been synthesized by hydrothermal reactions. The cluster anions are derived from the (V(18)O(42)) Keggin cluster shell by substitution of V=O(2+) "caps" by Ge(2)O(OH)(2)(4+) species. In Cs(8)[Ge(4)V(16)O(42)(OH)(4)].4.7H(2)O, 1, (monoclinic, space group C2/c (No. 15), Z = 8, a = 44.513(2) A, b = 12.7632(7) A, c = 22.923(1) A, beta = 101.376(1) degrees ) and (pipH(2))(4)(pipH)(4)[Ge(8)V(14)O(50).(H(2)O)] (pip = C(4)N(2)H(10)), 2 (tetragonal, space group P4(2)/nnm (No. 134), Z = 2, a = 14.9950(7) A, c = 18.408(1) A), two and four VO(2+) caps are replaced, respectively, and each cluster anion encapsulates a water molecule. In K(5)H(8)Ge(8)V(12)SO(52).10H(2)O, 3, (tetragonal, space group I4/m (No. 87), Z = 2, a = 15.573(1) A, c = 10.963(1) A), four VO(2+) caps are replaced by Ge(2)O(OH)(2)(4+) species, and an additional two are omitted. The cluster ion in 3 contains a sulfate anion disordered over two positions. The cluster anions are analogous to the vanadoarsenate anions [V(18)(-)(n)()As(2)(n)()O(42)(X)](m)(-) (X = SO(3), SO(4), Cl; n = 3, 4) previously reported. PMID:12793808

  11. Lack of CFTR in Skeletal Muscle Predisposes to Muscle Wasting and Diaphragm Muscle Pump Failure in Cystic Fibrosis Mice

    OpenAIRE

    Maziar Divangahi; Haouaria Balghi; Gawiyou Danialou; Comtois, Alain S.; Alexandre Demoule; Sheila Ernest; Christina Haston; Renaud Robert; Hanrahan, John W.; Danuta Radzioch; Petrof, Basil J

    2009-01-01

    Cystic fibrosis (CF) patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR) plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and co-localized with sarcoplasmic reticulum-associated proteins. CFTR-deficient myotubes exhibit augmented levels of intracellular calcium aft...

  12. Defective CFTR expression and function are detectable in blood monocytes : development of a new blood test for cystic fibrosis

    OpenAIRE

    Sorio, C.; Buffelli, M.; C. Angiari; Ettorre, M; Johansson, J; M. Vezzalini; Viviani, L; Ricciardi, M.; G. Verzè; Assael, B M; P. Melotti

    2011-01-01

    Background Evaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes. Description Western blot, PCR, immunofluorescence and cell membrane ...

  13. Development of allele-specific multiplex PCR to determine the length of poly-T in intron 8 of CFTR

    OpenAIRE

    Neng Chen; Prada, Anne E.

    2014-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation analysis has been implemented for Cystic Fibrosis (CF) carrier screening, and molecular diagnosis of CF and congenital bilateral absence of the vas deferens (CBAVD). Although poly-T allele analysis in intron 8 of CFTR is required when a patient is positive for R117H, it is not recommended for routine carrier screening. Therefore, commercial kits for CFTR mutation analysis were designed either to mask the poly-T allele re...

  14. Potentiometric anion selective sensors

    NARCIS (Netherlands)

    Antonisse, Martijn M.G.; Reinhoudt, David N.

    1999-01-01

    In comparison with selective receptors (and sensors) for cationic species, work on the selective complexation and detection of anions is of more recent date. There are three important components for a sensor, a transducer element, a membrane material that separates the transducer element and the aqu

  15. Anion Ordering in Bichalcogenides

    Directory of Open Access Journals (Sweden)

    Martin Valldor

    2016-07-01

    Full Text Available This review contains recent developments and new insights in the research on inorganic, crystalline compounds with two different chalcogenide ions (bichalcogenides. Anion ordering is used as a parameter to form structural dimensionalities as well as local- and global-electric polarities. The reason for the electric polarity is that, in the heterogeneous bichalcogenide lattice, the individual bond-lengths between cations and anions are different from those in a homogeneous anion lattice. It is also shown that heteroleptic tetrahedral and octahedral coordinations offer a multitude of new crystal fields and coordinations for involved cations. This coordination diversity in bichalcogenides seems to be one way to surpass electro-chemical redox potentials: three oxidation states of a single transition metal can be stabilized, e.g., Ba15V12S34O3. A new type of disproportionation, related to coordination, is presented and results from chemical pressure on the bichalcogenide lattices of (La,CeCrS2O, transforming doubly [CrS3/3S2/2O1/1]3− (5+1 into singly [CrS4/2S2/3]7/3− (6+0 and [CrS4/3O2/1]11/3− (4+2 coordinations. Also, magnetic anisotropy is imposed by the anion ordering in BaCoSO, where magnetic interactions via S or O occur along two different crystallographic directions. Further, the potential of the anion lattice is discussed as a parameter for future materials design.

  16. Na+/K+/2Cl- cotransporter and CFTR gill expression after seawater transfer in smolts (0+) of different Atlantic salmon (Salmo salar) families

    Science.gov (United States)

    Mackie, P.M.; Gharbi, K.; Ballantyne, J.S.; McCormick, S.D.; Wright, P.A.

    2007-01-01

    Smoltification involves morphological and physiological changes in the gills that prepare anadromous salmonids to osmoregulate efficiently in seawater. In a previous study, we found that different families of Atlantic salmon (Salmo salar) smolts vary in their ability to osmoregulate when abruptly transferred to cold seawater and that these differences are correlated with gill Na+/K+ ATPase activity. Here we extend these findings to test whether other key transport proteins, namely Na+/K+/2Cl- contransporter (NKCC) and the Cl- channel or cystic fibrosis transmembrane conductance regulator (CFTR), play a significant role in osmoregulatory differences between families. To facilitate molecular analysis of NKCC, we first isolated a gill cDNA containing the complete coding region (1147 aa) of an isoform previously reported as a partial sequence. Phylogenetic analysis showed that this isoform is most closely related to isoforms of the NKCC1a subfamily found in European eel and Mozambique tilapia. In a second step, we quantified NKCC protein abundance as well as mRNA expression levels for NKCC1a and two CFTR isoforms (CFTRI and CFTRII) in 0+ smolts from three families prior to and following seawater transfer. The family with the lowest salinity tolerance also showed significant increases in gill NKCC1a mRNA after seawater transfer. Taken together with our previous study, these data indicate that family differences in expression of transport proteins are in part related to salinity tolerance, although the best indicator of osmoregulatory performance between families may be gill Na+/K+ ATPase activity and CFTR I mRNA levels, rather than Na+/K+ ATPase and NKCC1a mRNA levels or NKCC protein abundance. ?? 2007 Elsevier B.V. All rights reserved.

  17. Gramicidin Channels: Versatile Tools

    Science.gov (United States)

    Andersen, Olaf S.; Koeppe, Roger E., II; Roux, Benoît

    Gramicidin channels are miniproteins in which two tryptophan-rich subunits associate by means of transbilayer dimerization to form the conducting channels. That is, in contrast to other ion channels, gramicidin channels do not open and close; they appear and disappear. Each subunit in the bilayer-spanning channel is tied to the bilayer/solution interface through hydrogen bonds that involve the indole NH groups as donors andwater or the phospholipid backbone as acceptors. The channel's permeability characteristics are well-defined: gramicidin channels are selective for monovalent cations, with no measurable permeability to anions or polyvalent cations; ions and water move through a pore whose wall is formed by the peptide backbone; and the single-channel conductance and cation selectivity vary when the amino acid sequence is varied, even though the permeating ions make no contact with the amino acid side chains. Given the plethora of available experimental information—for not only the wild-type channels but also for channels formed by amino acid-substituted gramicidin analogues—gramicidin channels continue to provide important insights into the microphysics of ion permeation through bilayer-spanning channels. For similar reasons, gramicidin channels constitute a system of choice for evaluating computational strategies for obtaining mechanistic insights into ion permeation through the more complex channels formed by integral membrane proteins.

  18. Ubiquitination and degradation of CFTR by the E3 ubiquitin ligase MARCH2 through its association with adaptor proteins CAL and STX6.

    Science.gov (United States)

    Cheng, Jie; Guggino, William

    2013-01-01

    Golgi-localized cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) and syntaxin 6 (STX6) regulate the abundance of mature, post-ER CFTR by forming a CAL/STX6/CFTR complex (CAL complex) that promotes CFTR degradation in lysosomes. However, the molecular mechanism underlying this degradation is unknown. Here we investigated the interaction of a Golgi-localized, membrane-associated RING-CH E3 ubiquitin ligase, MARCH2, with the CAL complex and the consequent binding, ubiquitination, and degradation of mature CFTR. We found that MARCH2 not only co-immunoprecipitated and co-localized with CAL and STX6, but its binding to CAL was also enhanced by STX6, suggesting a synergistic interaction. In vivo ubiquitination assays demonstrated the ubiquitination of CFTR by MARCH2, and overexpression of MARCH2, like that of CAL and STX6, led to a dose-dependent degradation of mature CFTR that was blocked by bafilomycin A1 treatment. A catalytically dead MARCH2 RING mutant was unable to promote CFTR degradation. In addition, MARCH2 had no effect on a CFTR mutant lacking the PDZ motif, suggesting that binding to the PDZ domain of CAL is required for MARCH2-mediated degradation of CFTR. Indeed, silencing of endogenous CAL ablated the effect of MARCH2 on CFTR. Consistent with its Golgi localization, MARCH2 had no effect on ER-localized ΔF508-CFTR. Finally, siRNA-mediated silencing of endogenous MARCH2 in the CF epithelial cell line CFBE-CFTR increased the abundance of mature CFTR. Taken together, these data suggest that the recruitment of the E3 ubiquitin ligase MARCH2 to the CAL complex and subsequent ubiquitination of CFTR are responsible for the CAL-mediated lysosomal degradation of mature CFTR.

  19. Ubiquitination and Degradation of CFTR by the E3 Ubiquitin Ligase MARCH2 through Its Association with Adaptor Proteins CAL and STX6

    OpenAIRE

    Jie Cheng; William Guggino

    2013-01-01

    Golgi-localized cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) and syntaxin 6 (STX6) regulate the abundance of mature, post-ER CFTR by forming a CAL/STX6/CFTR complex (CAL complex) that promotes CFTR degradation in lysosomes. However, the molecular mechanism underlying this degradation is unknown. Here we investigated the interaction of a Golgi-localized, membrane-associated RING-CH E3 ubiquitin ligase, MARCH2, with the CAL complex and the consequent bindin...

  20. CFTR suppresses tumor progression through miR-193b targeting urokinase plasminogen activator (uPA) in prostate cancer.

    Science.gov (United States)

    Xie, C; Jiang, X H; Zhang, J T; Sun, T T; Dong, J D; Sanders, A J; Diao, R Y; Wang, Y; Fok, K L; Tsang, L L; Yu, M K; Zhang, X H; Chung, Y W; Ye, L; Zhao, M Y; Guo, J H; Xiao, Z J; Lan, H Y; Ng, C F; Lau, K M; Cai, Z M; Jiang, W G; Chan, H C

    2013-05-01

    Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is expressed in the epithelial cells of a wide range of organs/tissues from which most cancers are derived. Although accumulating reports have indicated the association of cancer incidence with genetic variations in CFTR gene, the exact role of CFTR in cancer development and the possible underlying mechanism have not been elucidated. Here, we report that CFTR expression is significantly decreased in both prostate cancer cell lines and human prostate cancer tissue samples. Overexpression of CFTR in prostate cancer cell lines suppresses tumor progression (cell growth, adhesion and migration), whereas knockdown of CFTR leads to enhanced malignancies both in vitro and in vivo. In addition, we demonstrate that CFTR knockdown-enhanced cell proliferation, cell invasion and migration are significantly reversed by antibodies against either urokinase plasminogen activator (uPA) or uPA receptor (uPAR), which are known to be involved in various malignant traits of cancer development. More interestingly, overexpression of CFTR suppresses uPA by upregulating the recently described tumor suppressor microRNA-193b (miR-193b), and overexpression of pre-miR-193b significantly reverses CFTR knockdown-enhanced malignant phenotype and abrogates elevated uPA activity in prostate cancer cell line. Finally, we show that CFTR gene transfer results in significant tumor repression in prostate cancer xenografts in vivo. Taken together, the present study has demonstrated a previously undefined tumor-suppressing role of CFTR and its involvement in regulation of miR-193b in prostate cancer development. PMID:22797075

  1. Eicosanoid release is increased by membrane destabilization and CFTR inhibition in Calu-3 cells.

    Directory of Open Access Journals (Sweden)

    Florence Borot

    Full Text Available The antiinflammatory protein annexin-1 (ANXA1 and the adaptor S100A10 (p11, inhibit cytosolic phospholipase A2 (cPLA2alpha by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2alpha. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM induced by TNF-alpha. This was concomitant with increased IL-8 synthesis and cPLA2alpha activation, ultimately resulting in eicosanoid (PGE2 and LTB4 overproduction. DRM destabilizing agent methyl-beta-cyclodextrin induced further cPLA2alpha activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-alpha-induced relocalization to DRM. These results show that (i CFTR may form a complex with cPLA2alpha and ANXA1 via interaction with p11, (ii CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-alpha-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the

  2. Pathophysiologic consequences following inhibition of a CFTR-dependent developmental cascade in the lung

    Directory of Open Access Journals (Sweden)

    Larson Janet E

    2005-02-01

    Full Text Available Abstract Background Examination of late gestation developmental genes in vivo may be limited by early embryonic lethality and compensatory mechanisms. This problem is particularly apparent in evaluating the developmental role of the cystic fibrosis transmembrane conductance regulator (CFTR gene in the cystic fibrosis (CF phenotype. A previously described transient in utero knockout (TIUKO technology was used to address the developmental role of CFTR in the rat lung. Results Rat fetuses transiently treated with antisense cftr in utero developed pathology that replicated aspects of the human CF phenotype. The TIUKO CF rat developed lung fibrosis, chronic inflammation, reactive airway disease, and the CF Antigen (MRP8/14, a marker for CF in human patients, was expressed. Conclusions The transient in utero antisense technology can be used to evaluate genes that exhibit either early lethality or compensating gene phenotypes. In the lung CFTR is part of a developmental cascade for normal secretory cell differentiation. Absence of CFTR results in a constitutive inflammatory process that is involved in some aspects of CF pathophysiology.

  3. Ivacaftor treatment in patients with cystic fibrosis and the G551D-CFTR mutation

    Directory of Open Access Journals (Sweden)

    Isabelle Sermet-Gaudelus

    2013-03-01

    Full Text Available Cystic fibrosis (CF is an autosomal recessive lethal disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR gene that encodes for CFTR, an epithelial cell-surface expressed protein responsible for the transport of chloride (Cl-. Gating mutations associated with defective conductance can be modulated by CFTR potentiators. Ivacaftor is a CFTR potentiator approved for the treatment of CF patients >6 yrs of age with at least one copy of the G551D-CFTR mutation. Herein, the clinical trial development programme for ivacaftor will be reviewed, including two pivotal studies in adolescents/adults and in children. These studies report sustained improvements in lung function and sweat chloride concentrations, and a reduction in pulmonary exacerbations over a 48-week treatment period. In the era of personalised medicine, ivacaftor offers an effective and well-tolerated treatment for the clinical management of CF patients with the G551D mutation. A long-term, open-label study will report the effects of ivacaftor over a further 48 weeks.

  4. Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition.

    Science.gov (United States)

    Luciani, Alessandro; Villella, Valeria Rachela; Esposito, Speranza; Brunetti-Pierri, Nicola; Medina, Diego; Settembre, Carmine; Gavina, Manuela; Pulze, Laura; Giardino, Ida; Pettoello-Mantovani, Massimo; D'Apolito, Maria; Guido, Stefano; Masliah, Eliezer; Spencer, Brian; Quaratino, Sonia; Raia, Valeria; Ballabio, Andrea; Maiuri, Luigi

    2010-09-01

    Accumulation of unwanted/misfolded proteins in aggregates has been observed in airways of patients with cystic fibrosis (CF), a life-threatening genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Here we show how the defective CFTR results in defective autophagy and decreases the clearance of aggresomes. Defective CFTR-induced upregulation of reactive oxygen species (ROS) and tissue transglutaminase (TG2) drive the crosslinking of beclin 1, leading to sequestration of phosphatidylinositol-3-kinase (PI(3)K) complex III and accumulation of p62, which regulates aggresome formation. Both CFTR knockdown and the overexpression of green fluorescent protein (GFP)-tagged-CFTR(F508del) induce beclin 1 downregulation and defective autophagy in non-CF airway epithelia through the ROS-TG2 pathway. Restoration of beclin 1 and autophagy by either beclin 1 overexpression, cystamine or antioxidants rescues the localization of the beclin 1 interactome to the endoplasmic reticulum and reverts the CF airway phenotype in vitro, in vivo in Scnn1b-transgenic and Cftr(F508del) homozygous mice, and in human CF nasal biopsies. Restoring beclin 1 or knocking down p62 rescued the trafficking of CFTR(F508del) to the cell surface. These data link the CFTR defect to autophagy deficiency, leading to the accumulation of protein aggregates and to lung inflammation.

