Evidence for direct CFTR inhibition by CFTRinh-172 based on Arg347 mutagenesis

Caci, Emanuela; Caputo, Antonella; Hinzpeter, Alexandre; Arous, N.; Fanen, Pascale; Sonawane, N.D.; Verkman, A. S.; Ravazzolo, Roberto; Zegarra‐Moran, Olga; Galietta, Luis J. V.

doi:10.1042/bj20080029

Cited by 94 publications

(92 citation statements)

References 35 publications

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“…At the end of each experiment (Fig. 2, A-C), we added CFTR inh -172 (10 M), a selective CFTR inhibitor (Caci et al, 2008). This compound returned transepithelial current to values close to zero, thus indicating that forskolin-and potentiator-activated currents depended on CFTR activity.…”

Section: Resultsmentioning

confidence: 99%

“…CFTR activity was determined in transiently transfected COS-7 cells with use of the halide-sensitive yellow fluorescent protein (YFP)-H148Q/I152L (Galietta et al, 2001a) as also described previously (Caci et al, 2008). Cells were plated in 96-well microplates (25,000 cells/well) in 100 l of antibiotic-free culture medium and, after 6 h, cotransfected with plasmids carrying the coding sequence for CFTR and the halide-sensitive YFP.…”

Section: Methodsmentioning

confidence: 99%

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Mutation-Specific Potency and Efficacy of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Potentiators

Caputo

Hinzpeter

Caci

et al. 2009

J Pharmacol Exp Ther

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Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) ClϪ channel. The mutations G551D and G1349D, which affect the nucleotidebinding domains (NBDs) of CFTR protein, reduce channel activity. This defect can be corrected pharmacologically by small molecules called potentiators. CF mutations residing in the intracellular loops (ICLs), connecting the transmembrane segments of CFTR, may also reduce channel activity. We have investigated the extent of loss of function caused by ICL mutations and the sensitivity to pharmacological stimulation. We found that E193K and G970R (in ICL1 and ICL3, respectively) cause a severe loss of CFTR channel activity that can be rescued by the same potentiators that are effective on NBD mutations. We compared potency and efficacy of three different potentiators for E193K, G970R, and G551D. The 1,4-dihydropyridine felodipine and the phenylglycine PG-01 [2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylacetamide] were strongly effective on the three CFTR mutants. The efficacy of sulfonamide SF-01 [6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide], another CFTR potentiator, was instead significantly lower than felodipine and PG-01 for the E193K and G970R mutations, and almost abolished for G551D. Furthermore, SF-01 modified the response of G551D and G970R to the other two potentiators, an effect that may be explained by an allosteric antagonistic effect. Our results indicate that CFTR potentiators correct the basic defect caused by CF mutations residing in different CFTR domains. However, there are differences among potentiators, with felodipine and PG-01 having a wider pharmacological activity, and SF-01 being more mutation specific. Our observations are useful in the prioritization and development of drugs targeting the CF basic defect.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mutation-Specific Potency and Efficacy of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Potentiators

Caputo

Hinzpeter

Caci

et al. 2009

J Pharmacol Exp Ther

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“…CFTRinh-172 alters CFTR gating by voltage-independent binding to a cytoplasmic nucleotide binding domain (39) or, according to more recent evidence, by direct pore interaction (2). CFTRinh-172 inhibition of CFTR may alter direct or indirect interactions between CFTR and Slc26-like anion exchangers.…”

Section: Identification Of Apical CL ϫ /Hcomentioning

confidence: 99%

Functional coupling of apical Cl⁻/HCO₃⁻ exchange with CFTR in stimulated HCO₃⁻ secretion by guinea pig interlobular pancreatic duct

