Genistein potentiates wild-type and delta F508-CFTR channel activity

Hwang, Tzyh Chang; Wang, Fei; Yang, Iris Chun-Hui; Reenstra, William W.

doi:10.1152/ajpcell.1997.273.3.c988

Cited by 192 publications

(192 citation statements)

References 26 publications

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“…Genistein potentiates cAMP-dependent activation of DF508 CFTR [8]. The present results confirm that genistein induces the cAMP-dependent activation of DF508 CFTR and suggests further that it is able to increase the Cl -secretion achieved after incubation with 4PBA.…”

Section: Discussionsupporting

confidence: 85%

Activation of DeltaF508 CFTR in a cystic fibrosis respiratory epithelial cell line by 4-phenylbutyrate, genistein and CPX

Andersson¹,

Roomans²

2000

Eur Respir J

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The cellular basis of cystic fibrosis (CF) is a defect in a cyclic adenosine monophosphate (cAMP)‐activated chloride channel (CF transmembrane conductance regulator) in epithelial cells that leads to decreased chloride ion transport and impaired water transport across the cell membrane. This study investigated whether it was possible to activate the defective chloride channel in cystic fibrosis respiratory epithelial cells with 4‐phenylbutyrate (4PBA), genistein and 8‐cyclopentyl‐1,3‐dipropylxanthine (CPX). The CF bronchial epithelial cell line CFBE41o‐, which expresses the ΔF508 mutation, was treated with these agents and loss of Cl‐, indicating Cl‐ efflux, measured by X‐ray microanalysis. 8‐bromo‐cAMP alone did not induce Cl‐ efflux in CFBE41o‐ cells, but after incubation with 4PBA a significant efflux of Cl‐ occurred. Stimulation of cells with a combination of genistein and cAMP also induced Cl‐ efflux, whereas a combination of pretreatment with 4PBA and a combined stimulation with genistein and cAMP induced an even larger Cl‐ efflux. Cl‐ efflux could also be stimulated by CPX, but this effect was not enhanced by 4PBA pretreatment. The ΔF508 mutation leads to impaired processing of the cystic fibrosis transmembrane conductance regulator. The increased efflux of chloride after 4‐phenylbutyrate treatment can be explained by the fact that 4‐phenylbutyrate allows the ΔF508 cystic fibrosis transmembrane conductance regulator to escape degradation and to be transported to the cell surface. Genistein and 8‐cyclopentyl‐1,3‐dipropylxanthine act by stimulating chloride ion efflux by increasing the probability of the cystic fibrosis transmembrane conductance regulator being open. The combination of 4‐phenylbutyrate and genistein may be useful in a potential pharmacological therapy for cystic fibrosis patients with the ΔF508 mutation.

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Section: Discussionsupporting

confidence: 85%

Activation of DeltaF508 CFTR in a cystic fibrosis respiratory epithelial cell line by 4-phenylbutyrate, genistein and CPX

Andersson¹,

Roomans²

2000

Eur Respir J

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“…Furthermore, the sweat chloride level, a parameter directly reflecting CFTR activity in vivo and thus a widely used diagnostic tool for CF, in patients taking Vx-770 (19,20) were still higher than those found in patients with mild-form CF (19,20,40), suggesting that Vx-770 alone is insufficient to completely rectify the dysfunction associated with the G551D mutation in vivo. Furthermore, recent clinical trials suggest that treatments with drugs that can improve the trafficking of ΔF508-CFTR may not be sufficient to ameliorate the clinical symptoms in patient carrying the ΔF508 mutation (1) presumably because this most common pathogenic mutation causes deficits in both membrane expression and gating (41)(42)(43)(44)(45)(46). Thus, discovering new ways to enhance CFTR activity remains an outstanding goal in the field.…”

Section: Discussionmentioning

confidence: 99%

Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle

Jih

Hwang

2013

Proc. Natl. Acad. Sci. U.S.A.