  5. Cystic fibrosis transmembrane conductance regulator (CFTR gene abnormalities in Indian males with congenital bilateral absence of vas deferens & renal anomalies

    Directory of Open Access Journals (Sweden)

    Rahul Gajbhiye

    2016-01-01

    Results: Three potential regulatory CFTR gene variants (c.1540A>G, c.2694T>G and c.4521G>A were detected along with IVS8-5T mutation in three infertile males with CBAVD-URA. Five novel CFTR gene variants (c.621+91A>G, c.2752+106A>T, c.2751+85_88delTA, c.3120+529InsC and c.4375-69C>T, four potential regulatory CFTR gene variants (M470V, T854T, P1290P, Q1463Q and seven previously reported CFTR gene variants (c.196+12T>C, c.875+40A>G, c.3041-71G>C, c.3271+42A>T, c.3272-93T>C, c.3500-140A>C and c.3601-65C>A were detected in infertile men having CBAVD and renal anomalies Interpretation & conclusions: Based on our findings, we speculate that CBAVD-URA may also be attributed to CFTR gene mutations and can be considered as CFTR-related disorder (CFTR-RD. The CFTR gene mutation screening may be offered to CBAVD-URA men and their female partners undergoing ICSI. Further studies need to be done in a large sample to confirm the findings.

  6. The Delta F508 mutation shortens the biochemical half-life of plasma membrane CFTR in polarized epithelial cells.

    Science.gov (United States)

    Heda, G D; Tanwani, M; Marino, C R

    2001-01-01

    Although the biosynthetic arrest of the DeltaF508 mutant of cystic fibrosis transmembrane conductance regulator (CFTR) can be partially reversed by physical and chemical means, recent evidence suggests that the functional stability of the mutant protein after reaching the cell surface is compromised. To understand the molecular basis for this observation, the current study directly measured the half-life of Delta F508 and wild-type CFTR at the cell surface of transfected LLC-PK(1) cells. Plasma membrane CFTR expression over time was characterized biochemically and functionally in these polarized epithelial cells. Surface biotinylation, streptavidin extraction, and quantitative immunoblot analysis determined the biochemical half-life of plasma membrane DeltaF508 CFTR to be approximately 4 h, whereas the plasma membrane half-life of wild-type CFTR exceeded 48 h. This difference in biochemical stability correlated with CFTR-mediated transport function. These findings indicate that the Delta F508 mutation decreases the biochemical stability of CFTR at the cell surface. We conclude that the Delta F508 mutation triggers more rapid internalization of CFTR and/or its preferential sorting to a pathway of rapid degradation. PMID:11121388

  7. Altered intestinal bile salt biotransformation in a cystic fibrosis (Cftr(-/-)) mouse model with hepato-biliary pathology

    NARCIS (Netherlands)

    Bodewes, Frank A. J. A.; van der Wulp, Mariette Y. M.; Beharry, Satti; Doktorova, Marcela; Havinga, Rick; Boverhof, Renze; Phillips, M. James; Durie, Peter R.; Verkade, Henkjan J.

    2015-01-01

    Background: Cftr(-/-tm1UC) mice develop progressive hepato-biliary pathology. We hypothesize that this liver pathology is related to alterations' in biliary bile hydrophobicity and bile salt metabolism in Cftr(-/-tm1Unc) mice. Methods: We determined bile production, biliary and fecal bile salt- and

  8. The primary folding defect and rescue of ΔF508 CFTR emerge during translation of the mutant domain

    NARCIS (Netherlands)

    Hoelen, H.M.; Kleizen, B.; Schmidt, A.; Richardson, J.; Charitou, P.; Braakman, L.J.; Thomas, P. J.

    2010-01-01

    In the vast majority of cystic fibrosis (CF) patients, deletion of residue F508 from CFTR is the cause of disease. F508 resides in the first nucleotide binding domain (NBD1) and its absence leads to CFTR misfolding and degradation. We show here that the primary folding defect arises during synthesis

  9. Failure of the Cystic Fibrosis Transmembrane Conductance Regulator to Conduct ATP

    Science.gov (United States)

    Reddy, M. M.; Quinton, P. M.; Haws, C.; Wine, J. J.; Grygorczyk, R.; Tabcharani, J. A.; Hanrahan, J. W.; Gunderson, K. L.; Kopito, R. R.

    1996-03-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is chloride ion channel regulated by protein kinase A and adenosine triphosphate (ATP). Loss of CFTR-mediated chloride ion conductance from the apical plasma membrane of epithelial cells is a primary physiological lesion in cystic fibrosis. CFTR has also been suggested to function as an ATP channel, although the size of the ATP anion is much larger than the estimated size of the CFTR pore. ATP was not conducted through CFTR in intact organs, polarized human lung cell lines, stably transfected mammalian cell lines, or planar lipid bilayers reconstituted with CFTR protein. These findings suggest that ATP permeation through the CFTR is unlikely to contribute to the normal function of CFTR or to the pathogenesis of cystic fibrosis.

  10. First functional polymorphism in CFTR promoter that results in decreased transcriptional activity and Sp1/USF binding

    International Nuclear Information System (INIS)

    Growing evidences show that functionally relevant polymorphisms in various promoters alter both transcriptional activity and affinities of existing protein-DNA interactions, and thus influence disease progression in humans. We previously reported the -94G>T CFTR promoter variant in a female CF patient in whom any known disease-causing mutation has been detected. To investigate whether the -94G>T could be a regulatory variant, we have proceeded to in silico analyses and functional studies including EMSA and reporter gene assays. Our data indicate that the promoter variant decreases basal CFTR transcriptional activity in different epithelial cells and alters binding affinities of both Sp1 and USF nuclear proteins to the CFTR promoter. The present report provides evidence for the first functional polymorphism that negatively affects the CFTR transcriptional activity and demonstrates a cooperative role of Sp1 and USF transcription factors in transactivation of the CFTR gene promoter

  11. A novel CFTR mutation found in a Chinese patient with cystic fibrosis

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background Cystic fibrosis (CF) is rare in Chinese. We investigated the mutations in the gene of cystic fibrosis transmembrane conductance regulator (CFTR) in a Chinese CF patient and reviewed the clinical features, gene mutations in Chinese CF cases. Methods Blood samples were collected from a previously reported CF girl and her parents. The 24 coding exons of CFTR of the proband were amplified and sequenced. Results A Chinese girl of 16 years old was diagnosed as CF at the age of 14. She had recurrent productive cough with bronchiectasis in bilateral upper lobes, parasinusitis and otitis media, but without pancreatic involvement. Her sweat chloride was (108.9 ±3.3) mmol/L. A heterozygous novel missense mutation of 699 C→A which results in the amino acid change of N189K was identified in exon 5. In addition, a heterozygous 3821-3823 delT mutation in exon 19 was found in CFTR. The mutation 699C→A was inherited from her father, and the 3821-3823delT mutation was from her mother. Twenty patients with CF in Chinese reported from 1974 to 2004 were also reviewed. DelF508 mutation was not found in the nine cases whose CFTR mutations were analyzed. Conclusions The CF proband carries two heterozygous mutations (699C→A and 3821-3823delT) in CFTR. 699C→A mutation is a novel mutation which is not reported previously. Review of reported Chinese cases suggests that the genotype of Chinese CF may be different from those of white cases. More studies are needed to understand the spectra of CFTR and clinical CF features in Chinese.

  12. Resveratrol increases F508del-CFTR dependent salivary secretion in cystic fibrosis mice

    Directory of Open Access Journals (Sweden)

    Barbara Dhooghe

    2015-07-01

    Full Text Available Cystic fibrosis (CF is a fatal genetic disease associated with widespread exocrine gland dysfunction. Studies have suggested activating effects of resveratrol, a naturally-occurring polyphenol compound with antioxidant and anti-inflammatory properties, on CF transmembrane conductance regulator (CFTR protein function. We assayed, in F508del-CFTR homozygous (CF and in wild-type mice, the effect of resveratrol on salivary secretion in basal conditions, in response to inhibition by atropine (basal β-adrenergic-dependent component and to stimulation by isoprenaline (CFTR-dependent component. Both components of the salivary secretion were smaller in CF mice than in controls. Two hours after intraperitoneal administration of resveratrol (50 mg/kg dissolved in DMSO, the compound was detected in salivary glands. As in both CF and in wild-type mice, DMSO alone increased the response to isoprenaline in males but not in females, the effect of resveratrol was only measured in females. In wild-type mice, isoprenaline increased secretion by more than half. In CF mice, resveratrol rescued the response to isoprenaline, eliciting a 2.5-fold increase of β-adrenergic-stimulated secretion. We conclude that the salivary secretion assay is suitable to test DMSO-soluble CFTR modulators in female mice. We show that resveratrol applied in vivo to mice reaches salivary glands and increases β-adrenergic secretion. Immunolabelling of CFTR in human bronchial epithelial cells suggests that the effect is associated with increased CFTR protein expression. Our data support the view that resveratrol is beneficial for treating CF. The salivary secretion assay has a potential application to test efficacy of novel CF therapies.

  13. Impact of Cystic Fibrosis Transmembrane Regulator (CFTR gene mutations on male infertility

    Directory of Open Access Journals (Sweden)

    Jlenia Elia

    2014-09-01

    Full Text Available Objective. The aim of this study was to evaluate the prevalence of most common mutations and intron 8 5T (IVS8-5T polymorphism of CFTR gene in Italian: a azoospermic males; b non azoospermic subjects, male partners of infertile couples enrolled in assisted reproductive technology (ART programs. Material and methods. We studied 242 subjects attending our Andrology Unit (44 azoospermic subjects and 198 non azoospermic subjects, male partners of infertile couples enrolled in ART programs. Semen analysis, molecular analysis for CFTR gene mutations and genomic variant of IVS8-5T polymorphic tract, karyotype and chromosome Y microdeletions, hormonal profile (LH, FSH, Testosterone and seminal biochemical markers (fructose, citric acid and L-carnitine were carried out. Results. The prevalence of the common CFTR mutations and/or the IVS8-5T polymorphism was 12.9% (4/31 cases in secretory azoospermia, while in obstructive azoospermia was 84.6% (11/13 cases; in these, the most frequent mutations were the F508del, R117H and W1282X. Regarding the non azoospermic subjects, the prevalence of the CFTR and/or the IVS8-5T polymorphism was 11.1% (11/99 cases in severe dyspermia, 8.1% (6/74 cases in moderate dyspermia and finally 4.0% (1/25 cases in normospermic subjects. Conclusions. This study confirms the highly significant prevalence of CFTR mutations in males with bilateral absence of the vas deferens or ejaculatory ducts obstruction compared with subjects with secretory azoospermia. Moreover, the significant prevalence of mutations in severely dyspermic subjects may suggest the possible involvement of CFTR even in the spermatogenic process. This could explain the unsatisfactory recovery of sperm from testicular fine needle aspiration in patients affected by genital tract blockage.

  14. Identification of CFTR Gene Mutations in Chinese Patients with Congenital Obstructive Azoospermia

    Institute of Scientific and Technical Information of China (English)

    曾国华; 吴开俊; 梅骅; 庄广伦

    2001-01-01

    Objective To analyze the frequency and hot spot of CFTR gene mutations in Chinese patients with congenital obstructive azoospermia Materials & Methods Mutations in CFTR exon 2,3,4,5,6a,8,10,11,12,13,15A 17b, 19A,20,21and 23 were detected. PCR-single strand conformation poly-morphism (SSCP) and direct sequencing were performed on 32 patients with congenital bilateral absence of the vas deferens (CBAVD), 17 patients with congenital unilateral absence of the vas deferens (CUAVD) and 50 normal Chinese.Results No CFTR gene mutations were detected in 50 normal Chinese. One CBAVD patient exhibited an abnormal band on SSCP for exon 10 of the CFTR gene and subsequent DNA sequencing showed a 3 bp deletion at position 1 653~ 1 655, which caused the deletion of a single amino acid, phenyalanine, in codon 508, i. e. , △F 508. A shift mutation was detected in another CBAVD patient in exon 2, a 1 bp deletion at position 225, 225 delC. One CUAVD patient exhibited an abnormal band on SSCP for exon 17 b of CFTR gene. Subsequent DNA sequencing showed a C-to-A transversion at position 3 295, which led to a predicted change of Leusine (codon 1 055,CUU) to Isoleucine (codon AUU), L1055I.Conclusion CFTR mutation could be detected in Chinese patients with congenital obstructive azoospermia. But no hot spots of mutations are discovered. 225 delC and L1055I are identified as two novel mutations, which are found only in Chinese.

  15. VAMP-associated Proteins (VAP) as Receptors That Couple Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Proteostasis with Lipid Homeostasis.

    Science.gov (United States)

    Ernst, Wayne L; Shome, Kuntala; Wu, Christine C; Gong, Xiaoyan; Frizzell, Raymond A; Aridor, Meir

    2016-03-01

    Unesterified cholesterol accumulates in late endosomes in cells expressing the misfolded cystic fibrosis transmembrane conductance regulator (CFTR). CFTR misfolding in the endoplasmic reticulum (ER) or general activation of ER stress led to dynein-mediated clustering of cholesterol-loaded late endosomes at the Golgi region, a process regulated by ER-localized VAMP-associated proteins (VAPs). We hypothesized that VAPs serve as intracellular receptors that couple lipid homeostasis through interactions with two phenylalanines in an acidic track (FFAT) binding signals (found in lipid sorting and sensing proteins, LSS) with proteostasis regulation. VAPB inhibited the degradation of ΔF508-CFTR. The activity was mapped to the ligand-binding major sperm protein (MSP) domain, which was sufficient in regulating CFTR biogenesis. We identified mutations in an unstructured loop within the MSP that uncoupled VAPB-regulated CFTR biogenesis from basic interactions with FFAT. Using this information, we defined functional and physical interactions between VAPB and proteostasis regulators (ligands), including the unfolded protein response sensor ATF6 and the ER degradation cluster that included FAF1, VCP, BAP31, and Derlin-1. VAPB inhibited the degradation of ΔF508-CFTR in the ER through interactions with the RMA1-Derlin-BAP31-VCP pathway. Analysis of pseudoligands containing tandem FFAT signals supports a competitive model for VAP interactions that direct CFTR biogenesis. The results suggest a model in which VAP-ligand binding couples proteostasis and lipid homeostasis leading to observed phenotypes of lipid abnormalities in protein folding diseases.

  16. Lowest autodetachment state of the water anion

    Science.gov (United States)

    Houfek, Karel; Čížek, Martin

    2016-05-01

    The potential energy surface of the ground state of the water anion H2O- is carefully mapped using multireference CI calculations for a large range of molecular geometries. Particular attention is paid to a consistent description of both the O-+H2 and OH-+H asymptotes and to a relative position of the anion energy to the ground state energy of the neutral molecule. The autodetachment region, where the anion state crosses to the electronic continuum is identified. The local minimum in the direction of the O- + H2 channel previously reported by Werner et al. [J. Chem. Phys. 87, 2913 (1987)] is found to be slighly off the linear geometry and is separated by a saddle from the autodetachment region. The autodetachment region is directly accessible from the OH-+H asymptote. For the molecular geometries in the autodetachment region and in its vicinity we also performed fixed-nuclei electron-molecule scattering calculations using the R-matrix method. Tuning of consistency of a description of the correlation energy in both the multireference CI and R-matrix calculations is discussed. Two models of the correlation energy within the R-matrix method that are consistent with the quantum chemistry calculations are found. Both models yield scattering quantities in a close agreement. The results of this work will allow a consistent formulation of the nonlocal resonance model of the water anion in a future publication. Contribution to the Topical Issue "Advances in Positron and Electron Scattering", edited by Paulo Limao-Vieira, Gustavo Garcia, E. Krishnakumar, James Sullivan, Hajime Tanuma and Zoran Petrovic.

  17. A molecular switch in the scaffold NHERF1 enables misfolded CFTR to evade the peripheral quality control checkpoint.

    Science.gov (United States)

    Loureiro, Cláudia A; Matos, Ana Margarida; Dias-Alves, Ângela; Pereira, Joana F; Uliyakina, Inna; Barros, Patrícia; Amaral, Margarida D; Matos, Paulo

    2015-05-19

    The peripheral protein quality control (PPQC) checkpoint removes improperly folded proteins from the plasma membrane through a mechanism involving the E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70 interacting protein). PPQC limits the efficacy of some cystic fibrosis (CF) drugs, such as VX-809, that improve trafficking to the plasma membrane of misfolded mutants of the CF transmembrane conductance regulator (CFTR), including F508del-CFTR, which retains partial functionality. We investigated the PPQC checkpoint in lung epithelial cells with F508del-CFTR that were exposed to VX-809. The conformation of the scaffold protein NHERF1 (Na(+)/H(+) exchange regulatory factor 1) determined whether the PPQC recognized "rescued" F508del-CFTR (the portion that reached the cell surface in VX-809-treated cells). Activation of the cytoskeletal regulator Rac1 promoted an interaction between the actin-binding adaptor protein ezrin and NHERF1, triggering exposure of the second PDZ domain of NHERF1, which interacted with rescued F508del-CFTR. Because binding of F508del-CFTR to the second PDZ of NHERF1 precluded the recruitment of CHIP, the coexposure of airway cells to Rac1 activator nearly tripled the efficacy of VX-809. Interference with the NHERF1-ezrin interaction prevented the increase of efficacy of VX-809 by Rac1 activation, but the actin-binding domain of ezrin was not required for the increase in efficacy. Thus, rather than mainly directing anchoring of F508del-CFTR to the actin cytoskeleton, induction of ezrin activation by Rac1 signaling triggered a conformational change in NHERF1, which was then able to bind and stabilize misfolded CFTR at the plasma membrane. These insights into the cell surface stabilization of CFTR provide new targets to improve treatment of CF.

