scholarly journals Deletion of Slc26a6 alters the stoichiometry of apical Cl−/HCO3− exchange in mouse pancreatic duct

2012 ◽  
Vol 303 (8) ◽  
pp. C815-C824 ◽  
Author(s):  
Ying Song ◽  
Akiko Yamamoto ◽  
Martin C. Steward ◽  
Shigeru B. H. Ko ◽  
Andrew K. Stewart ◽  
...  
Keyword(s):  
Pancreatic Duct ◽  
Apical Membrane ◽  
Flux Ratio ◽  
Pancreatic Ducts ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Membrane Hyperpolarization ◽  
Disulphonic Acid ◽  
Cystic Fibrosis Transmembrane

To define the stoichiometry and molecular identity of the Cl−/HCO3− exchanger in the apical membrane of pancreatic duct cells, changes in luminal pH and volume were measured simultaneously in interlobular pancreatic ducts isolated from wild-type and Slc26a6-null mice. Transepithelial fluxes of HCO3− and Cl− were measured in the presence of anion gradients favoring rapid exchange of intracellular HCO3− with luminal Cl− in cAMP-stimulated ducts. The flux ratio of Cl− absorption/HCO3− secretion was ∼0.7 in wild-type ducts and ∼1.4 in Slc26a6−/− ducts where a different Cl−/HCO3− exchanger, most likely SLC26A3, was found to be active. Interactions between Cl−/HCO3− exchange and cystic fibrosis transmembrane conductance regulator (CFTR) in cAMP-stimulated ducts were examined by measuring the recovery of intracellular pH after alkali-loading by acetate prepulse. Hyperpolarization induced by luminal application of CFTRinh-172 enhanced HCO3− efflux across the apical membrane via SLC26A6 in wild-type ducts but significantly reduced HCO3− efflux in Slc26a6−/− ducts. In microperfused wild-type ducts, removal of luminal Cl−, or luminal application of dihydro-4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid to inhibit SLC26A6, caused membrane hyperpolarization, which was abolished in Slc26a6−/− ducts. In conclusion, we have demonstrated that deletion of Slc26a6 alters the apparent stoichiometry of apical Cl−/HCO3− exchange in native pancreatic duct. Our results are consistent with SLC26A6 mediating 1:2 Cl−/HCO3− exchange, and the exchanger upregulated in its absence, most probably SLC26A3, mediating 2:1 exchange.

scholarly journals Carbon monoxide-releasing molecules inhibit the cystic fibrosis transmembrane conductance regulator Cl− channel

2020 ◽  
Vol 319 (6) ◽  
pp. L997-L1009
Author(s):  
Mayuree Rodrat ◽  
Walailak Jantarajit ◽  
Demi R. S. Ng ◽  
Bartholomew S. J. Harvey ◽  
Jia Liu ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Carbon Monoxide ◽  
Current Flow ◽  
Open Channels ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Flow Through ◽  
Cystic Fibrosis Transmembrane

The gasotransmitter carbon monoxide (CO) regulates fluid and electrolyte movements across epithelial tissues. However, its action on anion channels is incompletely understood. Here, we investigate the direct action of CO on the cystic fibrosis transmembrane conductance regulator (CFTR) by applying CO-releasing molecules (CO-RMs) to the intracellular side of excised inside-out membrane patches from cells heterologously expressing wild-type human CFTR. Addition of increasing concentrations of tricarbonyldichlororuthenium(II) dimer (CORM-2) (1–300 μM) inhibited CFTR channel activity, whereas the control RuCl3 (100 μM) was without effect. CORM-2 predominantly inhibited CFTR by decreasing the frequency of channel openings and, hence, open probability ( Po). But, it also reduced current flow through open channels with very fast kinetics, particularly at elevated concentrations. By contrast, the chemically distinct CO-releasing molecule CORM-3 inhibited CFTR by decreasing Po without altering current flow through open channels. Neither depolarizing the membrane voltage nor raising the ATP concentration on the intracellular side of the membrane affected CFTR inhibition by CORM-2. Interestingly, CFTR inhibition by CORM-2, but not by CFTRinh-172, was prevented by prior enhancement of channel activity by the clinically approved CFTR potentiator ivacaftor. Similarly, when added after CORM-2, ivacaftor completely relieved CFTR inhibition. In conclusion, CORM-2 has complex effects on wild-type human CFTR consistent with allosteric inhibition and open-channel blockade. Inhibition of CFTR by CO-releasing molecules suggests that CO regulates CFTR activity and that the gasotransmitter has tissue-specific effects on epithelial ion transport. The action of ivacaftor on CFTR Cl− channels inhibited by CO potentially expands the drug’s clinical utility.

Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia

2000 ◽  
Vol 279 (5) ◽  
pp. L835-L841 ◽  
Author(s):  
Olafur Baldursson ◽  
Herbert A. Berger ◽  
Michael J. Welsh
Keyword(s):  
Functional Role ◽  
Transmembrane Conductance Regulator ◽  
Regulatory Domain ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Dependent Protein ◽  
Rat Thyroid ◽  
Cystic Fibrosis Transmembrane

The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl− current. Mutation of four in vivo phosphorylation sites, Ser660, Ser737, Ser795, and Ser813 (S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser660 or Ser813 alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser660 nor Ser813 alone increased current to wild-type levels; both residues were required. Changing Ser737 to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser737 may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines.

scholarly journals Influence of Cystic Fibrosis Transmembrane Conductance Regulator on Gene Expression in Response to Pseudomonas aeruginosa Infection of Human Bronchial Epithelial Cells

2005 ◽  
Vol 73 (10) ◽  
pp. 6822-6830 ◽  
Author(s):  
Nina Reiniger ◽  
Jeffrey K. Ichikawa ◽  
Gerald B. Pier
Keyword(s):  
Cystic Fibrosis ◽  
Cell Line ◽  
Cell Lines ◽  
The Other ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Bronchial Epithelial ◽  
Cystic Fibrosis Transmembrane ◽  
In Cells

ABSTRACT Chronic lung infection by Pseudomonas aeruginosa causes significant morbidity in cystic fibrosis patients initiated by the failure of innate immune responses. We used microarray analysis and real-time PCR to detect transcriptional changes associated with cytokine production in isogenic bronchial epithelial cell lines with either wild-type (WT) or mutant cystic fibrosis transmembrane conductance regulator (CFTR) in response to P. aeruginosa infection. The transcription of four NF-κB-regulated cytokine genes was maximal in the presence of WT CFTR: the interleukin-8 (IL-8), IL-6, CXCL1, and intracellular adhesion molecule 1 (ICAM-1) genes. Analysis of protein expression in two cell lines paired for wild-type and mutant CFTR with three P. aeruginosa strains showed IL-6 and IL-8 expressions were consistently enhanced by the presence of WT CFTR in both cell lines with all three strains of P. aeruginosa, although some strains gave small IL-8 increases in cells with mutant CFTR. CXCL1 production showed consistent enhancement in cells with WT CFTR using all three bacterial strains in one cell line, whereas in the other cell line, CXCL1 showed a significant increase in cells with either WT or mutant CFTR. ICAM-1 was unchanged at the protein level in one of the cell lines but did show mild enhancement with WT CFTR in the other cell pair. Inhibitions of NF-κB prior to infection indicated differing degrees of dependence on NF-κB for production of the cytokines, contingent on the cell line. Cytokine effectors of innate immunity to P. aeruginosa were found to be positively influenced by the presence of WT CFTR, indicating a role in resistance to P. aeruginosa infection.

scholarly journals Enhancement of alveolar epithelial sodium channel activity with decreased cystic fibrosis transmembrane conductance regulator expression in mouse lung

2011 ◽  
Vol 301 (4) ◽  
pp. L557-L567 ◽  
Author(s):  
Ahmed Lazrak ◽  
Asta Jurkuvenaite ◽  
Lan Chen ◽  
Kim M. Keeling ◽  
James F. Collawn ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Alveolar Epithelial ◽  
Cystic Fibrosis Transmembrane

We sought to establish whether the cystic fibrosis transmembrane conductance regulator (CFTR) regulates the activity of amiloride-sensitive sodium channels (ENaC) in alveolar epithelial cells of wild-type, heterozygous ( Cftr +/−), knockout ( Cftr −/−), and ΔF508-expressing mice in situ. RT-PCR studies confirmed the presence of CFTR message in freshly isolated alveolar type II (ATII) cells from wild-type mice. We patched alveolar type I (ATI) and ATII cells in freshly prepared lung slices from these mice and demonstrated the presence of 4-pS ENaC channels with the following basal open probabilities (Po): wild-type=0.21 ± 0.015: Cftr +/−=0.4 ± 0.03; ΔF508=0.55 ± 0.01; and Cftr −/−=and 0.81 ± 0.016 (means ± SE; n ≥ 9). Forskolin (5 μM) or trypsin (2 μM), applied in the pipette solution, increased the Po and number of channels in ATII cells of wild-type, Cftr +/−, and ΔF508, but not in Cftr −/− mice, suggesting that the latter were maximally activated. Western blot analysis showed that lungs of all groups of mice had similar levels of α-ENaC; however, lungs of Cftr +/− and Cftr −/− mice had significantly higher levels of an α-ENaC proteolytic fragment (65 kDa) that is associated with active ENaC channels. Our results indicate that ENaC activity is inversely correlated to predicted CFTR levels and that CFTR heterozygous and homozygous mice have higher levels of proteolytically processed ENaC fragments in their lungs. This is the first demonstration of functional ENaC-CFTR interactions in alveolar epithelial cells in situ.