187

229

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Vx-770 (Ivacaftor), a Food and Drug Administration (FDA)-approved drug for clinical application to patients with cystic fibrosis (CF), shifts the paradigm from conventional symptomatic treatments to therapeutics directly tackling the root of the disease: functional defects of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel caused by pathogenic mutations. The underlying mechanism for the action of Vx-770 remains elusive partly because this compound not only increases the activity of wild-type (WT) channels whose gating is primarily controlled by ATP binding/hydrolysis, but also improves the function of G551D-CFTR, a disease-associated mutation that abolishes CFTR's responsiveness to ATP. Here we provide a unified theory to account for this dual effect of Vx-770. We found that Vx-770 enhances spontaneous, ATP-independent activity of WT-CFTR to a similar magnitude as its effects on G551D channels, a result essentially explaining Vx-770's effect on G551D-CFTR. Furthermore, Vx-770 increases the open time of WT-CFTR in an [ATP]-dependent manner. This distinct kinetic effect is accountable with a newly proposed CFTR gating model depicting an [ATP]-dependent "reentry" mechanism that allows CFTR shuffling among different open states by undergoing multiple rounds of ATP hydrolysis. We further examined the effect of Vx-770 on R352C-CFTR, a unique mutant that allows direct observation of hydrolysis-triggered gating events. Our data corroborate that Vx-770 increases the open time of WT-CFTR by stabilizing a posthydrolytic open state and thereby fosters decoupling between the gating cycle and ATP hydrolysis cycle. The current study also suggests that this unique mechanism of drug action can be further exploited to develop strategies that enhance the function of CFTR.

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“…The ⌬F508-CFTR channels exhibit several physiological abnormalities; one of them is the low P o observed in intact cells due to a reduced opening rate (20,22). It was suggested that this low opening rate was due to a very slow phosphorylation rate (30) and not to an intrinsic defect in the gating mechanism.…”

Section: Discussionmentioning

confidence: 99%

Potentiation of Disease-associated Cystic Fibrosis Transmembrane Conductance Regulator Mutants by Hydrolyzable ATP Analogs

Miki

Zhou

et al. 2010

Journal of Biological Chemistry

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The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP-binding cassette transporter superfamily. CFTR is gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBDs), which dimerize in the presence of ATP to form two ATP-binding pockets (ABP1 and ABP2). Mutations reducing the activity of CFTR result in the genetic disease cystic fibrosis. Two of the most common mutations causing a severe phenotype are G551D and ⌬F508. Previously we found that the ATP analog N 6 -(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by ϳ7-fold. Here we show that 2-deoxy-ATP (dATP), but not 3-deoxy-ATP, increases the activity of G551D-CFTR by ϳ8-fold. We custom synthesized N 6 -(2-phenylethyl)-2-deoxy-ATP (P-dATP), an analog combining the chemical modifications in dATP and P-ATP. This new analog enhances G551D current by 36.2 ؎ 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Interestingly, P-dATP completely rectified the gating abnormality of ⌬F508-CFTR by increasing its activity by 19.5 ؎ 3.8-fold through binding to both ABPs. This result highlights the severity of the gating defect associated with ⌬F508, the most prevalent disease-associated mutation. The new analog P-dATP can be not only an invaluable tool to study CFTR gating, but it can also serve as a proof-of-principle that, by combining elements that potentiate the channel activity independently, the increase in chloride transport necessary to reach a therapeutic target is attainable. The cystic fibrosis transmembrane conductance regulator (CFTR)2 chloride channel is a major player in salt and water transport across epithelia. Like all members of the ATPbinding cassette (ABC) family, CFTR has two nucleotide-binding domains (NBDs), which contain the Walker A and B motifs and the highly conserved signature sequence. A regulatory (R) domain, unique to CFTR, needs to be phosphorylated by protein kinase A (PKA) for the channel to function. Experimental evidence suggests that the two NBDs of CFTR dimerize in a head-to-tail configuration (1), as in other ABC transporters, forming two ATP-binding pockets (ABPs) with two ATP molecules sandwiched in the interface. ABP1 is formed by the Walker A and B motifs of NBD1, and the signature sequence of NBD2 and ABP2 is formed by the Walker A and B motifs of NBD2 and the signature sequence of NBD1. Interestingly, two ABPs of CFTR assume distinct functional roles in controlling CFTR gating. Zhou et al. (2) showed that ABP2 is the site critical for channel opening by ATP, whereas the role of ABP1 is limited to help with the stabilization of the open channel conformation. Furthermore, although ABP1 seems unable to hydrolyze ATP, ATP hydrolysis at NBD2 is associat...

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Genistein potentiates wild-type and delta F508-CFTR channel activity

Cited by 192 publications

References 26 publications

Activation of DeltaF508 CFTR in a cystic fibrosis respiratory epithelial cell line by 4-phenylbutyrate, genistein and CPX

Activation of DeltaF508 CFTR in a cystic fibrosis respiratory epithelial cell line by 4-phenylbutyrate, genistein and CPX

Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle

Potentiation of Disease-associated Cystic Fibrosis Transmembrane Conductance Regulator Mutants by Hydrolyzable ATP Analogs

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