  18. Analysis of Y chromosome microdeletions and CFTR gene mutations as genetic markers of infertility in Serbian men

    Directory of Open Access Journals (Sweden)

    Dinić Jelena

    2007-01-01

    Full Text Available Background/Aim. Impaired fertility of a male partner is the main cause of infertility in up to one half of all infertile couples. At the genetic level, male infertility can be caused by chromosome aberrations or gene mutations. The presence and types of Y chromosome microdeletions and cystic fybrosis transmembrane conductance regulator (CFTR gene mutations as genetic cause of male infertility was tested in Serbian men. The aim of this study was to analyze CFTR gene mutations and Y chromosome microdelations as potential causes of male infertility in Serbian patients, as well as to test the hypothesis that CFTR mutations in infertile men are predominantly located in the several last exons of the gene. Methods. This study has encompassed 33 men with oligo- or azoospermia. The screening for Y chromosome microdeletions in the azoospermia factor (AZF region was performed by multiplex PCR analysis. The screening of the CFTR gene was performed by denaturing gradient gel electrophoresis (DGGE method. Results. Deletions on Y chromosome were detected in four patients, predominantly in AZFc region (four of total six deletions. Mutations in the CFTR gene were detected on eight out of 66 analyzed chromosomes of infertile men. The most common mutation was F508del (six of total eight mutations. Conclusion. This study confirmed that both Y chromosome microdeletions and CFTR gene mutations played important role in etiology of male infertility in Serbian infertile men. Genetic testing for Y chromosome microdeletions and CFTR gene mutations has been introduced in routine diagnostics and offered to couples undergoing assisted reproduction techniques. Considering that both the type of Y chromosome microdeletion and the type of CFTR mutation have a prognostic value, it is recommended that AZF and CFTR genotyping should not only be performed in patients with reduced sperm quality before undergoing assisted reproduction, but also for the purpose of preimplantation and

  19. Pseudomonas aeruginosa Reduces VX-809 Stimulated F508del-CFTR Chloride Secretion by Airway Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Bruce A Stanton

    Full Text Available P. aeruginosa is an opportunistic pathogen that chronically infects the lungs of 85% of adult patients with Cystic Fibrosis (CF. Previously, we demonstrated that P. aeruginosa reduced wt-CFTR Cl secretion by airway epithelial cells. Recently, a new investigational drug VX-809 has been shown to increase F508del-CFTR Cl secretion in human bronchial epithelial (HBE cells, and, in combination with VX-770, to increase FEV1 (forced expiratory volume in 1 second by an average of 3-5% in CF patients homozygous for the F508del-CFTR mutation. We propose that P. aeruginosa infection of CF lungs reduces VX-809 + VX-770- stimulated F508del-CFTR Cl secretion, and thereby reduces the clinical efficacy of VX-809 + VX-770.F508del-CFBE cells and primary cultures of CF-HBE cells (F508del/F508del were exposed to VX-809 alone or a combination of VX-809 + VX-770 for 48 hours and the effect of P. aeruginosa on F508del-CFTR Cl secretion was measured in Ussing chambers. The effect of VX-809 on F508del-CFTR abundance was measured by cell surface biotinylation and western blot analysis. PAO1, PA14, PAK and 6 clinical isolates of P. aeruginosa (3 mucoid and 3 non-mucoid significantly reduced drug stimulated F508del-CFTR Cl secretion, and plasma membrane F508del-CFTR.The observation that P. aeruginosa reduces VX-809 and VX-809 + VX-770 stimulated F508del CFTR Cl secretion may explain, in part, why VX-809 + VX-770 has modest efficacy in clinical trials.

  20. CFTR delivery to 25% of surface epithelial cells restores normal rates of mucus transport to human cystic fibrosis airway epithelium.

    Directory of Open Access Journals (Sweden)

    Liqun Zhang

    2009-07-01

    Full Text Available Dysfunction of CFTR in cystic fibrosis (CF airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL, mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE was used to test whether a human parainfluenza virus (PIV vector engineered to express CFTR (PIVCFTR could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl(- and Na(+ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%-65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients

  1. Funciones de los canales iónicos CFTR y ENAC en la fibrosis quística

    OpenAIRE

    Alejandra G. Palma; Basilio A. Kotsias; Gabriela I. Marino

    2014-01-01

    La fibrosis quística se debe a la ausencia o defecto del canal transmembrana regulador de la fibrosis quística (CFTR), un canal de cloruro codificado en el gen cftr que juega un papel clave en la homeostasis del agua e iones. El CFTR es activado por el AMPc y se localiza en las membranas apicales y basolaterales de las vías aéreas, intestino y glándulas exocrinas. Una de sus funciones primarias en los pulmones es mantener la capa de líquido superficial a través de su función de canal y regula...

  2. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cystic fibrosis transmembrane conductance... DEVICES Immunological Test Systems § 866.5900 Cystic fibrosis transmembrane conductance regulator (CFTR... intended as an aid in confirmatory diagnostic testing of individuals with suspected cystic fibrosis...

  3. Mutation Analysis of CFTR Gene in 70 Iranian Cystic Fibrosis Patients

    Directory of Open Access Journals (Sweden)

    Reza Alibakhshi Mahdi Zamani

    2006-03-01

    Full Text Available Cystic fibrosis (CF is the most common inherited disorder in Caucasian populations, with over 1400 cystic fibrosis transmembrane conductance regulator (CFTR mutations. The type of mutations and their distributions varies widely between different countries and/or ethnic groups. Seventy Iranian cystic fibrosis patients were screened for the CFTR gene mutation using ARMS/PCR (amplification refractory mutation system for the following mutations: ∆F508, N1303K, G542X, 1717-1G>A, R553X, W1282X, G551D, 621+1G>T, ∆I507 and R560T. Single strand conformation polymorphism (SSCP analysis of exons 3, 7, 10, 11 and 17b, including both the exon/intron junctions, of the CFTR gene was performed in patients in whom no mutation could be identified on one or both CFTR genes. As a result of this screening, only three mutations were found: ∆F508 mutation was found in 25 (17.8% alleles, N1303K in six (4.3% alleles and G542X in five (3.6% alleles. Thus, a total of 3 mutations cover 25.7% of CF alleles. These finding will be used for planning future screening and appropriate genetic counseling programs in Iranian CF patients.

  4. Analysis of mutations in the cystic fibrosis transmembrane regulator (CFTR gene in patients with obstructive azoospermia

    Directory of Open Access Journals (Sweden)

    Andrea L.F. Bernardino

    2003-01-01

    Full Text Available Congenital bilateral absence of the vas deferens (CBAVD accounts for 1%-2% of sterility in men. A high incidence of mutations, as well as the involvement of the 5T variant of the T tract length in intron 8 of the cystic fibrosis conductance regulator (CFTR gene, have been previously described in males with CBAVD. Herein we report the screening for mutations and for the 5T variant of the CFTR gene in 17 patients with CBAVD and three others with non-CABVD obstructive azoospermia. In the CBAVD group, three patients (15% were compound heterozygotes for mutations, and five patients (25% had a mutation in one allele and the 5T variant in the other; the 5T variant was also present in two other patients, one of them being homozygous. The most frequent mutation was DF508, present on five chromosomes (12.5%. A novel missense mutation (A399D was detected in a Japanese CBVAD patient. Our results yield further evidence for a strong association between male obstructive azoospermia caused by CBAVD and mutation/5T variant in the CFTR gene. The search for CFTR mutations in such patients is thus recommended for genetic counseling of couples who undergo assisted fertilization due to CBAVD.

  5. Comprehensive and accurate mutation scanning of the CFTR gene by two-dimensional DNA electrophoresis

    NARCIS (Netherlands)

    Wu, Y; Hofstra, RMW; Scheffer, H; Uitterlinden, AG; Mullaart, E; Buys, CHCM; Vijg, J

    1996-01-01

    The large number of possible disease causing mutations in the 27 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has severely limited direct diagnosis of cystic fibrosis (CF) patients and carriers by mutation detection. Here we show that in principle testing for mutation

  6. Cholic acid induces a Cftr dependent biliary secretion and liver growth response in mice

    NARCIS (Netherlands)

    Bodewes, Frank A. J. A.; Bijvelds, Marcel J.; de Vries, Willemien; Baller, Juul F. W.; Gouw, Annette S. H.; de Jonge, Hugo R.; Verkade, Henkjan J.

    2015-01-01

    The cause of Cystic fibrosis liver disease (CFLD), is unknown. It is well recognized that hepatic exposure to hydrophobic bile salts is associated with the development of liver disease. For this reason, we hypothesize that, CFTR dependent variations, in the hepatic handling of hydrophobic bile salts

  7. Cholic acid induces a Cftr dependent biliary secretion and liver growth response in mice

    NARCIS (Netherlands)

    F.A.J.A. Bodewes (Frank); M.J.C. Bijvelds (Marcel); De Vries, W. (Willemien); Baller, J.F.W. (Juul F. W.); A.S.H. Gouw (Annette); H.R. de Jonge (Hugo); H.J. Verkade (Henkjan)

    2015-01-01

    textabstractThe cause of Cystic fibrosis liver disease (CFLD), is unknown. It is well recognized that hepatic exposure to hydrophobic bile salts is associated with the development of liver disease. For this reason, we hypothesize that, CFTR dependent variations, in the hepatic handling of hydrophobi

  8. Personalized medicine in cystic fibrosis: genistein supplementation as a treatment option for patients with a rare S1045Y-CFTR mutation.

    Science.gov (United States)

    Arora, Kavisha; Yarlagadda, Sunitha; Zhang, Weiqiang; Moon, ChangSuk; Bouquet, Erin; Srinivasan, Saumini; Li, Chunying; Stokes, Dennis C; Naren, Anjaparavanda P

    2016-08-01

    Cystic fibrosis (CF) is a life-shortening disease caused by the mutations that generate nonfunctional CF transmembrane conductance regulator (CFTR) protein. A rare serine-to-tyrosine (S1045Y) CFTR mutation was earlier reported to result in CF-associated fatality. We identified an African-American patient with the S1045Y mutation in CFTR, as well as a stop-codon mutation, who has a mild CF phenotype. The underlying mechanism of CF caused by S1045Y-CFTR has not been elucidated. In this study, we determined that S1045Y-CFTR exhibits twofold attenuated function compared with wild-type (WT)-CFTR. We report that serine-to-tyrosine mutation leads to increased tyrosine phosphorylation of S1045Y-CFTR, followed by recruitment and binding of E3-ubiquitin ligase c-cbl, resulting in enhanced ubiquitination and passage of S1045Y-CFTR in the endosome/lysosome degradative compartments. We demonstrate that inhibition of tyrosine phosphorylation partially rescues S1045Y-CFTR surface expression and function. Based on our findings, it could be suggested that consuming genistein (a tyrosine phosphorylation inhibitor) would likely ameliorate CF symptoms in individuals with S1045Y-CFTR, providing a unique personalized therapy for this rare CF mutation. PMID:27261451

  9. Important role of platelets in modulating endotoxin-induced lung inflammation in CFTR-deficient mice.

    Directory of Open Access Journals (Sweden)

    Caiqi Zhao

    Full Text Available Mutation of CFTR (cystic fibrosis transmembrane conductance regulator leads to cystic fibrosis (CF. Patients with CF develop abnormalities of blood platelets and recurrent lung inflammation. However, whether CFTR-mutated platelets play a role in the development of lung inflammation is elusive. Therefore, we intratracheally challenged wildtype and F508del (a common type of CFTR mutation mice with LPS to observe changes of F508del platelets in the peripheral blood and indexes of lung inflammation (BAL neutrophils and protein levels. Furthermore, we investigated whether or not and how F508del platelets modulate the LPS-induced acute lung inflammation by targeting anti-platelet aggregation, depletion of neutrophils, reconstitution of bone marrow or neutrophils, blockade of P-selectin glycoprotein ligand-1 (PSGL-1, platelet activating factor (PAF, and correction of mutated CFTR trafficking. We found that LPS-challenged F508del mice developed severe thrombocytopenia and had higher levels of plasma TXB2 coincided with neutrophilic lung inflammation relative to wildtype control. Inhibition of F508del platelet aggregation or depletion of F508del neutrophils diminished the LPS-induced lung inflammation in the F508del mice. Moreover, wildtype mice reconstituted with either F508del bone marrow or neutrophils developed worse thrombocytopenia. Blocking PSGL-1, platelet activating factor (PAF, or rectifying trafficking of mutated CFTR in F508del mice diminished and alveolar neutrophil transmigration in the LPS-challenged F508del mice. These findings suggest that F508del platelets and their interaction with neutrophils are requisite for the development of LPS-induced lung inflammation and injury. As such, targeting platelets might be an emerging strategy for dampening recurrent lung inflammation in cystic fibrosis patients.

  10. The calpain, caspase 12, caspase 3 cascade leading to apoptosis is altered in F508del-CFTR expressing cells.

    Directory of Open Access Journals (Sweden)

    Mathieu Kerbiriou

    Full Text Available In cystic fibrosis (CF, the most frequent mutant variant of the cystic fibrosis transmembrane conductance regulator (CFTR, F508del-CFTR protein, is misfolded and retained in the endoplasmic reticulum (ER. We previously showed that the unfolded protein response (UPR may be triggered in CF. Since prolonged UPR activation leads to apoptosis via the calcium-calpain-caspase-12-caspase-3 cascade and because apoptosis is altered in CF, our aim was to compare the ER stress-induced apoptosis pathway between wild type (Wt and F508del-CFTR expressing cells. Here we show that the calcium-calpain-caspase-12-caspase-3 cascade is altered in F508del-CFTR expressing cells. We propose that this alteration is involved in the altered apoptosis triggering observed in CF.

  11. Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

    Science.gov (United States)

    He, Lihua; Kota, Pradeep; Aleksandrov, Andrei A; Cui, Liying; Jensen, Tim; Dokholyan, Nikolay V; Riordan, John R

    2013-02-01

    Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve ΔF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37°C (CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

  12. cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

    NARCIS (Netherlands)

    A.B. Vaandrager (Arie); S.M. Lohmann (Suzanne); H.R. de Jonge (Hugo); W.C. Poller; B.C. Tilly (Bernard); A. Smolenski; S. Schneider-Rasp; A.G. Bot (Alice); M.J. Edixhoven (Marcel); B.J. Scholte (Bob); T. Jarchau; U. Walter

    1997-01-01

    textabstractIn order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was ana

  13. Isotype-specific activation of cystic fibrosis transmembrane conductance regulator-chloride channels by cGMP-dependent protein kinase II

    NARCIS (Netherlands)

    P.J. French (Pim); J. Bijman (Jan); M.J. Edixhoven (Marcel); A.B. Vaandrager (Arie); B.J. Scholte (Bob); S.M. Lohmann (Suzanne); A.C. Nairn; H.R. de Jonge (Hugo)

    1995-01-01

    textabstractType II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type I alpha cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl- channel in excis

  14. Cystic fibrosis transmembrane conductance regulator (CFTR) gene abnormalities in Indian males with congenital bilateral absence of vas deferens & renal anomalies

    Science.gov (United States)

    Gajbhiye, Rahul; Kadam, Kaushiki; Khole, Aalok; Gaikwad, Avinash; Kadam, Seema; Shah, Rupin; Kumaraswamy, Rangaswamy; Khole, Vrinda

    2016-01-01

    Background & objectives: The role of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in congenital bilateral absence of vas deferens and unilateral renal agenesis (CBAVD-URA) has been controversial. Here, we report the cases of five Indian males with CBAVD-URA. The objective was to evaluate the presence or absence of CFTR gene mutations and variants in CBAVD-URA. The female partners of these males were also screened for cystic fibrosis (CF) carrier status. Methods: Direct DNA sequencing of CFTR gene was carried out in five Indian infertile males having CBAVD-URA. Female partners (n=5) and healthy controls (n=32) were also screened. Results: Three potential regulatory CFTR gene variants (c.1540A>G, c.2694T>G and c.4521G>A) were detected along with IVS8-5T mutation in three infertile males with CBAVD-URA. Five novel CFTR gene variants (c.621+91A>G, c.2752+106A>T, c.2751+85_88delTA, c.3120+529InsC and c.4375-69C>T), four potential regulatory CFTR gene variants (M470V, T854T, P1290P, Q1463Q) and seven previously reported CFTR gene variants (c.196+12T>C, c.875+40A>G, c.3041-71G>C, c.3271+42A>T, c.3272-93T>C, c.3500-140A>C and c.3601-65C>A) were detected in infertile men having CBAVD and renal anomalies Interpretation & conclusions: Based on our findings, we speculate that CBAVD-URA may also be attributed to CFTR gene mutations and can be considered as CFTR-related disorder (CFTR-RD). The CFTR gene mutation screening may be offered to CBAVD-URA men and their female partners undergoing ICSI. Further studies need to be done in a large sample to confirm the findings. PMID:27488005

  15. Phosphazene-promoted anionic polymerization

    KAUST Repository

    Zhao, Junpeng

    2014-01-01

    In the recent surge of metal-free polymerization techniques, phosphazene bases have shown their remarkable potential as organic promoters/catalysts for the anionic polymerization of various types of monomers. By complexation with the counterion (e.g. proton or lithium cation), phosphazene base significantly improve the nucleophilicity of the initiator/chain-end resulting in rapid and usually controlled anionic/quasi-anionic polymerization. In this review, we will introduce the general mechanism, i.e. in situ activation (of initiating sites) and polymerization, and summarize the applications of such a mechanism on macromolecular engineering toward functionalized polymers, block copolymers and complex macromolecular architectures.

  16. The Novel CFTR Mutation A457P in a Male with a Delayed Diagnosis of Cystic Fibrosis

    OpenAIRE

    Zuckerman, Jonathan B.; Yarmus, Lonny B.; Sosnay, Patrick R.; Cole, Kate H.

