scholarly journals Rescue of epithelial HCO3−secretion in murine intestine by apical membrane expression of the cystic fibrosis transmembrane conductance regulator mutant F508del

2012 ◽  
Vol 590 (21) ◽  
pp. 5317-5334 ◽  
Author(s):  
Fang Xiao ◽  
Junhua Li ◽  
Anurag Kumar Singh ◽  
Brigitte Riederer ◽  
Jiang Wang ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Apical Membrane ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Membrane Expression ◽  
Cystic Fibrosis Transmembrane

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.

scholarly journals Differential thermostability and response to cystic fibrosis transmembrane conductance regulator potentiators of human and mouse F508del-CFTR

2019 ◽  
Vol 317 (1) ◽  
pp. L71-L86 ◽  
Author(s):  
Samuel J. Bose ◽  
Marcel J. C. Bijvelds ◽  
Yiting Wang ◽  
Jia Liu ◽  
Zhiwei Cai ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Membrane Expression ◽  
The Impact ◽  
Cystic Fibrosis Transmembrane ◽  
Human And Mouse

Cross-species comparative studies have highlighted differences between human and mouse cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial Cl− channel defective in cystic fibrosis (CF). Here, we compare the impact of the most common CF mutation F508del on the function of human and mouse CFTR heterologously expressed in mammalian cells and their response to CFTR modulators using the iodide efflux and patch-clamp techniques. Once delivered to the plasma membrane, human F508del-CFTR exhibited a severe gating defect characterized by infrequent channel openings and was thermally unstable, deactivating within minutes at 37°C. By contrast, the F508del mutation was without effect on the gating pattern of mouse CFTR, and channel activity demonstrated thermostability at 37°C. Strikingly, at all concentrations tested, the clinically approved CFTR potentiator ivacaftor was without effect on the mouse F508del-CFTR Cl− channel. Moreover, eight CFTR potentiators, including ivacaftor, failed to generate CFTR-mediated iodide efflux from CHO cells expressing mouse F508del-CFTR. However, they all produced CFTR-mediated iodide efflux with human F508del-CFTR-expressing CHO cells, while fifteen CFTR correctors rescued the plasma membrane expression of both human and mouse F508del-CFTR. Interestingly, the CFTR potentiator genistein enhanced CFTR-mediated iodide efflux from CHO cells expressing either human or mouse F508del-CFTR, whereas it only potentiated human F508del-CFTR Cl− channels in cell-free membrane patches, suggesting that its action on mouse F508del-CFTR is indirect. Thus, the F508del mutation has distinct effects on human and mouse CFTR Cl− channels.

Deactivation of CFTR-Cl conductance by endogenous phosphatases in the native sweat duct

1996 ◽  
Vol 270 (2) ◽  
pp. C474-C480 ◽  
Author(s):  
M. M. Reddy ◽  
P. M. Quinton
Keyword(s):  
Cystic Fibrosis ◽  
Okadaic Acid ◽  
Apical Membrane ◽  
Transmembrane Conductance Regulator ◽  
Sweat Duct ◽  
Transmembrane Conductance ◽  
Phosphatase Inhibitors ◽  
Cystic Fibrosis Transmembrane ◽  
Cl Conductance

Cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated Cl channel. However, very little is known about the endogenous mechanism(s) of deactivation of CFTR-Cl conductance (CFTR-GCl) in vivo. We studied the action of endogenous phosphatases in regulation of the adenosine 3',5'-cyclic monophosphate (cAMP)- and ATP-induced CFTR-GCl in the apical membrane of microperfused preparations of basolaterally permeabilized native sweat duct. Activation of CFTR-GCl was monitored by measuring the apical Cl diffusion potentials and GCl, which spontaneously deactivated on removal of cAMP. This spontaneous loss of CFTR-GCl activity could be prevented by a cocktail of phosphatase inhibitors (fluoride, vanadate, and okadaic acid). We studied the effects of each of these phosphatase antagonists on the rate of deactivation of CFTR-GCl after cAMP washout. In contrast to vanadate or fluoride, okadaic acid virtually prevented deactivation of CFTR-GCl after cAMP washout. We conclude that either or both protein phosphatases 1 and 2A are responsible for the dephosphorylation deactivation of CFTR-GCl in vivo.

Expression of cystic fibrosis transmembrane conductance regulator in a model epithelium

1994 ◽  
Vol 266 (4) ◽  
pp. L405-L413 ◽  
Author(s):  
D. N. Sheppard ◽  
M. R. Carson ◽  
L. S. Ostedgaard ◽  
G. M. Denning ◽  
M. J. Welsh
Keyword(s):  
Cystic Fibrosis ◽  
Apical Membrane ◽  
Single Cells ◽  
Basolateral Membrane ◽  
Recombinant Vaccinia Virus ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Monovalent Ions ◽  
Rat Thyroid ◽  
Cystic Fibrosis Transmembrane

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphorylation and by intracellular nucleotides. The function of CFTR, like other recombinant ion channels, has generally been studied in single cells using voltage-clamp techniques. However, because CFTR is normally located in the apical membrane of epithelia we wanted to develop a system to study the function of recombinant CFTR expressed in an epithelium. We chose Fischer rat thyroid (FRT) epithelia for two reasons. First, when grown on permeable filter supports, FRT cells form polarized epithelia with a high transepithelial resistance. Second, they have no endogenous cAMP-regulated Cl- channels in their apical membrane. We expressed CFTR in FRT epithelia either transiently, using recombinant vaccinia virus, or stably, using a retrovirus. To measure apical membrane Cl- currents, we permeabilized the basolateral membrane to monovalent ions with nystatin and imposed a large transepithelial Cl- concentration gradient. cAMP agonists stimulated apical membrane Cl- currents in FRT epithelia infected with wild-type CFTR (vTF-CFTR) but not in FRT epithelia infected with either control virus (vTF7-3) or CFTR containing the delta F508 mutation (vTF-delta F508). These Cl- currents had properties similar to those of cAMP-activated Cl- currents in cells expressing endogenous or recombinant CFTR.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator in Polarized Intestinal Epithelial Cells

2009 ◽  
Vol 20 (8) ◽  
pp. 2337-2350 ◽  
Author(s):  
Mark R. Silvis ◽  
Carol A. Bertrand ◽  
Nadia Ameen ◽  
Franca Golin-Bisello ◽  
Michael B. Butterworth ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Expression System ◽  
Anion Channel ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
T84 Cells ◽  
Anion Secretion ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.

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