Processing and function of CFTR-ΔF508 are species-dependent

Ostedgaard, Lynda S.; Rogers, Christopher S.; Randak, Christoph O.; Vermeer, Daniel W.; Rokhlina, Tatiana; Karp, Philip H.; Welsh, Michael J.

doi:10.1073/pnas.0706974104

Cited by 109 publications

(154 citation statements)

References 63 publications

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“…There are also contradicting reports regarding the actual P o of ⌬F508-CFTR channels (17,21,(27)(28)(29)(30). It is important to note that, for a channel with very low P o , it is very difficult to obtain a quantitative understanding of the kinetic defects associated with ⌬F508-CFTR when it is uncertain how many channels are present in a patch.…”

Section: G551d-cftr Gating By 2ј-and 3ј-deoxy-atp-mentioning

confidence: 77%

“…Several studies found that in cell-attached patches the maximal P o of ⌬F508-CFTR was less than that of WT-CFTR, the difference being due to a prolonged closed time for ⌬F508-CFTR (20 -22). However, studies performed in cell-free systems, including excised inside-out patches and reconstituted lipid bilayers, quote very different maximal P o ranging from 0.1 to 0.4 for ⌬F508-CFTR (17,21,(27)(28)(29)(30)(31). It should be noted that these studies may be liable to quantitative errors due to technical difficulties in quantifying ⌬F508-CFTR gating.…”

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confidence: 99%

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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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Section: G551d-cftr Gating By 2ј-and 3ј-deoxy-atp-mentioning

confidence: 77%

mentioning

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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“…We also expressed a recombinant CMV promoter-driven CFTR-ΔF508 cDNA in primary human CFTR null airway epithelia (CFTR Q493X/S912X) using an adenovirus (Ad) vector (41). In this setting, CFTR-mediated Cl − current was restored only in epithelia pretreated with the miR-138 mimic or SIN3A DsiRNA (Fig.…”

Section: Resultsmentioning

confidence: 99%

A microRNA network regulates expression and biosynthesis of wild-type and ΔF508 mutant cystic fibrosis transmembrane conductance regulator

Ramachandran

Karp²,

Jiang³

et al. 2012

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

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110

126

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Production of functional proteins requires multiple steps, including gene transcription and posttranslational processing. MicroRNAs (miRNAs) can regulate individual stages of these processes. Despite the importance of the cystic fibrosis transmembrane conductance regulator (CFTR) channel for epithelial anion transport, how its expression is regulated remains uncertain. We discovered that miRNA-138 regulates CFTR expression through its interactions with the transcriptional regulatory protein SIN3A. Treating airway epithelia with an miR-138 mimic increased CFTR mRNA and also enhanced CFTR abundance and transepithelial Cl − permeability independent of elevated mRNA levels. An miR-138 anti-miR had the opposite effects. Importantly, miR-138 altered the expression of many genes encoding proteins that associate with CFTR and may influence its biosynthesis. The most common CFTR mutation, ΔF508, causes protein misfolding, protein degradation, and cystic fibrosis. Remarkably, manipulating the miR-138 regulatory network also improved biosynthesis of CFTR-ΔF508 and restored Cl − transport to cystic fibrosis airway epithelia. This miRNA-regulated network directs gene expression from the chromosome to the cell membrane, indicating that an individual miRNA can control a cellular process more broadly than recognized previously. This discovery also provides therapeutic avenues for restoring CFTR function to cells affected by the most common cystic fibrosis mutation.

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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.

191

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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Processing and function of CFTR-ΔF508 are species-dependent

Cited by 109 publications

References 63 publications

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

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

A microRNA network regulates expression and biosynthesis of wild-type and ΔF508 mutant cystic fibrosis transmembrane conductance regulator

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

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