    2011-01-01

    Cystic fibrosis (CF) is an autosomal recessive disease that may be caused by more than 1000 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We describe the case of a CF patient who was initially diagnosed at 16 years of age after presenting with mild respiratory compromise and pancreatic sufficiency. When genetic testing was first performed using a CF mutation panel, only a single F508del CFTR allele was identified. We subsequently performed testing...

  17. Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis

    OpenAIRE

    Sonawane Nitin D; Previs Stephen F; Jiang Dechen; Ruddy Jennifer; Manson Mary E; West Richard H; Fang Danjun; Burgess James D; Kelley Thomas J

    2010-01-01

    Abstract Background Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis. Methods Electrochemical detectio...

  18. Tetramethylpyrazine stimulates cystic fibrosis transmembrane conductance regulator-mediated anion secretion in distal colon of rodents

    Institute of Scientific and Technical Information of China (English)

    Qiong He; Jin-Xia Zhu; Ying Xing; Lai-Ling Tsang; Ning Yang; Dewi Kenneth Rowlands; Yiu-Wa Chung; Hsiao-Chang Chan

    2005-01-01

    AIM: To investigate the effect of tetramethylpyrazine (TMP), an active compound from Ligustiun Wollichii Franchat, on electrolyte transport across the distal colon of rodents and the mechanism involved.METHODS: The short-circuit current (ISC) technique in conjunction with pharmacological agents and specific inhibitors were used in analyzing the electrolyte transport across the distal colon of rodents. The underlying cellular signaling mechanism was investigated by radioimmunoassay analysis (RIA) and a special mouse model of cystic fibrosis.RESULTS: TMP stimulated a concentration-dependent rise in ISC, which was dependent on both Cl- and HCO3-, and inhibited by apical application of diphenylamine-2,2'-dicarboxylic acid (DPC) and glibenclamide, but resistant to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS). Removal of Na+ from basolateral solution almost completely abolished the ISC response to TMP, but it was insensitive to apical Na+ replacement or apical Na+channel blocker, amiloride. Pretreatment of colonic mucosa with BAPTA-AM, a membrane-permeable selective Ca2+chelator, did not significantly alter the TMP-induced ISC. No additive effect of forskolin and 3-isobutyl-1-methylxanthine (IBMX) was observed on the TMP-induced ISc, but it was significantly reduced by a protein kinase A inhibitor, H89.RIA results showed that TMP (1 mmol/L) elicited a significant increase in cellular cAMP production, which was similar to that elicited by the adenylate cyclase activator, forskolin (10 μmol/L). The TMP-elicited ISC as well as forskolin- or IBMX-induced ISC were abolished in mice with homozygous mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) presenting defective CFTR functions and secretions.CONCLUSION: TMP may stimulate cAMP-dependent and CFTR-mediated Cl- and HCO3- secretion. This may have implications in the future development of alternative treatment for constipation.

  19. Lack of CFTR in skeletal muscle predisposes to muscle wasting and diaphragm muscle pump failure in cystic fibrosis mice.

    Directory of Open Access Journals (Sweden)

    Maziar Divangahi

    2009-07-01

    Full Text Available Cystic fibrosis (CF patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and co-localized with sarcoplasmic reticulum-associated proteins. CFTR-deficient myotubes exhibit augmented levels of intracellular calcium after KCl-induced depolarization, and exposure to an inflammatory milieu induces excessive NF-kB translocation and cytokine/chemokine gene upregulation. To determine the effects of an inflammatory environment in vivo, sustained pulmonary infection with Pseudomonas aeruginosa was produced, and under these conditions diaphragmatic force-generating capacity is selectively reduced in Cftr(-/- mice. This is associated with exaggerated pro-inflammatory cytokine expression as well as upregulation of the E3 ubiquitin ligases (MuRF1 and atrogin-1 involved in muscle atrophy. We conclude that an intrinsic alteration of function is linked to the absence of CFTR from skeletal muscle, leading to dysregulated calcium homeostasis, augmented inflammatory/atrophic gene expression signatures, and increased diaphragmatic weakness during pulmonary infection. These findings reveal a previously unrecognized role for CFTR in skeletal muscle function that may have major implications for the pathogenesis of cachexia and respiratory muscle pump failure in CF patients.

  20. Acute administration of ivacaftor to people with cystic fibrosis and a G551D-CFTR mutation reveals smooth muscle abnormalities

    Science.gov (United States)

    Adam, Ryan J.; Hisert, Katherine B.; Dodd, Jonathan D.; Grogan, Brenda; Launspach, Janice L.; Barnes, Janel K.; Gallagher, Charles G.; Sieren, Jered P.; Gross, Thomas J.; Fischer, Anthony J.; Cavanaugh, Joseph E.; Hoffman, Eric A.; Singh, Pradeep K.; Welsh, Michael J.; McKone, Edward F.; Stoltz, David A.

    2016-01-01

    BACKGROUND. Airflow obstruction is common in cystic fibrosis (CF), yet the underlying pathogenesis remains incompletely understood. People with CF often exhibit airway hyperresponsiveness, CF transmembrane conductance regulator (CFTR) is present in airway smooth muscle (ASM), and ASM from newborn CF pigs has increased contractile tone, suggesting that loss of CFTR causes a primary defect in ASM function. We hypothesized that restoring CFTR activity would decrease smooth muscle tone in people with CF. METHODS. To increase or potentiate CFTR function, we administered ivacaftor to 12 adults with CF with the G551D-CFTR mutation; ivacaftor stimulates G551D-CFTR function. We studied people before and immediately after initiation of ivacaftor (48 hours) to minimize secondary consequences of CFTR restoration. We tested smooth muscle function by investigating spirometry, airway distensibility, and vascular tone. RESULTS. Ivacaftor rapidly restored CFTR function, indicated by reduced sweat chloride concentration. Airflow obstruction and air trapping also improved. Airway distensibility increased in airways less than 4.5 mm but not in larger-sized airways. To assess smooth muscle function in a tissue outside the lung, we measured vascular pulse wave velocity (PWV) and augmentation index, which both decreased following CFTR potentiation. Finally, change in distensibility of <4.5-mm airways correlated with changes in PWV. CONCLUSIONS. Acute CFTR potentiation provided a unique opportunity to investigate CFTR-dependent mechanisms of CF pathogenesis. The rapid effects of ivacaftor on airway distensibility and vascular tone suggest that CFTR dysfunction may directly cause increased smooth muscle tone in people with CF and that ivacaftor may relax smooth muscle. FUNDING. This work was funded in part from an unrestricted grant from the Vertex Investigator-Initiated Studies Program. PMID:27158673

  1. Pentaarylfullerenes as noncoordinating cyclopentadienyl anions

    NARCIS (Netherlands)

    Bouwkamp, Marco W.; Meetsma, Auke

    2009-01-01

    The first example of an early-transition-metal complex involving a pentaarylfullerene was prepared. Instead of half-sandwich complexes, solvent separated ion pairs were obtained in which the pentaarylfullerene moiety acts as noncoordinating cyclopentadienyl anion.

  2. Anion Transport with Chalcogen Bonds.

    Science.gov (United States)

    Benz, Sebastian; Macchione, Mariano; Verolet, Quentin; Mareda, Jiri; Sakai, Naomi; Matile, Stefan

    2016-07-27

    In this report, we introduce synthetic anion transporters that operate with chalcogen bonds. Electron-deficient dithieno[3,2-b;2',3'-d]thiophenes (DTTs) are identified as ideal to bind anions in the focal point of the σ holes on the cofacial endocyclic sulfur atoms. Anion binding in solution and anion transport across lipid bilayers are found to increase with the depth of the σ holes of the DTT anionophores. These results introduce DTTs and related architectures as a privileged motif to engineer chalcogen bonds into functional systems, complementary in scope to classics such as 2,2'-bipyrroles or 2,2'-bipyridines that operate with hydrogen bonds and lone pairs, respectively. PMID:27433964

  3. Differential expression of CFTR gene in the mouse intestinal tissues%CFTR 基因在小鼠肠道组织中的差异表达

    Institute of Scientific and Technical Information of China (English)

    王月影; 韩莹倩; 查光明; 汪新建; 李和平

    2014-01-01

    Object This experiment was conducted to study the relationship between CFTR gene expression in the intestinal tissues and secretory diarrhea.Methods Twenty-four Kunming mice were selected, half male and half female, and were randomly divided into 3 groups ( n=8 in each group):control group with intraperitoneal injection of 0.2 mL nor-mal saline, and the experimental group of mice by intraperitoneal injection of lipopolysaccharide(LPS) (6 mg/kg· bw). The mental state and intestinal morphology of the mice at 1 h and 8 h after LPS injection were observed to assess whether the secretory diarrhea model was successfully established.The expression of CFTR gene segments of intestine tissue was de-tected by fluorescence quantitative PCR.Results LPS induced secretory diarrhea.CFTR gene was expressed in the mouse duodenum, jejunum, ileum and colon tissues with different expression abundance.It was highest in the colon, but the difference was not significant between intestinal segments.Compared with the control group, LPS up-regulated the tran-scription level of CFTR gene in the duodenum, jejunum and ileum, and down-regulated the transcription of CFTR gene in the colon.Conclusions The results of our study suggest that the changes of the transcriptional level of CFTR gene are closely related with the diarrhea induced by LPS and the effects in different intestinal segments on the diarrhea is different. The jejunum plays a crucial role and the colon plays a least role in the Cl-secretion.%目的:研究肠道组织CFTR基因表达与分泌性腹泻发生的关系。方法选取KM小鼠24只,雌雄各半,随机分为3组(每组8只):对照组经小鼠腹腔注射0.2 mL生理盐水,实验组小鼠经腹腔注射LPS[6 mg/(kg· bw)]分别作用1 h、8 h,于注射后通过小鼠精神状态、肠道组织形态学判定分泌性腹泻模型的建立,利用荧光定量PCR法检测各段肠道组织CFTR基因的表达。结果 LPS成功诱导小鼠发生了分泌

  4. Bound anionic states of adenine

    Energy Technology Data Exchange (ETDEWEB)

    Haranczyk, Maciej; Gutowski, Maciej S; Li, Xiang; Bowen, Kit H

    2007-03-20

    Anionic states of nucleic acid bases are involved in DNA damage by low-energy electrons and in charge transfer through DNA. Previous gas phase studies of free, unsolvated nucleic acid base parent anions probed only dipole-bound states, which are not present in condensed phase environments, but did not observe valence anionic states, which for purine bases, are thought to be adiabatically unbound. Contrary to this expectation, we have demonstrated that some thus far ignored tautomers of adenine, which result from enamine-imine transformations, support valence anionic states with electron vertical detachment energies as large as 2.2 eV, and at least one of these anionic tautomers is adiabatically bound. Moreover, we predict that the new anionic tautomers should also dominate in solutions and should be characterized by larger values of electron vertical detachment energy than the canonical valence anion. All of the new-found anionic tautomers might be formed in the course of dissociative electron attachment followed by a hydrogen atom attachment to a carbon atom, and they might affect the structure and properties of DNA and RNA exposed to low-energy electrons. The discovery of these valence anionic states of adenine was facilitated by the development of: (i) a new experimental method for preparing parent anions of nucleic acid bases for photoelectron experiments, and (ii) a new combinatorial/ quantum chemical approach for identification of the most stable tautomers of organic molecules. The computational portion of this work was supported by the: (i) Polish State Committee for Scientific Research (KBN) Grants: DS/8000-4-0140-7 (M.G.) and N204 127 31/2963 (M.H.), (ii) European Social Funds (EFS) ZPORR/2.22/II/2.6/ARP/U/2/05 (M.H.), and (iii) US DOE Office of Biological and Environmental Research, Low Dose Radiation Research Program (M.G.). M.H. holds the Foundation for Polish Science (FNP) award for young scientists. The calculations were performed at the Academic

  5. CFTR depletion results in changes in fatty acid composition and promotes lipogenesis in intestinal Caco 2/15 cells.

    Directory of Open Access Journals (Sweden)

    Geneviève Mailhot

    Full Text Available BACKGROUND: Abnormal fatty acid composition (FA in plasma and tissue lipids frequently occurs in homozygous and even in heterozygous carriers of cystic fibrosis transmembrane conductance regulator (CFTR mutations. The mechanism(s underlying these abnormalities remained, however, poorly understood despite the potentially CFTR contributing role. METHODOLOGY/PRINCIPAL FINDINGS: The aim of the present study was to investigate the impact of CFTR depletion on FA uptake, composition and metabolism using the intestinal Caco-2/15 cell line. shRNA-mediated cftr gene silencing induced qualitative and quantitative modifications in FA composition in differentiated enterocytes as determined by gas-liquid chromatography. With the cftr gene disruption, there was a 1,5 fold increase in the total FA amount, largely attributable to monounsaturated and saturated FA compared to controls. The activity of delta-7 desaturase, estimated by the 16:1(n-7/16:0, was significantly higher in knockdown cells and consistent with the striking elevation of the n-7 FA family. When incubated with [14C]-oleic acid, CFTR-depleted cells were capable of quick incorporation and export to the medium concomitantly with the high protein expression of L-FABP known to promote intracellular FA trafficking. Accordingly, lipoprotein vehicles (CM, VLDL, LDL and HDL, isolated from CFTR knockdown cells, exhibited higher levels of radiolabeled FA. Moreover, in the presence of [14C]-acetate, knockdown cells exhibited enhanced secretion of newly synthesized phospholipids, triglycerides, cholesteryl esters and free FA, thereby suggesting a stimulation of the lipogenic pathway. Conformably, gene expression of SREBP-1c, a key lipogenic transcription factor, was increased while protein expression of the phosphorylated and inactive form of acetylCoA carboxylase was reduced, confirming lipogenesis induction. Finally, CFTR-depleted cells exhibited lower gene expression of transcription factors (PPARalpha

  6. Nanostructure-controlled anion exchange membranes for fuel cell applications by high-energy heavy-ion irradiation. Preparation and characterization of anion exchange membranes

    International Nuclear Information System (INIS)

    Heavy ions at kinetic energies typically from several hundreds of MeV to a few GeV passing through a polymer substrate induce a continuous trail of excitations and ionizations called latent tracks. We used a direct ion-track grafting method for preparation of anion exchange membranes for fuel cells. The functional anion exchange groups were introduced inside the latent tracks, thereby achieving OH--conductive channels through the thickness. These straight channels increased conductivities, while the isolated cylindrical structure of tracks restricted the water uptake. (author)

  7. F508del-CFTR rescue: a matter of cell stress response.

    Science.gov (United States)

    Nieddu, Erika; Pollarolo, Benedetta; Merello, Luisa; Schenone, Silvia; Mazzei, Mauro

    2013-01-01

    Cystic fibrosis (CF) is a common inherited fatal disease affecting 70,000 people worldwide, with a median predicted age of survival of approximately 38 years. The deletion of Phenylalanine in position 508 of the Cystic Fibrosis Transmembrane conductance Regulator (F508del-CFTR) is the most common mutation in CF patients: the deleted protein, not properly folded, is degraded. To date no commercial drugs are available. Low temperature, some osmolytes and conditions able to induce heat shock protein 70 (Hsp70) expression and heat shock cognate 70 (Hsc70) inhibition result in F508del-CFTR rescue, hence restoring its physiological function: this review sheds light on the correlation between these several evidences. Interestingly, all these approaches have a role in the cell stress response (CSR), a set of cell reactions to stress. In addition, unpredictably, F508del-CFTR rescue has to be considered in the frame of CSR: entities that induce - or are induced during - the CSR are, in general, also able to correct trafficking defect of CFTR. Specifically, the low temperature induces, by definition, a CSR; osmolytes, such as glycerol and trimethylamine N-oxide (TMAO), are products of the CSR; pharmacological correctors, such as Matrine and 4-phenylbutirric acid (4PBA), down-regulate the constitutive Hsc70 in favor of an up-regulation of the inducible chaperone Hsp70, another component of the CSR. The identification of a common mechanism of action for different types of correctors could drive the discovery of new active molecules in CF, overcoming methods clinically inapplicable, such as the low temperature. PMID:23331026

  8. Inflammation and CFTR: Might Neutrophils be the Key in Cystic Fibrosis?

    OpenAIRE

    Witko-Sarsat, V; Sermet-Gaudelus, I; Lenoir, G.; Descamps-Latscha, B

    1999-01-01

    The aim of this hypothesis is to provide new insights into the still unclear mechanisms governing airway inflammation in cystic fibrosis. Although the genetic basis of cystic fibrosis as well as the molecular structure of cystic fibrosis transmembrane regulator (CFTR), the mutated protein which causes the disease, have been well defined, a clear relationship between the genetic defect and the pulmonary pathophysiology, especially chronic infections and neutrophil-dominated airway inflammation...

  9. Phosphorylation of CFTR and other membrane proteins: a role in biogenesis and traffic?

    OpenAIRE

    Romeiras, Francisco Maria de Sousa de Macedo Malta, 1986-

    2009-01-01

    Tese de mestrado, Bioquímica (Bioquímica Médica), Universidade de Lisboa, Faculdade de Ciências, 2009 Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder among Caucasians, is caused by mutations in the gene that encodes for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). With more than 1600 mutations reported, a single mutation– the deletion of phenylalanine residue at position 508 – accounts for more than 70% of chromosomes worldwide. The presence of ...

  10. Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in allergic bronchopulmonary aspergillosis.