scholarly journals Na+/H+ Exchanger Regulatory Factor 1 Overexpression-dependent Increase of Cytoskeleton Organization Is Fundamental in the Rescue of F508del Cystic Fibrosis Transmembrane Conductance Regulator in Human Airway CFBE41o- Cells

2010 ◽  
Vol 21 (1) ◽  
pp. 73-86 ◽  
Author(s):  
Maria Favia ◽  
Lorenzo Guerra ◽  
Teresa Fanelli ◽  
Rosa Angela Cardone ◽  
Stefania Monterisi ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Apical Membrane ◽  
Rho Kinase ◽  
Chloride Secretion ◽  
Dominant Negative ◽  
Apical Region ◽  
Transmembrane Conductance Regulator ◽  
Regulatory Factor ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

We have demonstrated that Na+/H+ exchanger regulatory factor 1 (NHERF1) overexpression in CFBE41o- cells induces a significant redistribution of F508del cystic fibrosis transmembrane conductance regulator (CFTR) from the cytoplasm to the apical membrane and rescues CFTR-dependent chloride secretion. Here, we observe that CFBE41o- monolayers displayed substantial disassembly of actin filaments and that overexpression of wild-type (wt) NHERF1 but not NHERF1-Δ Ezrin-Radixin-Moesin (ERM) increased F-actin assembly and organization. Furthermore, the dominant-negative band Four-point one, Ezrin, Radixin, Moesin homology (FERM) domain of ezrin reversed the wt NHERF1 overexpression-induced increase in both F-actin and CFTR-dependent chloride secretion. wt NHERF1 overexpression enhanced the interaction between NHERF1 and both CFTR and ezrin and between ezrin and actin and the overexpression of wt NHERF1, but not NHERF1-ΔERM, also increased the phosphorylation of ezrin in the apical region of the cell monolayers. Furthermore, wt NHERF1 increased RhoA activity and transfection of constitutively active RhoA in CFBE41o- cells was sufficient to redistribute phospho-ezrin to the membrane fraction and rescue both the F-actin content and the CFTR-dependent chloride efflux. Rho kinase (ROCK) inhibition, in contrast, reversed the wt NHERF1 overexpression-induced increase of membrane phospho-ezrin, F-actin content, and CFTR-dependent secretion. We conclude that NHERF1 overexpression in CFBE41o- rescues CFTR-dependent chloride secretion by forming the multiprotein complex RhoA-ROCK-ezrin-actin that, via actin cytoskeleton reorganization, tethers F508del CFTR to the cytoskeleton stabilizing it on the apical membrane.

Dynasore inhibits removal of wild-type and ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) from the plasma membrane

2009 ◽  
Vol 421 (3) ◽  
pp. 377-385 ◽  
Author(s):  
Andrew Young ◽  
Martina Gentzsch ◽  
Cynthia Y. Abban ◽  
Ying Jia ◽  
Patricio I. Meneses ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Small Molecule ◽  
Short Exposure ◽  
Growth Media ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Cystic Fibrosis Transmembrane

Dynasore, a small molecule inhibitor of dynamin, was used to probe the role of dynamin in the endocytosis of wild-type and mutant CFTR (cystic fibrosis transmembrane conductance regulator). Internalization of both wild-type and ‘temperature-corrected’ ΔF508 CFTR was markedly inhibited by a short exposure to dynasore, implicating dynamin as a key element in the endocytic internalization of both wild-type and mutant CFTR. The inhibitory effect of dynasore was readily reversible upon washout of dynasore from the growth media. Corr-4 ({2-(5-chloro-2-methoxy-phenylamino)-4′-methyl-[4,5′]-bithiazolyl-2′-yl}-phenyl-methanonone), a pharmacological corrector of ΔF508 CFTR biosynthesis, caused a marked increase in the cell surface expression of mutant CFTR. Co-incubation of ΔF508 CFTR expressing cells with Corr-4 and dynasore caused a significantly greater level of cell surface CFTR than that observed in the presence of Corr-4 alone. These results argue that inhibiting the endocytic internalization of mutant CFTR provides a novel therapeutic target for augmenting the benefits of small molecule correctors of mutant CFTR biosynthesis.

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