Stewart

Yamamoto²,

Nakakuki³

et al. 2009

American Journal of Physiology-Gastrointestinal and Liver Physi

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Ϫ -rich fluid in response to stimulation by the adenylyl cyclase-coupled hormone secretin. The Cl Ϫ -rich secretion of pancreatic acinar cells is modified as it flows along the pancreatic ductal system, ultimately producing pancreatic juice with final concentrations of ϳ140 mM HCO 3 Ϫ and ϳ20 mM Cl Ϫ (31). In the guinea pig pancreatic duct, the uptake of bicarbonate across the basolateral membrane is mediated by Na ϩ -HCO 3 Ϫ cotransport (NBC1) and by Na ϩ /H ϩ exchange (19). HCO 3 Ϫ efflux across the apical membrane has been proposed to be mediated by CFTR, in concert with apical Cl Ϫ /HCO 3 Ϫ exchanger activity (9). Proximal pancreatic duct HCO 3 Ϫ secretion by apical Cl Ϫ / HCO 3 Ϫ exchange is favored by the high luminal Cl Ϫ concentrations resulting from pancreatic acinar Cl Ϫ secretion. However, since this proximal fluid flows distally toward the ampullary terminus of the duct, its Cl Ϫ concentration progressively decreases in parallel with a gradual increase in its HCO 3 Ϫ concentration. These inverse changes in Cl Ϫ and HCO 3 Ϫ concentrations are predicted to alter relative contributions of apical membrane Cl Ϫ /HCO 3 Ϫ exchange and CFTR-mediated HCO 3 Ϫ conductance and have been suggested to reflect axial variation in expression and regulation of these transport activities (18). The Cl Ϫ /HCO 3 Ϫ exchangers Slc26a3 and Slc26a6 have been detected in mouse pancreatic duct and in the human pancreatic cell line CFPAC-1 (10, 22), leading to the proposal that Cl Ϫ -dependent HCO 3 Ϫ secretion by pancreatic proximal ducts represents combined activities of Slc26a6 and Slc26a3 anion exchangers, positively regulated by activated CFTR, with reciprocal positive regulation of CFTR by the anion exchangers (4, 5, 22, 23). However, a study in native mouse pancreatic duct suggests negative regulation of CFTR by Slc26a6 (43).Secretin-stimulated HCO 3 Ϫ secretion in guinea pig pancreatic duct is not dependent on elevated luminal [Cl Ϫ ] (21) (brackets denote concentration) and occurs even in the presence of luminal fluid containing 125 mM HCO 3 Ϫ and 23 mM Cl Ϫ (17). As luminal [Cl Ϫ ] falls to or below this level (with corresponding elevation of [HCO 3 Ϫ ]), CFTR Cl Ϫ permeability may fall while that for HCO 3 Ϫ may rise (34). Measurements of membrane potential (V m ) and intracellular pH (pH i ) in luminally perfused interlobular ducts from guinea pig pancreas suggest that CFTR HCO 3 Ϫ conductance can account for observed levels of stimulated bicarbonate secretion (15,18,20). A computational model of pancreatic duct HCO 3 Ϫ secretion predicted for similar conditions that ϳ94% of HCO 3 Ϫ efflux across the apical membrane was mediated by HCO 3 Ϫ conductance (36), although the model did not consider contributions from electrogenic Cl Ϫ /HCO 3 Ϫ exchange. Thus the HCO 3 Ϫ conductance of CFTR could provide the main route for apical HCO 3 Ϫ secretion in distal pancreatic ducts from species in which, like human and guinea pig, pancreatic juice contains high concentrations of HCO 3 Ϫ (ϳ140 mM). Studies of the role of SLC26-media...

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“…The amplitudes of the CFTR currents were obtained from the total current blocked by the specific CFTR inhibitor CFTR inh -172. 31 The best clone, FRTcl7, responded to the cAMP agonist with a short-circuit current increase of ca. 18 mA cm À2 ( Figure 6).…”

Section: Cftr Expression From the Human Locus In Bac Vectorsmentioning

confidence: 99%

CFTR expression and activity from the human CFTR locus in BAC vectors, with regulatory regions, isolated by a single-step procedure

et al. 2010

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We have assembled two BAC vectors containing a single fragment spanning the entire CFTR locus and including the upstream and downstream regions. The two vectors differ in size of the upstream region, and were recovered in Escherichia coli, with intact BAC DNAs prepared for structural and functional analyses. Sequence analysis allowed precise mapping of the inserts. We show that the CFTR gene was wild type and is categorized as the most frequent haplotype in Caucasian populations, identified by the following polymorphisms: (GATT) 7 in intron 6a; (TG) 11 T 7 in intron 8; V470 at position 470. CFTR expression and activity were analyzed in model cells by RT-PCR, quantitative real-time PCR, western blotting, indirect immunofluorescence and electrophysiological methods, which show the presence of an active CFTR Cl À channel. Finally, and supporting the hypothesis that CFTR functions as a receptor for Pseudomonas aeruginosa, we show that CFTR-expressing cells internalized more bacteria than parental cells that do not express CFTR. Overall, these data demonstrate that the BAC vectors contain a functional CFTR fragment and have unique features, including derivation from a single fragment, availability of a detailed genomic map and the possibility to use standard extraction procedures for BAC DNA preparations.

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Evidence for direct CFTR inhibition by CFTRinh-172 based on Arg347 mutagenesis

Cited by 94 publications

References 35 publications

Mutation-Specific Potency and Efficacy of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Potentiators

Mutation-Specific Potency and Efficacy of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Potentiators

Functional coupling of apical Cl⁻/HCO₃⁻ exchange with CFTR in stimulated HCO₃⁻ secretion by guinea pig interlobular pancreatic duct

CFTR expression and activity from the human CFTR locus in BAC vectors, with regulatory regions, isolated by a single-step procedure

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Evidence for direct CFTR inhibition by CFTRinh-172 based on Arg347 mutagenesis

Cited by 94 publications

References 35 publications

Mutation-Specific Potency and Efficacy of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Potentiators

Mutation-Specific Potency and Efficacy of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Potentiators

Functional coupling of apical Cl−/HCO3− exchange with CFTR in stimulated HCO3− secretion by guinea pig interlobular pancreatic duct

CFTR expression and activity from the human CFTR locus in BAC vectors, with regulatory regions, isolated by a single-step procedure

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Functional coupling of apical Cl⁻/HCO₃⁻ exchange with CFTR in stimulated HCO₃⁻ secretion by guinea pig interlobular pancreatic duct