    OpenAIRE

    Miller, P. W.; Hamosh, A.; Macek, M.; Greenberger, P. A.; MacLean, J; Walden, S M; Slavin, R G; Cutting, G R

    1996-01-01

    The etiology of allergic bronchopulmonary aspergillosis (ABPA) is not well understood. A clinical phenotype resembling the pulmonary disease seen in cystic fibrosis (CF) patients can occur in some individuals with ABPA. Reports of familial occurrence of ABPA and increased incidence in CF patients suggest a possible genetic basis for the disease. To test this possibility, the entire coding region of the cystic fibrosis transmembrane regulator (CFTR) gene was analyzed in 11 individuals who met ...

  11. Impact of Cystic Fibrosis Transmembrane Regulator (CFTR) gene mutations on male infertility

    OpenAIRE

    Jlenia Elia; Rossella Mazzilli; Michele Delfino; Maria Piane; Cristina Bozzao; Vincenzo Spinosa; Luciana Chessa; Fernando Mazzilli

    2014-01-01

    Objective. The aim of this study was to evaluate the prevalence of most common mutations and intron 8 5T (IVS8-5T) polymorphism of CFTR gene in Italian: a) azoospermic males; b) non azoospermic subjects, male partners of infertile couples enrolled in assisted reproductive technology (ART) programs. Material and methods. We studied 242 subjects attending our Andrology Unit (44 azoospermic subjects and 198 non azoospermic subjects, male partners of infertile couples enrolled in ART programs). S...

  12. Chemically modified peptide scaffolds target the CFTR-associated ligand PDZ domain.

    Directory of Open Access Journals (Sweden)

    Jeanine F Amacher

    Full Text Available PDZ domains are protein-protein interaction modules that coordinate multiple signaling and trafficking pathways in the cell and that include active therapeutic targets for diseases such as cancer, cystic fibrosis, and addiction. Our previous work characterized a PDZ interaction that restricts the apical membrane half-life of the cystic fibrosis transmembrane conductance regulator (CFTR. Using iterative cycles of peptide-array and solution-binding analysis, we targeted the PDZ domain of the CFTR-Associated Ligand (CAL, and showed that an engineered peptide inhibitor rescues cell-surface expression of the most common CFTR disease mutation ΔF508. Here, we present a series of scaffolds containing chemically modifiable side chains at all non-motif positions along the CAL PDZ domain binding cleft. Concordant equilibrium dissociation constants were determined in parallel by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance techniques, confirming robust affinity for each scaffold and revealing an enthalpically driven mode of inhibitor binding. Structural studies demonstrate a conserved binding mode for each peptide, opening the possibility of combinatorial modification. Finally, we diversified one of our peptide scaffolds with halogenated substituents that yielded modest increases in binding affinity. Overall, this work validates our approach and provides a stereochemical foundation for further CAL inhibitor design and screening.

  13. Detection of phospho-sites generated by protein kinase CK2 in CFTR: mechanistic aspects of Thr1471 phosphorylation.

    Directory of Open Access Journals (Sweden)

    Andrea Venerando

    Full Text Available By mass spectrometry analysis of mouse Cystic Fibrosis Transmembrane-conductance Regulator (mCFTR expressed in yeast we have detected 21 phosphopeptides accounting for 22 potential phospho-residues, 12 of which could be unambiguously assigned. Most are conserved in human CFTR (hCFTR and the majority cluster in the Regulatory Domain, lying within consensus sequences for PKA, as identified in previous mammalian studies. This validates our yeast expression model. A number of phospho-residues were novel and human conserved, notably mouse Ser670, Ser723, Ser737, and Thr1467, that all lie in acidic sequences, compatible with their phosphorylation by protein kinase CK2. Thr1467 is localized in the C-terminal tail, embedded in a functionally important and very acidic sequence (EETEEE which displays an optimal consensus for protein kinase CK2. Herein, we show that Thr1467, homologous to human Thr1471 is readily phosphorylated by CK2. Indeed a 42 amino acid peptide encompassing the C-terminal segment of human CFTR is readily phosphorylated at Thr1471 with favorable kinetics (Km 1.7 µM by CK2 holoenzyme, but neither by its isolated catalytic subunit nor by other acidophilic Ser/Thr kinases (CK1, PLK2/3, GCK/FAM20C. Our finding that by treating CFTR expressing BHK cells with the very specific CK2 inhibitor CX4945, newly synthesized wild type CFTR (and even more its Phe508del mutant accumulates more abundantly than in the absence of CK2 inhibitor, supports the conclusion that phosphorylation of CFTR by CK2 correlates with decreased stability of the protein.

  14. ΔF508 CFTR surface stability is regulated by DAB2 and CHIP-mediated ubiquitination in post-endocytic compartments.

    Science.gov (United States)

    Fu, Lianwu; Rab, Andras; Tang, Li ping; Bebok, Zsuzsa; Rowe, Steven M; Bartoszewski, Rafal; Collawn, James F

    2015-01-01

    The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2) and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2) on low-temperature rescued ΔF508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ΔF508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ΔF508 CFTR by ~18% and ~91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ΔF508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ΔF508 CFTR. To determine where and when the ubiquitination occurs during ΔF508 CFTR turnover, we monitored the ubiquitination of rescued ΔF508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ΔF508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a post-endocytic compartment. This post-endocytic ubiquination of ΔF508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ΔF508 CFTR.

  15. The GAP Portion of Pseudomonas Aeruginosa Type III Secreted Toxin ExoS Upregulates Total and Surface Levels of Wild Type CFTR

    Directory of Open Access Journals (Sweden)

    Deepali N. Tukaye

    2013-02-01

    Full Text Available Background: Pseudomonas aeruginosa (PA infections account for a large percentage of fatal hospital acquired pneumonias. One of the PA Type III secreted toxin (TTST ExoS, a bifunctional protein with N-terminal GTPase activating protein (GAP and C-terminal ADP rybosyl transferase (ADPRT activities, significantly contributes to PA virulence by targeting small molecular weight G-proteins (SMWGP. In this study, we have looked at one of the mechanisms by which the GAP portion of ExoS (ExoS-GAP mediates cellular toxicity. Methods: The effects of ExoS-GAP on CFTR trafficking were studied in CFBE41o- Kir 2.2 and MDCK cell lines stably expressing CFTR using a transient transfection system. Results: Transient transfection of ExoS-GAP increased the total and surface protein levels of mature wild type CFTR in epithelial cells stably expressing wild type (WT CFTR. The effect of ExoS-GAP was specific to CFTR in bronchial epithelial cells since it did not affect the total protein levels of Na+/K+ATPase, another membrane protein. A point mutation in the ExoS GAP domain (R146K, known to disrupt its catalytic GAP activity, abolished the effect of ExoS-GAP on WT CFTR. Lysosomal inhibition studies with Bafilomycin A1 indicate that ExoS-GAP decreased lysosomal degradation of the mature WT CFTR with concomitant increase in the total levels of mature WT CFTR. However, ExoS-GAP did not increase the total protein levels of ∆F508CFTR. Conclusion: The GAP portion of the PA TTST ExoS increases the total and surface levels of wild type CFTR in vitro mammalian cell system. The effect of ExoS-GAP on WT CFTR total protein levels provides new insight into understanding the virulent pathophysiology of PA infections.

  16. Action spectroscopy of gas-phase carboxylate anions by multiple photon IR electron detachment/attachment

    CERN Document Server

    Steill, Jeffrey D

    2008-01-01

    We report on a form of gas-phase anion action spectroscopy based on infrared multiple photon electron detachment and subsequent capture of the free electrons by a neutral electron scavenger in a Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer. This method allows one to obtain background-free spectra of strongly bound anions, for which no dissociation channels are observed. The first gas-phase spectra of the acetate and propionate anions are presented using SF6 as electron scavenger and a free electron laser as source of intense and tunable infrared radiation. To validate the method, we compare infrared spectra obtained through multiple photon electron detachment/attachment and multiple photon dissociation for the benzoate anion. In addition, different electron acceptors are used, comparing both associative and dissociative electron capture. The relative energies of dissociation (by CO2 loss) and electron detachment are investigated for all three anions by DFT and CCSD(T) methods. DFT calcu...

  17. Pu Anion Exchange Process Intensification

    Energy Technology Data Exchange (ETDEWEB)

    Taylor-Pashow, K. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

    2015-10-08

    This project seeks to improve the efficiency of the plutonium anion-exchange process for purifying Pu through the development of alternate ion-exchange media. The objective of the project in FY15 was to develop and test a porous foam monolith material that could serve as a replacement for the current anion-exchange resin, Reillex® HPQ, used at the Savannah River Site (SRS) for purifying Pu. The new material provides advantages in efficiency over the current resin by the elimination of diffusive mass transport through large granular resin beads. By replacing the large resin beads with a porous foam there is much more efficient contact between the Pu solution and the anion-exchange sites present on the material. Several samples of a polystyrene based foam grafted with poly(4-vinylpyridine) were prepared and the Pu sorption was tested in batch contact tests.

  18. MARCH2 regulates autophagy by promoting CFTR ubiquitination and degradation and PIK3CA-AKT-MTOR signaling.

    Science.gov (United States)

    Xia, Dan; Qu, Liujing; Li, Ge; Hongdu, Beiqi; Xu, Chentong; Lin, Xin; Lou, Yaxin; He, Qihua; Ma, Dalong; Chen, Yingyu

    2016-09-01

    MARCH2 (membrane-associated RING-CH protein 2), an E3 ubiquitin ligase, is mainly associated with the vesicle trafficking. In the present study, for the first time, we demonstrated that MARCH2 negatively regulates autophagy. Our data indicated that overexpression of MARCH2 impaired autophagy, as evidenced by attenuated levels of LC3B-II and impaired degradation of endogenous and exogenous autophagic substrates. By contrast, loss of MARCH2 expression had the opposite effects. In vivo experiments demonstrate that MARCH2 knockout mediated autophagy results in an inhibition of tumorigenicity. Further investigation revealed that the induction of autophagy by MARCH2 deficiency was mediated through the PIK3CA-AKT-MTOR signaling pathway. Additionally, we found that MARCH2 interacts with CFTR (cystic fibrosis transmembrane conductance regulator), promotes the ubiquitination and degradation of CFTR, and inhibits CFTR-mediated autophagy in tumor cells. The functional PDZ domain of MARCH2 is required for the association with CFTR. Thus, our study identified a novel negative regulator of autophagy and suggested that the physical and functional connection between the MARCH2 and CFTR in different conditions will be elucidated in the further experiments. PMID:27308891

  19. Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR.

    Science.gov (United States)

    Stevers, Loes M; Lam, Chan V; Leysen, Seppe F R; Meijer, Femke A; van Scheppingen, Daphne S; de Vries, Rens M J M; Carlile, Graeme W; Milroy, Lech G; Thomas, David Y; Brunsveld, Luc; Ottmann, Christian

    2016-03-01

    Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein-protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3-CFTR interface might offer an approach for cystic fibrosis therapeutics.

  20. A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR.

    Science.gov (United States)

    Tosco, A; De Gregorio, F; Esposito, S; De Stefano, D; Sana, I; Ferrari, E; Sepe, A; Salvadori, L; Buonpensiero, P; Di Pasqua, A; Grassia, R; Leone, C A; Guido, S; De Rosa, G; Lusa, S; Bona, G; Stoll, G; Maiuri, M C; Mehta, A; Kroemer, G; Maiuri, L; Raia, V

    2016-08-01

    We previously reported that the combination of two safe proteostasis regulators, cysteamine and epigallocatechin gallate (EGCG), can be used to improve deficient expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients homozygous for the CFTR Phe508del mutation. Here we provide the proof-of-concept that this combination treatment restored CFTR function and reduced lung inflammation (Ptreatment in vitro. We assessed individual responses to cysteamine plus EGCG in a single-centre, open-label phase-2 trial. The combination treatment decreased sweat chloride from baseline, increased both CFTR protein and function in nasal cells, restored autophagy in such cells, decreased CXCL8 and TNF-α in the sputum, and tended to improve respiratory function. These positive effects were particularly strong in patients carrying Phe508del CFTR mutations in homozygosity or heterozygosity. However, a fraction of patients bearing other CFTR mutations failed to respond to therapy. Importantly, the same patients whose primary nasal brushed cells did not respond to cysteamine plus EGCG in vitro also exhibited deficient therapeutic responses in vivo. Altogether, these results suggest that the combination treatment of cysteamine plus EGCG acts 'on-target' because it can only rescue CFTR function when autophagy is functional (in mice) and improves CFTR function when a rescuable protein is expressed (in mice and men). These results should spur the further clinical development of the combination treatment. PMID:27035618

  1. The many ways of making anionic clays

    Indian Academy of Sciences (India)

    Michael Rajamathi; Grace S Thomas; P Vishnu Kamath

    2001-10-01

    Together with hydrotalcite-like layered double hydroxides, bivalent and trivalent metal hydroxides and their hydroxy salts are actually anionic clays consisting of positively charged hydroxide layers with anions intercalated in the interlayer region. The anionic clays exhibit anion sorption, anion diffusion and exchange properties together with surface basicity making them materials of importance for many modern applications. In this article, we discuss many different ways of making anionic clays and compare and contrast the rich diversity of this class of materials with the better-known cationic clays.

  2. Plant guard cell anion channel SLAC1 regulates stomatal closure

    OpenAIRE

    Vahisalu, Triin

    2010-01-01

    Plants are rooted to their growth place; therefore it is important that they react adequately to changes in environmental conditions. Stomatal pores, which are formed of a pair of guard cells in leaf epidermis, regulate plant gas-exchange. Importantly, guard cells protect the plant from desiccation in drought conditions by reducing the aperture of the stomatal pore. They serve also as the first barrier against the major air pollutant ozone, but the behaviour of guard cells during ozone expo...

  3. Serum- and glucocorticoid-induced protein kinase 1 (SGK1) increases the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells by phosphorylating Shank2E protein.

    Science.gov (United States)

    Koeppen, Katja; Coutermarsh, Bonita A; Madden, Dean R; Stanton, Bruce A

    2014-06-13

    The glucocorticoid dexamethasone increases cystic fibrosis transmembrane conductance regulator (CFTR) abundance in human airway epithelial cells by a mechanism that requires serum- and glucocorticoid-induced protein kinase 1 (SGK1) activity. The goal of this study was to determine whether SGK1 increases CFTR abundance by phosphorylating Shank2E, a PDZ domain protein that contains two SGK1 phosphorylation consensus sites. We found that SGK1 phosphorylates Shank2E as well as a peptide containing the first SGK1 consensus motif of Shank2E. The dexamethasone-induced increase in CFTR abundance was diminished by overexpression of a dominant-negative Shank2E in which the SGK1 phosphorylation sites had been mutated. siRNA-mediated reduction of Shank2E also reduced the dexamethasone-induced increase in CFTR abundance. Taken together, these data demonstrate that the glucocorticoid-induced increase in CFTR abundance requires phosphorylation of Shank2E at an SGK1 consensus site.

  4. Anion binding in biological systems

    Science.gov (United States)

    Feiters, Martin C.; Meyer-Klaucke, Wolfram; Kostenko, Alexander V.; Soldatov, Alexander V.; Leblanc, Catherine; Michel, Gurvan; Potin, Philippe; Küpper, Frithjof C.; Hollenstein, Kaspar; Locher, Kaspar P.; Bevers, Loes E.; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

    2009-11-01

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L3 (2p3/2) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  5. Anion binding in biological systems

    Energy Technology Data Exchange (ETDEWEB)

    Feiters, Martin C [Department of Organic Chemistry, Institute for Molecules and Materials, Faculty of Science, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen (Netherlands); Meyer-Klaucke, Wolfram [EMBL Hamburg Outstation at DESY, Notkestrasse 85, D-22607 Hamburg (Germany); Kostenko, Alexander V; Soldatov, Alexander V [Faculty of Physics, Southern Federal University, Sorge 5, Rostov-na-Donu, 344090 (Russian Federation); Leblanc, Catherine; Michel, Gurvan; Potin, Philippe [Centre National de la Recherche Scientifique and Universite Pierre et Marie Curie Paris-VI, Station Biologique de Roscoff, Place Georges Teissier, BP 74, F-29682 Roscoff cedex, Bretagne (France); Kuepper, Frithjof C [Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll PA37 1QA, Scotland (United Kingdom); Hollenstein, Kaspar; Locher, Kaspar P [Institute of Molecular Biology and Biophysics, ETH Zuerich, Schafmattstrasse 20, Zuerich, 8093 (Switzerland); Bevers, Loes E; Hagedoorn, Peter-Leon; Hagen, Wilfred R, E-mail: m.feiters@science.ru.n [Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft (Netherlands)

    2009-11-15

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L{sub 3} (2p{sub 3/2}) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  6. Application Progress in CFTR Inhibitors in the Treatment of Secretory Diarrhea%囊性纤维化跨膜传导调节因子抑制药在分泌性腹泻中的应用研究进展

    Institute of Scientific and Technical Information of China (English)

    徐倩倩; 王玉波; 王艳萍; 郭时金; 张志美; 沈志强

    2015-01-01

    Secretory diarrhea provides a major health challenge worldwide, which is one of the most important reasons for children morbidity and death. The activation of Cl- channels in intestinal epithelial cells resulting in the excessive fluid secretion in the intestine is the main reason of diarrhea caused by enterotoxins. In diarrhea caused by cholera and the other bacterial enterotoxins, cystic fibrosis transmembrane conductance regulator ( CFTR) is the main cAMP-control Cl- channel to promote the fluid secretion in epithelial cells. Therefore, CFTR inhibitors are the new choices for secretory diarrhea. CFTR inhibitors include thiazolidinone, glycine hydrazide and quinoxalinedione chemical classes, and some components from natural plants also exhibit CFTR inhibition activity, however, further studies should be done.%分泌性腹泻威胁着全球健康,是儿童发病和死亡的主要原因之一。肠上皮细胞腔内Cl-通道激活导致肠道液体过度分泌是肠毒素引起腹泻的主要原因。在霍乱及其他细菌肠毒素引发的腹泻中,囊性纤维化跨膜传导调节因子( cystic fibrosis transmembrane conductance regulator, CFTR)是主要的cAMP-调节Cl-通道,其主要功能是促进上皮细胞液体分泌。利用肠上皮细胞CFTR抑制药抗分泌性腹泻是一种新途径。已有的CFTR抑制药有噻唑啉酮类、甘氨酸酰肼类和喹喔啉二酮等。同时,从天然植物中提取分离的一些成分也具有CFTR抑制作用,但研究还不够深入。因此,对CFTR抑制药的研究进展进行了综述。

  7. pH-Regulated Nonelectrogenic Anion Transport by Phenylthiosemicarbazones.

