scholarly journals The chemical chaperone CFcor-325 repairs folding defects in the transmembrane domains of CFTR-processing mutants

2006 ◽  
Vol 395 (3) ◽  
pp. 537-542 ◽  
Author(s):  
Tip W. Loo ◽  
M. Claire Bartlett ◽  
Ying Wang ◽  
David M. Clarke
Keyword(s):  
Cystic Fibrosis ◽  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Hot Spot ◽  
Cross Linking ◽  
Cysteine Residues ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Chemical Chaperone

Most patients with CF (cystic fibrosis) express a CFTR [CF TM (transmembrane) conductance regulator] processing mutant that is not trafficked to the cell surface because it is retained in the endoplasmic reticulum due to altered packing of the TM segments. CL4 (cytoplasmic loop 4) connecting TMs 10 and 11 is a ‘hot-spot’ for CFTR processing mutations. The chemical chaperone CFcor-325 (4-cyclohexyloxy-2-{1-[4-(4-methoxy-benezenesulphonyl)piperazin-1-yl]-ethyl}-quinazoline) rescued most CL4 mutants. To test if CFcor-325 promoted correct folding of the TMDs (TM domains), we selected two of the CL4 mutants (Q1071P and H1085R) for disulphide cross-linking analysis. Pairs of cysteine residues that were cross-linked in mature wild-type CFTR were introduced into mutants Q1071P and H1085R. The cross-linking patterns of the Q1071P or H1085R double cysteine mutants rescued with CFcor-325 were similar to those observed with mature wild-type double cysteine proteins. These results show that CFcor-325 rescued CFTR mutants by repairing the folding defects in the TMDs.

Correctors promote folding of the CFTR in the endoplasmic reticulum

2008 ◽  
Vol 413 (1) ◽  
pp. 29-36 ◽  
Author(s):  
Tip W. Loo ◽  
M. Claire Bartlett ◽  
David M. Clarke
Keyword(s):  
Cystic Fibrosis ◽  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Cross Linking ◽  
Transmembrane Conductance Regulator ◽  
Native Structure ◽  
Transmembrane Conductance ◽  
Mutant Proteins ◽  
Regulator Protein ◽  
Cystic Fibrosis Transmembrane

Cystic fibrosis (CF) is most commonly caused by deletion of a residue (ΔF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) protein. The misfolded mutant protein is retained in the ER (endoplasmic reticulum) and is not trafficked to the cell surface (misprocessed mutant). Corrector molecules such as corr-2b or corr-4a are small molecules that increase the amount of functional CFTR at the cell surface. Correctors may function by stabilizing CFTR at the cell surface or by promoting folding in the ER. To test whether correctors promoted folding of CFTR in the ER, we constructed double-cysteine CFTR mutants that would be retained in the ER and only undergo cross-linking when the protein folds into a native structure. The mature form, but not the immature forms, of M348C(TM6)/T1142C(TM12) (where TM is transmembrane segment), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants were efficiently cross-linked. Mutations to the COPII (coatamer protein II) exit motif (Y563KDAD567) were then made in the cross-linkable cysteine mutants to prevent the mutant proteins from leaving the ER. Membranes were prepared from the mutants expressed in the absence or presence of correctors and subjected to disulfide cross-linking analysis. The presence of correctors promoted folding of the mutants as the efficiency of cross-linking increased from approx. 2–5% to 22–35%. The results suggest that correctors interact with CFTR in the ER to promote folding of the protein into a native structure.

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.

In vivo activation of CFTR-dependent chloride transport in murine airway epithelium by CNP

1997 ◽  
Vol 273 (5) ◽  
pp. L1065-L1072 ◽  
Author(s):  
Thomas J. Kelley ◽  
Calvin U. Cotton ◽  
Mitchell L. Drumm
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Transport ◽  
Nasal Epithelium ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Cyclic Monophosphate ◽  
Δf508 Cftr ◽  
Membrane Bound

Inhibitors of guanosine 3′,5′-cyclic monophosphate (cGMP)-inhibited phosphodiesterases stimulate Cl− transport across the nasal epithelia of cystic fibrosis mice carrying the ΔF508 mutation [cystic fibrosis transmembrane conductance regulator (CFTR) (ΔF/ΔF)], suggesting a role for cGMP in regulation of epithelial ion transport. Here we show that activation of membrane-bound guanylate cyclases by C-type natriuretic peptide (CNP) stimulates hyperpolarization of nasal epithelium in both wild-type and ΔF508 CFTR mice in vivo but not in nasal epithelium of mice lacking CFTR [CFTR(−/−)]. With the use of a nasal transepithelial potential difference (TEPD) assay, CNP was found to hyperpolarize lumen negative TEPD by 6.1 ± 0.6 mV in mice carrying wild-type CFTR. This value is consistent with that obtained with 8-bromoguanosine 3′,5′-cyclic monophosphate (6.2 ± 0.9 mV). A combination of the adenylate cyclase agonist forskolin and CNP demonstrated a synergistic ability to induce Cl− secretion across the nasal epithelium of CFTR(ΔF/ΔF) mice. No effect on TEPD was seen with this combination when used on CFTR(−/−) mice, implying that the CNP-induced change in TEPD in CFTR(ΔF/ΔF) mice is CFTR dependent.

