scholarly journals Locating a Plausible Binding Site for an Open-Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator

2012 ◽  
Vol 82 (6) ◽  
pp. 1042-1055 ◽  
Author(s):  
Yohei Norimatsu ◽  
Anthony Ivetac ◽  
Christopher Alexander ◽  
Nicolette O'Donnell ◽  
Leah Frye ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Binding Site ◽  
Open Channel ◽  
Channel Blocker ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
Open Channel Blocker

scholarly journals Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl- channel by two closely related arylaminobenzoates.

1993 ◽  
Vol 102 (1) ◽  
pp. 1-23 ◽  
Author(s):  
N A McCarty ◽  
S McDonough ◽  
B N Cohen ◽  
J R Riordan ◽  
N Davidson ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Binding Site ◽  
Open Channel ◽  
Transmembrane Conductance Regulator ◽  
Channel Block ◽  
Transmembrane Conductance ◽  
Open Channel Block ◽  
Whole Cell ◽  
Voltage Dependent ◽  
Cystic Fibrosis Transmembrane

The gene defective in cystic fibrosis encodes a Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is blocked by diphenylamine-2-carboxylate (DPC) when applied extracellularly at millimolar concentrations. We studied the block of CFTR expressed in Xenopus oocytes by DPC or by a closely related molecule, flufenamic acid (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by FFA, both at 200 microM, requires several minutes to reach full effect. Blockade is voltage dependent, suggesting open-channel block: currents at positive potentials are not affected but currents at negative potentials are reduced. The binding site for both drugs senses approximately 40% of the electric field across the membrane, measured from the inside. In single-channel recordings from excised patches without blockers, the conductance was 8.0 +/- 0.4 pS in symmetric 150 mM Cl-. A subconductance state, measuring approximately 60% of the main conductance, was often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hyperpolarizing voltages, bursts were interrupted by brief closures. Either DPC or FFA (50 microM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at Vm = -100 mV and not at Vm = +100 mV, in agreement with whole-cell experiments. DPC induced a higher frequency of flickers from the cytoplasmic side than the extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (approximately 1.2 ms) at -100 mV with application from either side. In cell-attached patch recordings with DPC or FFA applied to the bath, there was flickery block at Vm = -100 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a single binding site for both drugs, reached from either end of the channel. Open-channel block by DPC or FFA may offer tools for use with site-directed mutagenesis to describe the permeation pathway.

CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands

1999 ◽  
Vol 277 (4) ◽  
pp. L694-L699 ◽  
Author(s):  
Stephen T. Ballard ◽  
Laura Trout ◽  
Zsuzsa Bebök ◽  
E. J. Sorscher ◽  
Angela Crews
Keyword(s):  
Cystic Fibrosis ◽  
Channel Blocker ◽  
Polyclonal Antibodies ◽  
Surface Epithelium ◽  
Transmembrane Conductance Regulator ◽  
Submucosal Gland ◽  
Transmembrane Conductance ◽  
Active Secretion ◽  
Submucosal Glands ◽  
Cystic Fibrosis Transmembrane

Previous studies demonstrated that ACh-induced liquid secretion by porcine bronchi is driven by active Cl− and H[Formula: see text] secretion. The present study was undertaken to determine whether this process was localized to submucosal glands and mediated by the cystic fibrosis transmembrane conductance regulator (CFTR). When excised, cannulated, and treated with ACh, porcine bronchi secreted 15.6 ± 0.6 μl ⋅ cm−2 ⋅ h−1. Removal of the surface epithelium did not significantly affect the rate of secretion, indicating that the source of the liquid was the submucosal glands. Pretreatment with diphenylamine-2-carboxylate, a relatively nonselective Cl−-channel blocker, significantly reduced liquid secretion by 86%, whereas pretreatment with DIDS, which inhibits a variety of Cl− channels but not CFTR, had no effect. When bronchi were pretreated with glibenclamide or 5-nitro-2-(3-phenylpropylamino)benzoic acid (both inhibitors of CFTR), the rate of ACh-induced liquid secretion was significantly reduced by 39 and 91%, respectively, compared with controls. Agents that blocked liquid secretion also caused disproportionate reductions in H[Formula: see text] secretion. Polyclonal antibodies to the CFTR bound preferentially to submucosal gland ducts and the surface epithelium, suggesting that this channel was localized to these sites. These data suggest that ACh-induced gland liquid secretion by porcine bronchi is driven by active secretion of both Cl− and H[Formula: see text] and is mediated by the CFTR.

scholarly journals A Peptide Nucleic Acid (PNA) Masking the miR-145-5p Binding Site of the 3′UTR of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mRNA Enhances CFTR Expression in Calu-3 Cells

Molecules ◽  
2020 ◽  
Vol 25 (7) ◽  
pp. 1677 ◽  
Author(s):  
Shaiq Sultan ◽  
Andrea Rozzi ◽  
Jessica Gasparello ◽  
Alex Manicardi ◽  
Roberto Corradini ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Binding Site ◽  
Peptide Nucleic Acid ◽  
Therapeutic Strategy ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Treatment Protocols ◽  
Cftr Protein ◽  
Cystic Fibrosis Transmembrane ◽  
Different Levels

Peptide nucleic acids (PNAs) have been demonstrated to be very useful tools for gene regulation at different levels and with different mechanisms of action. In the last few years the use of PNAs for targeting microRNAs (anti-miRNA PNAs) has provided impressive advancements. In particular, targeting of microRNAs involved in the repression of the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis (CF), is a key step in the development of new types of treatment protocols. In addition to the anti-miRNA therapeutic strategy, inhibition of miRNA functions can be reached by masking the miRNA binding sites present within the 3′UTR region of the target mRNAs. The objective of this study was to design a PNA masking the binding site of the microRNA miR-145-5p present within the 3′UTR of the CFTR mRNA and to determine its activity in inhibiting miR-145-5p function, with particular focus on the expression of both CFTR mRNA and CFTR protein in Calu-3 cells. The results obtained support the concept that the PNA masking the miR-145-5p binding site of the CFTR mRNA is able to interfere with miR-145-5p biological functions, leading to both an increase of CFTR mRNA and CFTR protein content.

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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