    Science.gov (United States)

    Howe, Ethan N W; Busschaert, Nathalie; Wu, Xin; Berry, Stuart N; Ho, Junming; Light, Mark E; Czech, Dawid D; Klein, Harry A; Kitchen, Jonathan A; Gale, Philip A

    2016-07-01

    Gated ion transport across biological membranes is an intrinsic process regulated by protein channels. Synthetic anion carriers (anionophores) have potential applications in biological research; however, previously reported examples are mostly nonspecific, capable of mediating both electrogenic and electroneutral (nonelectrogenic) transport processes. Here we show the transmembrane Cl(-) transport studies of synthetic phenylthiosemicarbazones mimicking the function of acid-sensing (proton-gated) ion channels. These anionophores have remarkable pH-switchable transport properties with up to 640-fold increase in transport efficacy on going from pH 7.2 to 4.0. This "gated" process is triggered by protonation of the imino nitrogen and concomitant conformational change of the anion-binding thiourea moiety from anti to syn. By using a combination of two cationophore-coupled transport assays, with either monensin or valinomycin, we have elucidated the fundamental transport mechanism of phenylthiosemicarbazones which is shown to be nonelectrogenic, inseparable H(+)/Cl(-) cotransport. This study demonstrates the first examples of pH-switchable nonelectrogenic anion transporters. PMID:27299473

  8. Tripodal Receptors for Cation and Anion Sensors

    NARCIS (Netherlands)

    Kuswandi, Bambang; Nuriman,; Verboom, Willem; Reinhoudt, David N.

    2006-01-01

    This review discusses different types of artificial tripodal receptors for the selectiverecognition and sensing of cations and anions. Examples on the relationship between structure andselectivity towards cations and anions are described. Furthermore, their applications as potentiometricion sensing

  9. Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis

    Directory of Open Access Journals (Sweden)

    Sonawane Nitin D

    2010-05-01

    Full Text Available Abstract Background Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis. Methods Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age. Results Membrane cholesterol measurements are elevated in both R117H and ΔF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTRinh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility. Conclusions The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.

  10. Could a defective epithelial sodium channel lead to bronchiectasis

    Directory of Open Access Journals (Sweden)

    Hubert Dominique

    2008-05-01

    Full Text Available Abstract Background Bronchiectasis is defined as a permanent dilation of the airways arising from chronic bronchial inflammation/infection. In 50% of cases, no etiology can be identified. Recently, the role of the epithelial sodium channel ENaC has been pointed out in the pathophysiology of cystic fibrosis, a disease due to mutations in the CFTR gene and causing bronchiectasis in the airways. Moreover, it was found that transgenic mice overexpressing ENaCβ present cystic fibrosis-like lung disease symptoms. Our aim was to evaluate if a defective ENaC protein could be involved in the development of bronchiectasis. Methods We extensively analysed ENaCβ and γ genes in 55 patients with idiopathic bronchiectasis and without two mutations in the coding regions of CFTR. Thirty-eight patients presented functional abnormalities suggesting impaired sodium transport (abnormal sweat chloride concentration or nasal potential difference measurement, and 17 had no such evidence. Results Sequencing of the exons and flanking introns of the ENaCβ and γ gene identified five different amino-acid changes (p.Ser82Cys, p.Pro369Thr, p.Asn288Ser in ENaCβ ; and p.Gly183Ser, p.Glu197Lys in ENaCγ in heterozygous state in 8 patients. The p.Ser82Cys amino-acid change was found in 3 unrelated patients who were also heterozygous for a CFTR mutation or variant (1 p.F508del, 1 IVS8-5T, and 1 IVS8-5T:1716G>A (p.E528E. The other mutations were found in patients without CFTR mutation, the p.Glu197Lys mutation in 2 patients and the other variants in single patients. Among the 8 patients bearing an ENaC mutation, 5 had functional abnormalities suggesting impaired sodium transport. Conclusion Our results suggest that several variants in ENaCβ and γ genes might be deleterious for ENaC function and lead to bronchiectasis, especially in patients who are trans-heterozygotes for ENaCβ/CFTR mutations or variants.

  11. 乙醇对于豚鼠胰腺导管上皮细胞腔面膜侧 CFTR 介导的 HCO3-分泌的影响%Effect of ethanol on HCO 3- secretion mediated by CFTR from guinea - pig pancreatic duct cells. SONG Ying1,DING Xiang - fu2,WANG

    Institute of Scientific and Technical Information of China (English)

    宋莹; 丁相福; 王晓东; 段瑞峰; 石黑洋; 刘佰纯

    2016-01-01

    Objective To investigate the effect of ethanol on HCO3 - secretion induced by CFTR from guinea - pig pancreatic duct cells and to analyse the mechanisms of alcoholic pancreatitis. Methods Interlobular duct segments( diameter,100 ~ 150 μm)were microdissected under a dissection microscope using sharp needles. The lumen of the interlobular duct segment was microperfused. The bath and luminal solutions were modified separately. Intracellular pH(pHi)in the duct cells was estimated by microfluorometry using the pH - sensitive fluoroprobe BCECF. Transepithelial fluxes of HCO3 - were estimated under the anion gradients favoring rapid exchange of intracellular HCO3 - . Results ①When in vitro pancreatic duct pipe cavity,when filled with high concentration of sodium bicarbonate buffer micro pHi from epithelial cells after alkaline pH unit recovery rate was was 0. 048 ± 0. 009 pH unit·min - 1(n = 9),add 1 mm when ethanol pHi decline accelerated significantly(0. 066 ± 0. 005 pH unit·min - 1 ,n = 7,t = 0. 0009,P < 0. 05). ②Luminal application of CFTRinh - 172(10 μM)significantly( P < 0. 05)inhibited apical HCO3 - secretion(0. 039 ± 0. 010 pH unit·min - 1 ,n = 7,t = 0. 001). ③When the duct was stimulated with ethanol,luminal application of CFTRinh - 172 decrease the rate of pHi(0. 054 ± 0. 008 pH unit·min - 1 ,n = 7,t = 0. 0015,P < 0. 05). Conclusion Ethanol stimulated api-cal HCO3 - secretion of guinea - pig pancreatic ducts. The augmentation by ethanol appears to be mediated by CFTR.%目的:通过研究乙醇对于豚鼠胰腺导管上皮细胞腔面膜侧囊性纤维化转膜传导因子( CFTR)介导的碳酸氢根离子(HCO3-)分泌的影响,探讨酒精性胰腺炎的发病机制。方法运用显微解剖技术分离豚鼠胰腺导管(直径100~150μm),使用微灌流技术置换管腔内液体。采用荧光显微测定技术检测细胞内 pH 值变化,并计算出腔面膜侧HCO3-分泌率,进而探讨乙醇在 CFTR 介导的 HCO3-

  12. Efficient Amide Based Halogenide Anion Receptors

    Institute of Scientific and Technical Information of China (English)

    Hong Xing WU; Feng Hua LI; Hai LIN; Shou Rong ZHU; Hua Kuan LIN

    2005-01-01

    In this paper, we present the synthesis and anion recognition properties of the amide based phenanthroline derivatives 1, 2 and 3. In all cases 1:1 receptor: anion complexes were observed. The receptors were found to be selective for fluoride and chloride respectively over other putative anionic guest species.

  13. Distribution of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR Mutations in a Cohort of Patients Residing in Palestine.

    Directory of Open Access Journals (Sweden)

    Issa Siryani

    Full Text Available Cystic fibrosis (CF is an autosomal recessive inherited life-threatening disorder that causes severe damage to the lungs and the digestive system. In Palestine, mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR that contributes to the clinical presentation of CF are ill defined. A cohort of thirty three clinically diagnosed CF patients from twenty one different Palestinian families residing in the central and southern part of Palestine were incorporated in this study. Sweat chloride testing was performed using the Sweat Chek Conductivity Analyzer (ELITECH Group, France to confirm the clinical diagnosis of CF. In addition, nucleic acid from the patients' blood samples was extracted and the CFTR mutation profiles were assessed by direct sequencing of the CFTR 27 exons and the intron-exon boundaries. For patient's DNA samples where no homozygous or two heterozygous CFTR mutations were identified by exon sequencing, DNA samples were tested for deletions or duplications using SALSA MLPA probemix P091-D1 CFTR assay. Sweat chloride testing confirmed the clinical diagnosis of CF in those patients. All patients had NaCl conductivity >60 mmol/l. In addition, nine different CFTR mutations were identified in all 21 different families evaluated. These mutations were c.1393-1G>A, F508del, W1282X, G85E, c.313delA, N1303K, deletion exons 17a-17b-18, deletion exons 17a-17b and Q1100P. c.1393-1G>A was shown to be the most frequent occurring mutation among tested families. We have profiled the underling mutations in the CFTR gene of a cohort of 21 different families affected by CF. Unlike other studies from the Arab countries where F508del was reported to be the most common mutation, in southern/central Palestine, the c.1393-1G>A appeared to be the most common. Further studies are needed per sample size and geographic distribution to account for other possible CFTR genetic alterations and their frequencies. Genotype

  14. CFTR and cystic fibrosis%CFTR与囊性纤维化

    Institute of Scientific and Technical Information of China (English)

    王瑞; 李学军

    2006-01-01

    囊性纤维化跨膜传导调节因子(CFTR)是一种cAMP激活的ATP门控性氯离子通道,表达于气道,消化道和生殖道上皮细胞的顶部质膜中.囊性纤维化(CF)是白人中最常见的遗传性疾病之一,由CFTR基因突变造成.对CFTR基因的破译使人们进一步了解CF的发病机制,并为该疾病的诊断提供了新的线索.

  15. Development of ARMS PCR tests for detection of common CFTR gene mutations

    OpenAIRE

    Livshits L. A.; Pampukha V. M.; Soloviov O. O.

    2010-01-01

    Aim. To develop diagnostic assays, based on the amplification refractory mutation system (ARMS) principle, for detection of common mutations in the CFTR gene using two approaches: standard PCR with further gel-electrophoresis and Real-Time PCR with SYBR Green. Materials. For this study we have chosen the following mutations: dF508, W1282X, R117H, 621 + 1G > T, 2143delT with the frequencies in Ukraine: dF508 – 43.3 %; 2143delT – 1.38 %; W1282X – 1.1 %; R117H, 621 + 1G > T – < 0.6 %. For the de...

  16. Anion Solvation in Carbonate Electrolytes

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Zhengcheng

    2015-11-16

    With the correlation between Li+ solvation and interphasial chemistry on anodes firmly established in Li-ion batteries, the effect of cation–solvent interaction has gone beyond bulk thermodynamic and transport properties and become an essential element that determines the reversibility of electrochemistry and kinetics of Li-ion intercalation chemistries. As of now, most studies are dedicated to the solvation of Li+, and the solvation of anions in carbonate-based electrolytes and its possible effect on the electrochemical stability of such electrolytes remains little understood. As a mirror effort to prior Li+ solvation studies, this work focuses on the interactions between carbonate-based solvents and two anions (hexafluorophosphate, PF6–, and tetrafluoroborate, BF4–) that are most frequently used in Li-ion batteries. The possible correlation between such interaction and the interphasial chemistry on cathode surface is also explored.

  17. Polymerization of anionic wormlike micelles.

    Science.gov (United States)

    Zhu, Zhiyuan; González, Yamaira I; Xu, Hangxun; Kaler, Eric W; Liu, Shiyong

    2006-01-31

    Polymerizable anionic wormlike micelles are obtained upon mixing the hydrotropic salt p-toluidine hydrochloride (PTHC) with the reactive anionic surfactant sodium 4-(8-methacryloyloxyoctyl)oxybenzene sulfonate (MOBS). Polymerization captures the cross-sectional radius of the micelles (approximately 2 nm), induces micellar growth, and leads to the formation of a stable single-phase dispersion of wormlike micellar polymers. The unpolymerized and polymerized micelles were characterized using static and dynamic laser light scattering, small-angle neutron scattering, 1H NMR, and stopped-flow light scattering. Stopped-flow light scattering was also used to measure the average lifetime of the unpolymerized wormlike micelles. A comparison of the average lifetime of unpolymerized wormlike micelles with the surfactant monomer propagation rate was used to elucidate the mechanism of polymerization. There is a significant correlation between the ratio of the average lifetime to the monomer propagation rate and the average aggregation number of the polymerized wormlike micelles. PMID:16430253

  18. Laser Cooling of Molecular Anions

    CERN Document Server

    Yzombard, Pauline; Gerber, Sebastian; Doser, Michael; Comparat, Daniel

    2015-01-01

    We propose a scheme for laser cooling of negatively charged molecules. We briefly summarise the requirements for such laser cooling and we identify a number of potential candidates. A detailed computation study with C$\\_2^-$, the most studied molecular anion, is carried out. Simulations of 3D laser cooling in a gas phase show that this molecule could be cooled down to below 1 mK in only a few tens of milliseconds, using standard lasers. Sisyphus cooling, where no photo-detachment process is present, as well as Doppler laser cooling of trapped C$\\_2^-$, are also simulated. This cooling scheme has an impact on the study of cold molecules, molecular anions, charged particle sources and antimatter physics.

  19. Anion-induced urea deprotonation.

    Science.gov (United States)

    Boiocchi, Massimo; Del Boca, Laura; Esteban-Gómez, David; Fabbrizzi, Luigi; Licchelli, Maurizio; Monzani, Enrico

    2005-05-01

    The urea-based receptor 1 (1-(7-nitrobenzo[1,2,5]oxadiazol-4-yl)-3-(4-nitrophenyl)urea, L--H), interacts with X- ions in MeCN, according to two consecutive steps: 1) formation of a hydrogen-bond complex [L--H...X]-; 2) deprotonation of L--H to give L- and [HX2]-, as shown by spectrophotometric and 1H NMR titration experiments. Step 2) takes place with more basic anions (fluoride, carboxylates, dihydrogenphosphate), while less basic anions (Cl-, NO2-, NO3-) do not induce proton transfer. On crystallisation from a solution containing L--H and excess Bu4NF, the tetrabutylammonium salt of the deprotonated urea derivative (Bu4N[L]) was isolated and its crystal and molecular structure determined. PMID:15770711

  20. The activation effect of nobiletin on cystic fibrosis transmembrane conductance regulator chloride channel%川陈皮素对囊性纤维化跨膜传导调节因子的激活作用

    Institute of Scientific and Technical Information of China (English)

    杨爽; 于波; 张耀方; 王雪; 杨红

    2013-01-01

    Aim of the present study is to investigate activation effect of nobiletin on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity.CFTR-mediated iodide influx assay and patch-clamp tests were done on FRT cells stably co-transfected with human CFTR and EYFP/H148Q.Nobiletin potently activated CFTR chloride channel activity in a dose-and time-dependent manner.The CFTR blocker CFTRinh-172 could completely reverse the effect.Preliminary mechanism study indicated that nobiletin activated CFTR chloride channel through a direct binding way.In addition,ex vivo tests done on mice trachea showed that nobiletin time-dependently stimulated submucosal gland fluid secretion.Nobiletin may be a therapeutic lead compound in treating CFTR-related diseases including disseminated bronchiectasis.%本实验利用荧光淬灭实验和膜片钳技术在稳定表达人CFTR和荧光绿蛋白突变体EYFP/H148Q的Fischer大鼠甲状腺上皮细胞(Fischer rat thyroid,FRT)上,测定川陈皮素(nobiletin)对囊性纤维化跨膜传导因子(cystic fibrosis transmembrane conductance regulator,CFTR)氯离子通道的激活作用.结果发现,川陈皮素以剂量依赖的方式激活CFTR氯离子通道的C1-转运活性,且这种活性是快速、可逆的,并能够被CFTR特异性抑制剂CFTRinh-172完全抑制.初步的分子机制研究表明,川陈皮素是以与CFTR直接作用来激活通道活性的.进一步的研究结果显示,川陈皮素能够有效刺激小鼠气管黏膜下腺液体分泌速度.因此,川陈皮素可能发展成为治疗包括支气管扩张在内的CFTR相关疾病的先导药物.

  1. Functional modifications of acid-sensing ion channels by ligand-gated chloride channels.

    Directory of Open Access Journals (Sweden)

    Xuanmao Chen

    Full Text Available Together, acid-sensing ion channels (ASICs and epithelial sodium channels (ENaC constitute the majority of voltage-independent sodium channels in mammals. ENaC is regulated by a chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR. Here we show that ASICs were reversibly inhibited by activation of GABA(A receptors in murine hippocampal neurons. This inhibition of ASICs required opening of the chloride channels but occurred with both outward and inward GABA(A receptor-mediated currents. Moreover, activation of the GABA(A receptors modified the pharmacological features and kinetic properties of the ASIC currents, including the time course of activation, desensitization and deactivation. Modification of ASICs by open GABA(A receptors was also observed in both nucleated patches and outside-out patches excised from hippocampal neurons. Interestingly, ASICs and GABA(A receptors interacted to regulate synaptic plasticity in CA1 hippocampal slices. The activation of glycine receptors, which are similar to GABA(A receptors, also modified ASICs in spinal neurons. We conclude that GABA(A receptors and glycine receptors modify ASICs in neurons through mechanisms that require the opening of chloride channels.