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.

scholarly journals ΔF508 CFTR Pool in the Endoplasmic Reticulum Is Increased by Calnexin Overexpression

2004 ◽  
Vol 15 (2) ◽  
pp. 563-574 ◽  
Author(s):  
Tsukasa Okiyoneda ◽  
Kazutsune Harada ◽  
Motohiro Takeya ◽  
Kaori Yamahira ◽  
Ikuo Wada ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Endoplasmic Reticulum ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Cystic Fibrosis Transmembrane ◽  
Erad Pathway

The most common cystic fibrosis transmembrane conductance regulator (CFTR) mutant in cystic fibrosis patients, ΔF508 CFTR, is retained in the endoplasmic reticulum (ER) and is consequently degraded by the ubiquitin-proteasome pathway known as ER-associated degradation (ERAD). Because the prolonged interaction of ΔF508 CFTR with calnexin, an ER chaperone, results in the ERAD of ΔF508 CFTR, calnexin seems to lead it to the ERAD pathway. However, the role of calnexin in the ERAD is controversial. In this study, we found that calnexin overexpression partially attenuated the ERAD of ΔF508 CFTR. We observed the formation of concentric membranous bodies in the ER upon calnexin overexpression and that the ΔF508 CFTR but not the wild-type CFTR was retained in the concentric membranous bodies. Furthermore, we observed that calnexin overexpression moderately inhibited the formation of aggresomes accumulating the ubiquitinated ΔF508 CFTR. These findings suggest that the overexpression of calnexin may be able to create a pool of ΔF508 CFTR in the ER.

scholarly journals Correctors Promote Maturation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-processing Mutants by Binding to the Protein

2007 ◽  
Vol 282 (46) ◽  
pp. 33247-33251 ◽  
Author(s):  
Ying Wang ◽  
Tip W. Loo ◽  
M. Claire Bartlett ◽  
David M. Clarke
Keyword(s):  
Cystic Fibrosis ◽  
Channel Blocker ◽  
Cross Linking ◽  
Transmembrane Conductance Regulator ◽  
Mature Protein ◽  
Transmembrane Conductance ◽  
P Glycoprotein ◽  
Δf508 Cftr ◽  
Cross Linker ◽  
Cystic Fibrosis Transmembrane

The most common cause of cystic fibrosis (CF) is defective folding of a cystic fibrosis transmembrane conductance regulator (CFTR) mutant lacking Phe508 (ΔF508). The ΔF508 protein appears to be trapped in a prefolded state with incomplete packing of the transmembrane (TM) segments, a defect that can be repaired by expression in the presence of correctors such as corr-4a, VRT-325, and VRT-532. To determine whether the mechanism of correctors involves direct interactions with CFTR, our approach was to test whether correctors blocked disulfide cross-linking between cysteines introduced into the two halves of a Cys-less CFTR. Although replacement of the 18 endogenous cysteines of CFTR with Ser or Ala yields a Cys-less mutant that does not mature at 37 °C, we found that maturation could be restored if Val510 was changed to Ala, Cys, Ser, Thr, Gly, Ala, or Asp. The V510D mutation also promoted maturation of ΔF508 CFTR. The Cys-less/V510A mutant was used for subsequent cross-linking analysis as it yielded relatively high levels of mature protein that was functional in iodide efflux assays. We tested for cross-linking between cysteines introduced into TM6 and TM7 of Cys-less CFTR/V510A because cross-linking between TM6 and TM7 of P-glycoprotein, the sister protein of CFTR, was inhibited with the corrector VRT-325. Cys-less CFTR/V510A mutant containing cysteines at I340C(TM6) and S877C(TM7) could be cross-linked with a homobifunctional cross-linker. Correctors and the CFTR channel blocker benzbromarone, but not P-glycoprotein substrates, inhibited cross-linking of mutant I340C(TM6)/S877C(TM7). These results suggest that corrector molecules such as corr-4a interact directly with CFTR.

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