  2. Chloride Channels: Often enigmatic, rarely predictable

    Science.gov (United States)

    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

  3. The zebrafish Kupffer's vesicle as a model system for the molecular mechanisms by which the lack of Polycystin-2 leads to stimulation of CFTR

    Directory of Open Access Journals (Sweden)

    Mónica Roxo-Rosa

    2015-11-01

    Full Text Available In autosomal dominant polycystic kidney disease (ADPKD, cyst inflation and continuous enlargement are associated with marked transepithelial ion and fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR. Indeed, the inhibition or degradation of CFTR prevents the fluid accumulation within cysts. The in vivo mechanisms by which the lack of Polycystin-2 leads to CFTR stimulation are an outstanding challenge in ADPKD research and may bring important biomarkers for the disease. However, hampering their study, the available ADPKD in vitro cellular models lack the three-dimensional architecture of renal cysts and the ADPKD mouse models offer limited access for live-imaging experiments in embryonic kidneys. Here, we tested the zebrafish Kupffer's vesicle (KV as an alternative model-organ. KV is a fluid-filled vesicular organ, lined by epithelial cells that express both CFTR and Polycystin-2 endogenously, being each of them easily knocked-down. Our data on the intracellular distribution of Polycystin-2 support its involvement in the KV fluid-flow induced Ca2+-signalling. Mirroring kidney cysts, the KV lumen inflation is dependent on CFTR activity and, as we clearly show, the knockdown of Polycystin-2 results in larger KV lumens through overstimulation of CFTR. In conclusion, we propose the zebrafish KV as a model organ to study the renal cyst inflation. Favouring its use, KV volume can be easily determined by in vivo imaging offering a live readout for screening compounds and genes that may prevent cyst enlargement through CFTR inhibition.

  4. Development of rAAV2-CFTR: History of the First rAAV Vector Product to be Used in Humans.

    Science.gov (United States)

    Loring, Heather S; ElMallah, Mai K; Flotte, Terence R

    2016-04-01

    The first human gene therapy trials using recombinant adeno-associated virus (rAAV) vectors were performed in cystic fibrosis (CF) patients. Over 100 CF patients were enrolled in 5 separate trials of rAAV2-CFTR administration via nasal, endobronchial, maxillary sinus, and aerosol delivery. Recombinant AAV vectors were designed to deliver the CF transmembrane regulator (CFTR) gene and correct the basic CFTR defect by restoring chloride transport and reverting the upregulation of proinflammatory cytokines. However, vector DNA expression was limited in duration because of the low incidence of integration and natural airway epithelium turnover. In addition, repeated administration of AAV-CFTR vector resulted in a humoral immune response that prevented effective gene transfer from subsequent doses of vector. AAV serotype 2 was used in human trials before the comparison with other serotypes and determination that serotypes 1 and 5 not only possess higher tropism for the airway epithelium, but also are capable of bypassing the binding and trafficking processes-both were important hindrances to the effectiveness of rAAV2. Although rAAV-CFTR gene therapy does not appear likely to supplant newer small-molecule CFTR modulators in the near future, early work with rAAV-CFTR provided an important foundation for later use of rAAV in humans. PMID:26895204

  5. Dynamics of anion-molecule reactions at low energy

    International Nuclear Information System (INIS)

    Anion-molecule reactions must find their way through deeply bound entrance and exit channel complexes separated by a central barrier. This results in low reaction rates and rich dynamics since direct pathways compete with the formation of transient intermediates. In this thesis we examine the probability of proton transfer to a small anion and transient lifetimes of a thermoneutral bimolecular nucleophilic substitution (SN2) reaction at well defined variable temperature down to 8 Kelvin in a multipole trap. The observed strong inverse temperature dependence is attributed to the deficit of available quantum states in the entrance channel at decreasing temperature. Furthermore we investigate scattering dynamics of SN2 reactions at defined relative energy between 0.4 and 10 eV by crossed beam slice imaging. A weakly exothermic reaction with high central barrier proceeds via an indirect, complex-mediated mechanism at low relative energies featuring high internal product excitation in excellent quantitative agreement with a statistical model. In contrast, direct backward scattering prevails for higher energies with product velocities close to the kinematical cutoff. For a strongly exothermic reaction, competing SN2-, dihalide- and proton transfer-channels are explored which proceed by complex mediation for low energy and various rebound-, grazing- and collision induced bond rupture-mechanisms at higher energy. From our data and a collaboration with theory we identify a new indirect roundabout SN2 mechanism involving CH3-rotation. (orig.)

  6. Dynamics of anion-molecule reactions at low energy

    Energy Technology Data Exchange (ETDEWEB)

    Mikosch, J.

    2007-11-15

    Anion-molecule reactions must find their way through deeply bound entrance and exit channel complexes separated by a central barrier. This results in low reaction rates and rich dynamics since direct pathways compete with the formation of transient intermediates. In this thesis we examine the probability of proton transfer to a small anion and transient lifetimes of a thermoneutral bimolecular nucleophilic substitution (S{sub N}2) reaction at well defined variable temperature down to 8 Kelvin in a multipole trap. The observed strong inverse temperature dependence is attributed to the deficit of available quantum states in the entrance channel at decreasing temperature. Furthermore we investigate scattering dynamics of S{sub N}2 reactions at defined relative energy between 0.4 and 10 eV by crossed beam slice imaging. A weakly exothermic reaction with high central barrier proceeds via an indirect, complex-mediated mechanism at low relative energies featuring high internal product excitation in excellent quantitative agreement with a statistical model. In contrast, direct backward scattering prevails for higher energies with product velocities close to the kinematical cutoff. For a strongly exothermic reaction, competing S{sub N}2-, dihalide- and proton transfer-channels are explored which proceed by complex mediation for low energy and various rebound-, grazing- and collision induced bond rupture-mechanisms at higher energy. From our data and a collaboration with theory we identify a new indirect roundabout S{sub N}2 mechanism involving CH{sub 3}-rotation. (orig.)

  7. Anion

    Directory of Open Access Journals (Sweden)

    A. Vadivel Murugan

    2003-01-01

    . Its characterization is investigated by Fourier Transform Infrared Spectroscopy (FT-IR and Scanning Electron Microscopy (SEM. The hybrid material presents predominantly high electronic conductivities of around 2.0 and 7.0 S cm-1 at 300 and 400K respectively.

  8. Aquaporins with anion/monocarboxylate permeability: mechanisms, relevance for pathogen-host interactions

    Directory of Open Access Journals (Sweden)

    Janis eRambow

    2014-09-01

    Full Text Available Classically, aquaporins are divided based on pore selectivity into water specific, orthodox aquaporins and solute-facilitating aquaglyceroporins, which conduct e.g. glycerol and urea. However, more aquaporin-passing substrates have been identified over the years, such as the gases ammonia and carbon dioxide or the water-related hydrogen peroxide, and it became apparent that not all aquaporins clearly fit into one of only two subfamilies. Furthermore, certain aquaporins from both major subfamilies have been reported to conduct inorganic anions, such as chloride, or monoacids/monocarboxylates, such as lactic acid/lactate. Here, we summarize the findings on aquaporin anion transport, analyze the pore layout of such aquaporins in comparison to prototypical non-selective anion channels, monocarboxylate transporters, and formate-nitrite transporters, and discuss in which scenarios anion conducting aquaporins may be of physiological relevance.

  9. Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in allergic bronchopulmonary aspergillosis

    Energy Technology Data Exchange (ETDEWEB)

    Miller, P.W.; Hamosh, A.; Macek, M. Jr. [John Hopkins Univ. School of Medicine, Baltimore, MD (United States)] [and others

    1996-07-01

    The etiology of allergic bronchopulmonary aspergillosis (ABPA) is not well understood. A clinical phenotype resembling the pulmonary disease seen in cystic fibrosis (CF) patients can occur in some individuals with ABPA. Reports of familial occurrence of ABPA and increased incidence in CF patients suggest a possible genetic basis for the disease. To test this possibility, the entire coding region of the cystic fibrosis transmembrane regulator (CFTR) gene was analyzed in 11 individuals who met strict criteria for the diagnosis of ABPA and had normal sweat electrolytes ({le}40 mmol/liter). One patient carried two CF mutations ({Delta}F508/R347H), and five were found to carry one CF mutation (four {Delta}F508; one R117H). The frequency of the {Delta}F508 mutation in patients with ABPA was significantly higher than in 53 Caucasian patients with chronic bronchitis (P < .0003) and the general population (P < .003). These results suggest that CFTR plays an etiologic role in a subset of ABPA patients. 54 refs., 2 tabs.

  10. Cystic fibrosis and the role of gastrointestinal outcome measures in the new era of therapeutic CFTR modulation

    NARCIS (Netherlands)

    Bodewes, Frank A J A; Verkade, Henkjan J; Taminiau, Jan A J M; Borowitz, Drucy; Wilschanski, Michael

    2015-01-01

    With the development of new drugs that directly affect CFTR protein function, clinical trials are being designed or initiated for a growing number of patients with cystic fibrosis. The currently available and accepted clinical endpoints, FEV1 and BMI, have limitations. The aim of this report is to d

  11. Heterogeneity of phenotype in two cystic fibrosis patients homozygous for the CFTR exon 11 mutation G551D.

    OpenAIRE

    Parad, R B

    1996-01-01

    In the heterozygous state, the cystic fibrosis transmembrane conductance regulator (CFTR) exon 11 mutation G551D has been described as "severe," causing pancreatic insufficiency. Two cystic fibrosis (CF) patients homozygous for this mutation showed a mild rather than severe pancreatic phenotype and a variable pulmonary phenotype.

  12. Ursodeoxycholate modulates bile flow and bile salt pool independently from the cystic fibrosis transmembrane regulator (Cftr) in mice

    NARCIS (Netherlands)

    Bodewes, Frank A. J. A.; Wouthuyzen-Bakker, Marjan; Bijvelds, Marcel J.; Havinga, Rick; de Jonge, Hugo R.; Verkade, Henkjan J.

    2012-01-01

    Bodewes FAJA, Wouthuyzen-Bakker M, Bijvelds MJ, Havinga R, de Jonge HR, Verkade HJ. Ursodeoxycholate modulates bile flow and bile salt pool independently from the cystic fibrosis transmembrane regulator (Cftr) in mice. Am J Physiol Gastrointest Liver Physiol 302: G1035-G1042, 2012. First published F

  13. Development of allele-specific multiplex PCR to determine the length of poly-T in intron 8 of CFTR

    Directory of Open Access Journals (Sweden)

    Neng Chen

    2014-07-01

    Full Text Available Cystic fibrosis transmembrane conductance regulator (CFTR gene mutation analysis has been implemented for Cystic Fibrosis (CF carrier screening, and molecular diagnosis of CF and congenital bilateral absence of the vas deferens (CBAVD. Although poly-T allele analysis in intron 8 of CFTR is required when a patient is positive for R117H, it is not recommended for routine carrier screening. Therefore, commercial kits for CFTR mutation analysis were designed either to mask the poly-T allele results, unless a patient is R117H positive, or to have the poly-T analysis as a standalone reflex test using the same commercial platform. There are other standalone assays developed to detect poly-T alleles, such as heteroduplex analysis, High Resolution Melting (HRM curve analysis, allele-specific PCR (AS-PCR and Sanger sequencing. In this report, we developed a simple and easy-to-implement multiplex AS-PCR assay using unlabeled standard length primers, which can be used as a reflex or standalone test for CFTR poly-T track analysis. Out of 115 human gDNA samples tested, results from our new AS-PCR matched to the previous known poly-T results or results from Sanger sequencing.

  14. RFI channels

    Science.gov (United States)

    Mceliece, R. J.

    1980-01-01

    A class of channel models is presented which exhibit varying burst error severity much like channels encountered in practice. An information-theoretic analysis of these channel models is made, and conclusions are drawn that may aid in the design of coded communication systems for realistic noisy channels.

  15. Modeling Donnan Dialysis Separation for Carboxylic Anion Recovery

    DEFF Research Database (Denmark)

    Prado Rubio, Oscar Andres; Møllerhøj, Martin; Jørgensen, Sten Bay;

    2010-01-01

    dynamic model for transport of multiple ions through an anion exchange membrane is derived based on an irreversible thermodynamics approach. This model accounts for the convective transport of the dissociated and undissociated species in the channels with diffusion and migration across the boundary...... boundary layers and membranes. The model consists of a system of partial differential equations that are solved numerically. The aim of this paper is to corroborate this general model for several monoprotic carboxylic acids reported in the literature. The model reproduces satisfactorily experimental fluxes...

  16. Environmental behavior of inorganic anions

    International Nuclear Information System (INIS)

    Recent efforts have addressed two aspects of anion behavior in the soil/plant system. The first involves evaluation of the gaseous component of the terrestrial iodine cycle in soils and plants. Field analyses of 129I in soils and vegetation adjacent to a fuels reprocessing facility, which was idle for 10 years prior to the study, indicated that there may be a significant gaseous component to the terrestrial iodine cycle. Soil substrates, including a silt-sand, organic forest soil, quartz sand, and a sterilized soil, were amended with radioiodide, and the rates and quality of the volatile components evaluated

  17. Cystic fibrosis transmembrane conductance regulator (CFTR allelic variants relate to shifts in faecal microbiota of cystic fibrosis patients.

    Directory of Open Access Journals (Sweden)

    Serena Schippa

    Full Text Available INTRODUCTION: In this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF. CFTR mutations (F508del is the most common lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age. METHODS: Thirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure. RESULTS: Patients were classified by two different criteria: 1 presence/absence of F508del mutation; 2 disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum were reduced. CONCLUSIONS: This is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a 'systemic disease', linking the lung and the gut in a joined axis.

  18. Defective fluid secretion from submucosal glands of nasal turbinates from CFTR-/- and CFTR (ΔF508/ΔF508 pigs.

    Directory of Open Access Journals (Sweden)

    Hyung-Ju Cho

    Full Text Available BACKGROUND: Cystic fibrosis (CF, caused by reduced CFTR function, includes severe sinonasal disease which may predispose to lung disease. Newly developed CF pigs provide models to study the onset of CF pathophysiology. We asked if glands from pig nasal turbinates have secretory responses similar to those of tracheal glands and if CF nasal glands show reduced fluid secretion. METHODOLOGY/PRINCIPAL FINDINGS: Unexpectedly, we found that nasal glands differed from tracheal glands in five ways, being smaller, more numerous (density per airway surface area, more sensitive to carbachol, more sensitive to forskolin, and nonresponsive to Substance P (a potent agonist for pig tracheal glands. Nasal gland fluid secretion from newborn piglets (12 CF and 12 controls in response to agonists was measured using digital imaging of mucus bubbles formed under oil. Secretion rates were significantly reduced in all conditions tested. Fluid secretory rates (Controls vs. CF, in pl/min/gland were as follows: 3 µM forskolin: 9.2±2.2 vs. 0.6±0.3; 1 µM carbachol: 143.5±35.5 vs. 52.2±10.3; 3 µM forskolin + 0.1 µM carbachol: 25.8±5.8 vs. CF 4.5±0.9. We also compared CF(ΔF508/ΔF508 with CFTR(-/- piglets and found significantly greater forskolin-stimulated secretion rates in the ΔF508 vs. the null piglets (1.4±0.8, n = 4 vs. 0.2±0.1, n = 7. An unexpected age effect was also discovered: the ratio of secretion to 3 µM forskolin vs. 1 µM carbachol was ∼4 times greater in adult than in neonatal nasal glands. CONCLUSIONS/SIGNIFICANCE: These findings reveal differences between nasal and tracheal glands, show defective fluid secretion in nasal glands of CF pigs, reveal some spared function in the ΔF508 vs. null piglets, and show unexpected age-dependent differences. Reduced nasal gland fluid secretion may predispose to sinonasal and lung infections.

  19. Cell cycle-dependent activity of the volume- and Ca2+-activated anion currents in Ehrlich lettre ascites cells

    DEFF Research Database (Denmark)

    Klausen, Thomas Kjaer; Bergdahl, Andreas; Christophersen, Palle;

    2007-01-01

    Recent evidence implicates the volume-regulated anion current (VRAC) and other anion currents in control or modulation of cell cycle progression; however, the precise involvement of anion channels in this process is unclear. Here, Cl- currents in Ehrlich Lettre Ascites (ELA) cells were monitored......+ in the pipette), was unaltered from G0 to G1, but decreased in early S phase. A novel high-affinity anion channel inhibitor, the acidic di-aryl-urea NS3728, which inhibited both VRAC and CaCC, attenuated ELA cell growth, suggesting a possible mechanistic link between cell cycle progression and cell cycle......-dependent changes in the capacity for conductive Cl- transport. It is suggested that in ELA cells, entrance into the S phase requires an increase in VRAC activity and/or an increased potential for regulatory volume decrease (RVD), and at the same time a decrease in CaCC magnitude....

  20. Adsorption affinity of anions on metal oxyhydroxides

    Science.gov (United States)

    Pechenyuk, S. I.; Semushina, Yu. P.; Kuz'mich, L. F.

    2013-03-01

    The dependences of anion (phosphate, carbonate, sulfate, chromate, oxalate, tartrate, and citrate) adsorption affinity anions from geometric characteristics, acid-base properties, and complex forming ability are generalized. It is shown that adsorption depends on the nature of both the anions and the ionic medium and adsorbent. It is established that anions are generally grouped into the following series of adsorption affinity reduction: PO{4/3-}, CO{3/2-} > C2O{4/2-}, C(OH)(CH2)2(COO){3/3-}, (CHOH)2(COO){2/2-} > CrO{4/2-} ≫ SO{4/2-}.

  1. Lipoxin A4 and platelet activating factor are involved in E. coli or LPS-induced lung inflammation in CFTR-deficient mice.

    Directory of Open Access Journals (Sweden)

    Haiya Wu

    Full Text Available CFTR (cystic fibrosis transmembrane conductance regulator is expressed by both neutrophils and platelets. Lack of functional CFTR could lead to severe lung infection and inflammation. Here, we found that mutation of CFTR (F508del or inhibition of CFTR in mice led to more severe thrombocytopenia, alveolar neutrocytosis and bacteriosis, and lower lipoxin A4/MIP-2 (macrophage inhibitory protein-2 or lipoxin A4/neutrophil ratios in the BAL (bronchoalveolar lavage during acute E. coli pneumonia. In vitro, inhibition of CFTR promotes MIP-2 production in LPS-stimulated neutrophils; however, lipoxin A4 could dose-dependently suppress this effect. In LPS-induced acute lung inflammation, blockade of PSGL-1 (P-selectin glycoprotein ligand-1 or P-selectin, antagonism of PAF by WEB2086, or correction of mutated CFTR trafficking by KM11060 could significantly increase plasma lipoxin A4 levels in F508del relevant to wildtype mice. Concurrently, F508del mice had higher plasma platelet activating factor (PAF levels and PAF-AH activity compared to wildtype under LPS challenge. Inhibiting hydrolysis of PAF by a specific PAF-AH (PAF-acetylhydrolase inhibitor, MAFP, could worsen LPS-induced lung inflammation in F508del mice compared to vehicle treated F508del group. Particularly, depletion of platelets in F508del mice could significantly decrease plasma lipoxin A4 and PAF-AH activity and deteriorate LPS-induced lung inflammation compared to control F508del mice. Taken together, lipoxin A4 and PAF are involved in E. coli or LPS-induced lung inflammation in CFTR-deficient mice, suggesting that lipoxin A4 and PAF might be therapeutic targets for ameliorating CFTR-deficiency deteriorated lung inflammation.

  2. NF kappaB expression increases and CFTR and MUC1 expression decreases in the endometrium of infertile patients with hydrosalpinx: a comparative study

    Directory of Open Access Journals (Sweden)

    Song Yong

    2012-10-01

    Full Text Available Abstract Background Hydrosalpinx are associated with infertility, due to reduced rates of implantation and increased abortion rates. The aims of this study were to investigate the expression of cystic fibrosis transmembrane conductance regulator (CFTR, nuclear factor kappa B (NF KappaB and mucin-1 (MUC-1, and analyze the correlation between the expression of CFTR and NF KappaB or MUC1, in the endometrium of infertile women with and without hydrosalpinx. Methods Thirty-one infertile women with laparoscopy-confirmed unilateral or bilateral hydrosalpinx and 20 infertile women without hydrosalpinx or pelvic inflammatory disease (control group were recruited. Endometrial biopsy samples were collected and the expression of CFTR, NF KappaB and MUC1 were analyzed using immunohistochemistry and quantitative real-time PCR. Results CFTR, NF KappaB and MUC1 mRNA and protein expression tended to increase in the secretory phase compared to the proliferative phase in both groups; however, these differences were not significantly different. The endometrium of infertile patients with hydrosalpinx had significantly higher NF KappaB mRNA and protein expression, and significantly lower CFTR and MUC1 mRNA and protein expression, compared to control infertile patients. A positive correlation was observed between CFTR and MUC1 mRNA expression (r = 0.65, P CFTR mRNA and NF KappaB mRNA expression (r = −0.59, P Conclusions Increased NF KappaB expression and decreased CFTR and MUC1 expression in the endometrium of infertile patients with hydrosalpinx reinforce the involvement of a molecular mechanism in the regulation of endometrial receptivity.

  3. Increased efficacy of VX-809 in different cellular systems results from an early stabilization effect of F508del-CFTR.

    Science.gov (United States)

    Farinha, Carlos M; Sousa, Marisa; Canato, Sara; Schmidt, André; Uliyakina, Inna; Amaral, Margarida D

    2015-08-01

    Cystic fibrosis (CF), the most common recessive autosomal disease among Caucasians, is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to CFTR impaired plasma membrane trafficking. Therapies modulating CFTR basic defect are emerging, such as VX-809, a corrector of F508del-CFTR traffic which just succeeded in a Phase III clinical trial. We recently showed that VX-809 is additive to two other correctors (VRT-325 and compound 4a). Here, we aimed to determine whether the differential rescuing by these compounds results from cell-specific factors or rather from distinct effects at the early biogenesis and/or processing. The rescuing efficiencies of the above three correctors were first compared in different cellular models (primary respiratory cells, cystic fibrosis bronchial epithelial and baby hamster kidney [BHK] cell lines) by functional approaches: micro-Ussing chamber and iodide efflux. Next, biochemical methods (metabolic labeling, pulse-chase and immunoprecipitation) were used to determine their impact on CFTR biogenesis / processing. Functional analyses revealed that VX-809 has the greatest rescuing efficacy and that the relative efficiencies of the three compounds are essentially maintained in all three cellular models tested. Nevertheless, biochemical data show that VX-809 significantly stabilizes F508del-CFTR immature form, an effect that is not observed for C3 nor C4. VX-809 and C3 also significantly increase accumulation of immature CFTR. Our data suggest that VX-809 increases the stability of F508del-CFTR immature form at an early phase of its biogenesis, thus explaining its increased efficacy when inducing its rescue. PMID:26171232

  4. Ursodeoxycholic acid and superoxide anion

    Institute of Scientific and Technical Information of China (English)

    Predrag Ljubuncic; Omar Abu-Salach; Arieh Bomzon

    2005-01-01

    AIM: To investigate the ability of ursodeoxycholic acid (UDCA) to scavenge superoxide anion (O2-).METHODS: We assessed the ability of UDCA to scavenge (O2-) generated by xanthine-xanthine oxidase (X-XO) in a cell-free system and its effect on the rate of O2--induced ascorbic acid (AA) oxidation in hepatic post-mitochondrial supernatants.RESULTS: UDCA at a concentration as high as 1 mmol/Ldid not impair the ability of the X-XO system to generate O2-, but could scavenge O2- at concentrations of 0.5 and 1 mmol/L, and decrease the rate of AA oxidation at a concentration of 100 μmol/L.CONCLUSION: UDCA can scavenge O2-, an action that may be beneficial to patients with primary biliary cirrhosis.

  5. Marine Toxins Targeting Ion Channels

    Directory of Open Access Journals (Sweden)

    Hugo R. Arias

    2006-04-01

    Full Text Available Abstract: This introductory minireview points out the importance of ion channels for cell communication. The basic concepts on the structure and function of ion channels triggered by membrane voltage changes, the so-called voltage-gated ion channels (VGICs, as well as those activated by neurotransmitters, the so-called ligand-gated ion channel (LGICs, are introduced. Among the most important VGIC superfamiles, we can name the voltage-gated Na+ (NaV, Ca2+ (CaV, and K+ (KV channels. Among the most important LGIC super families, we can include the Cys-loop or nicotinicoid, the glutamate-activated (GluR, and the ATP-activated (P2XnR receptor superfamilies. Ion channels are transmembrane proteins that allow the passage of different ions in a specific or unspecific manner. For instance, the activation of NaV, CaV, or KV channels opens a pore that is specific for Na+, Ca2+, or K+, respectively. On the other hand, the activation of certain LGICs such as nicotinic acetylcholine receptors, GluRs, and P2XnRs allows the passage of cations (e.g., Na+, K+, and/or Ca2+, whereas the activation of other LGICs such as type A γ-butyric acid and glycine receptors allows the passage of anions (e.g., Cl− and/or HCO3−. In this regard, the activation of NaV and CaV as well as ligand-gated cation channels produce membrane depolarization, which finally leads to stimulatory effects in the cell, whereas the activation of KV as well as ligand-gated anion channels induce membrane hyperpolarization that finally leads to inhibitory effects in the cell. The importance of these ion channel superfamilies is emphasized by considering their physiological functions throughout the body as well as their pathophysiological implicance in several neuronal diseases. In this regard, natural molecules, and especially marine toxins, can be potentially used as modulators (e.g., inhibitors or prolongers of ion channel functions to treat or to alleviate a specific

  6. CFTR genotype-related body water and electrolyte balance during a marathon.

    Science.gov (United States)

    Del Coso, J; Lara, B; Salinero, J J; Areces, F; Ruiz-Vicente, D; Gallo-Salazar, C; Abián-Vicén, J; Cacabelos, R

    2016-09-01

    The aim of this investigation was to determine the influence of CFTR genotype on body water and electrolyte balance during a marathon. Fifty-one experienced runners completed a marathon race. Before and after the race, body mass and a sample of venous blood were obtained. During the race, sweat samples were collected using sweat patches, and fluid and electrolyte intake were obtained using self-reported questionnaires. Thirty-eight participants (74.5% of the total) were 7T/7T homozygotes, 11 (21.6%) were 7T/9T heterozygotes, and one participant presented the rare genotype 5T/7T. Another participant with 9T/9T presented the mutation p.L206W. Participants with 7T/7T showed higher sweat sodium concentrations (42.2 ± 21.6 mmol/L) than 7T/9T (29.0 ± 24.7 mmol/L; P = 0.04). The runner with the 5T/7T genotype (10.2 mmol/L) and the participant with the p.L206W mutation (20.5 mmol/L) exhibited low-range sweat sodium concentrations. However, post-race serum sodium concentration was similar in 7T/7T and 7T/9T (142.1 ± 1.3 and 142.4 ± 1.6 mmol/L, respectively; P = 0.27) and did not show abnormalities in participants with the 5T/7T genotype (140.0 mmol/L) and the p.L206W mutation (143.0 mmol/L). Runners with the CFTR-7T/7T genotype exhibited increased sweat sodium concentrations during a marathon. However, this phenotype was not related with increased likelihood of suffering body water and electrolyte imbalances during real competitions.

  7. Simultaneous anion and cation mobility in polypyrrole

    DEFF Research Database (Denmark)

    Skaarup, Steen; Bay, Lasse; Vidanapathirana, K.;

    2003-01-01

    Polypyrrole (PPy) polymer films permanently doped with large, immobile anion dodecyl benzene sulfonate (DBS) have been characterized by cyclic voltammetry in order to clarify the roles of cations and anions in the aqueous electrolyte as mobile ions in the film. Aqueous solutions of 0.05-0.1 M alk...

  8. Tripodal Receptors for Cation and Anion Sensors

    Directory of Open Access Journals (Sweden)

    David N. Reinhoudt

    2006-08-01

    Full Text Available This review discusses different types of artificial tripodal receptors for the selectiverecognition and sensing of cations and anions. Examples on the relationship between structure andselectivity towards cations and anions are described. Furthermore, their applications as potentiometricion sensing are emphasised, along with their potential applications in optical sensors or optodes.

  9. Test procedure for anion exchange chromatography

    International Nuclear Information System (INIS)

    Plutonium from stored nitrate solutions will be sorbed onto anion exchange resins and converted to storable plutonium dioxide. Useful information will be simultaneously gained on the thermal stability and ion exchange capacity of four commercially available anion exchange resins over several years and under severe degradative conditions. This information will prove useful in predicting the safe and efficient lifetimes of these resins

  10. Cell swelling activates separate taurine and chloride channels in Ehrlich mouse ascites tumor cells

    DEFF Research Database (Denmark)

    Lambert, Ian Henry; Hoffmann, Else Kay

    1994-01-01

    The taurine efflux from Ehrlich ascites tumor cells is stimulated by hypotonic cell swelling. The swelling-activated taurine efflux is unaffected by substitution of gluconate for extracellular Cl– but inhibited by addition of MK196 (anion channel blocker) and 4,4 -diisothiocyanostilbene-2......,2 -disulfonic acid (DIDS; anion channel and anion exchange blocker) and by depolarization of the cell membrane. This is taken to indicate that taurine does not leave the osmotically swollen Ehrlich cells in exchange for extracellular Cl–, i.e., via the anion exchanger but via a MK196- and DIDS-sensitive channel...... that is potential dependent. An additional stimulation of the swelling-activated taurine efflux is seen after addition of arachidonic acid and oleic acid. Cell swelling also activates a Mini Cl– channel. The Cl– efflux via this Cl– channel, in contrast to the swelling-activated taurine efflux, is...

  11. Counterintuitive interaction of anions with benzene derivatives

    Science.gov (United States)

    Quiñonero, David; Garau, Carolina; Frontera, Antonio; Ballester, Pau; Costa, Antonio; Deyà, Pere M.

    2002-06-01

    Ab initio calculations were carried out on complexes between 1,3,5-trinitrobenzene (TNB) and anions, where the anion is positioned over the ring along the C3 axis. This study combines crystallographic and computational evidences to demonstrate an attractive interaction between the anion and the π-cloud of TNB. This interaction is rationalized based on the important role of the quadrupole moment of TNB and the anion-induced polarization. In addition, this study has been extended to 1,3,5-trifluorobenzene (TFB), which possesses a very small quadrupole moment. As a result, minimum energy complexes have been found between TFB and both anions and cations due to the stabilization obtained from the ion-induced polarization.

  12. Formation and Decay of the Dehydrogenated Parent Anion upon Electron Attachment to Dialanine

    OpenAIRE

    Gschliesser, David; Vizcaino, Violaine; Probst, Michael; Scheier, Paul; Denifl, Stephan

    2012-01-01

    Abstract The dehydrogenated parent anion [M−H]− is one of the most dominant anions formed in dissociative electron attachment to various small biomolecules like nucleobases and single amino acids. In the present study, we investigate the [M−H]− channel for the dipeptide dialanine by utilizing an electron monochromator and a two-sector-field mass spectrometer. At electron energies below 2 eV, the measured high-resolution ion-efficiency curve has a different shape to that for the single amino a...

  13. Calcium homeostasis modulator (CALHM) ion channels.

    Science.gov (United States)

    Ma, Zhongming; Tanis, Jessica E; Taruno, Akiyuki; Foskett, J Kevin

    2016-03-01

    Calcium homeostasis modulator 1 (CALHM1), formerly known as FAM26C, was recently identified as a physiologically important plasma membrane ion channel. CALHM1 and its Caenorhabditis elegans homolog, CLHM-1, are regulated by membrane voltage and extracellular Ca(2+) concentration ([Ca(2+)]o). In the presence of physiological [Ca(2+)]o (∼1.5 mM), CALHM1 and CLHM-1 are closed at resting membrane potentials but can be opened by strong depolarizations. Reducing [Ca(2+)]o increases channel open probability, enabling channel activation at negative membrane potentials. Together, voltage and Ca(2+) o allosterically regulate CALHM channel gating. Through convergent evolution, CALHM has structural features that are reminiscent of connexins and pannexins/innexins/LRRC8 (volume-regulated anion channel (VRAC)) gene families, including four transmembrane helices with cytoplasmic amino and carboxyl termini. A CALHM1 channel is a hexamer of CALHM1 monomers with a functional pore diameter of ∼14 Å. CALHM channels discriminate poorly among cations and anions, with signaling molecules including Ca(2+) and ATP able to permeate through its pore. CALHM1 is expressed in the brain where it plays an important role in cortical neuron excitability induced by low [Ca(2+)]o and in type II taste bud cells in the tongue that sense sweet, bitter, and umami tastes where it functions as an essential ATP release channel to mediate nonsynaptic neurotransmitter release. CLHM-1 is expressed in C. elegans sensory neurons and body wall muscles, and its genetic deletion causes locomotion defects. Thus, CALHM is a voltage- and Ca(2+) o-gated ion channel, permeable to large cations and anions, that plays important roles in physiology. PMID:26603282

  14. Analysis of the CFTR gene in Venezuelan cystic fibrosis patients, identification of six novel cystic fibrosis-causing genetic variants

    OpenAIRE

    Sánchez K; de Mendonca E; Matute X; Chaustre I; Villalón M; Takiff H

    2016-01-01

    Karen Sánchez,1 Elizabeth de Mendonca,1 Xiorama Matute,2 Ismenia Chaustre,2 Marlene Villalón,3 Howard Takiff4 1Unit of Genetic and Forensic Studies, Venezuelan Institute for Scientific Research (IVIC), 2Hospital JM de los Ríos, 3Hospital José Ignacio Baldo, Algodonal, National Reference Unit, 4Laboratory of Molecular Genetics, Venezuelan Institute for Scientific Research (IVIC), Caracas, Venezuela. Abstract: The mutations in the CFTR gene found in patients with cy...

  15. Analysis of the CFTR gene in Venezuelan cystic fibrosis patients, identification of six novel cystic fibrosis-causing genetic variants

    OpenAIRE

    Sánchez, Karen

    2016-01-01

    Karen Sánchez,1 Elizabeth de Mendonca,1 Xiorama Matute,2 Ismenia Chaustre,2 Marlene Villalón,3 Howard Takiff4 1Unit of Genetic and Forensic Studies, Venezuelan Institute for Scientific Research (IVIC), 2Hospital JM de los Ríos, 3Hospital José Ignacio Baldo, Algodonal, National Reference Unit, 4Laboratory of Molecular Genetics, Venezuelan Institute for Scientific Research (IVIC), Caracas, Venezuela. Abstract: The mutations in the CFTR gene found in ...

  16. Frequency of CFTR, SPINK1, and Cathe