ATP alters current fluctuations of cystic fibrosis transmembrane conductance regulator: evidence for a three-state activation mechanism.

Venglarik, Charles J.; Schultz, Bruce D.; Frizzell, Raymond A.; Bridges, Robert J.

doi:10.1085/jgp.104.1.123

Cited by 78 publications

(113 citation statements)

References 66 publications

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“…Additionally, kinetic behavior of the channels in control conditions (see Figs. 4 and 5) was indistinguishable from our previous characterization of CFTR when evaluated in this system (21,22).…”

Section: Methodscontrasting

confidence: 53%

“…To determine the mechanism of this inhibition, the effect of ibuprofen on CFTR Cl Ϫ channels in excised, inside-out patches from mouse L cells recombinantly expressing CFTR (19) was determined. We characterized previously the kinetics of nucleotide-dependent gating (21,22) and sulfonylurea-dependent block of CFTR (23,31) in these cells in detail. The results of one experiment with ibuprofen are shown in Fig.…”

Section: Effect Of Ibuprofen On Cftr In Excised Inside-out Patchesmentioning

confidence: 99%

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Ibuprofen inhibits cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion.

Devor

Schultz²

1998

J. Clin. Invest.

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We evaluated the acute effects of ibuprofen and salicylic acid on cAMP-mediated Cl Ϫ secretion (I sc ) in both colonic and airway epithelia. In T84 cells, ibuprofen inhibited the forskolin-dependent I sc in a concentration-dependent manner, having an apparent K i of 142 M. Salicylic acid inhibited I sc with an apparent K i of 646 M. We determined whether ibuprofen would also inhibit the forskolin-stimulated I sc in primary cultures of mouse trachea epithelia (MTE) and human bronchial epithelia (HBE). Similar to our results in T84 cells, ibuprofen (500 M) inhibited the forskolin-induced I sc in MTEs and HBEs by 59 Ϯ 4% ( n ϭ 11) and 39 Ϯ 6% ( n ϭ 8), respectively. Nystatin was employed to selectively permeabilize the basolateral or apical membrane to determine the effect of ibuprofen on apical Cl Ϫ (I Cl ) and basolateral K ϩ (I K ) currents after stimulation by forskolin. After forskolin stimulation, ibuprofen (500 M) reduced both the I Cl and I K ; reducing I Cl and I K by 60 and 15%, respectively. To determine whether this inhibition of I Cl was due to the inhibition of CFTR, the effects of ibuprofen and salicylic acid on CFTR Cl Ϫ channels in excised, inside-out patches from L-cells were evaluated. Ibuprofen (300 M) reduced CFTR Cl Ϫ current by 60 Ϯ 16% and this was explained by a short-lived block ( ‫ف‬ 1.2 ms) which causes an apparent reduction in single channel amplitude from 1.07 Ϯ 0.04 pA to 0.59 Ϯ 0.04 pA ( n ϭ 3). Similarly, salicylic acid (3 mM) reduced CFTR Cl Ϫ current by 50 Ϯ 8% with an apparent reduction in single channel amplitude from 1.08 Ϯ 0.03 pA to 0.48 Ϯ 0.06 pA ( n ϭ 4). Based on these results, we conclude that the NSAIDs ibuprofen and salicylic acid inhibit cAMP-mediated Cl Ϫ secretion in human colonic and airway epithelia via a direct inhibition of CFTR Cl Ϫ channels as well as basolateral membrane K ϩ channels. This may reduce their efficacy in conjunction with other therapeutic strategies designed to increase CFTR expression and/or function in secretory epithelia. ( J. Clin. Invest.

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Section: Methodscontrasting

confidence: 53%

Section: Effect Of Ibuprofen On Cftr In Excised Inside-out Patchesmentioning

confidence: 99%

Ibuprofen inhibits cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion.

Devor

Schultz²

1998

J. Clin. Invest.

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“…CFTR channels require ATP and cAMP to open. In oocytes, resting ATP levels are sufficient (29,30); however, cAMP levels need to increase, and experimentally this can be accomplished by adding extracellular forskolin to stimulate adenylyl cylase activity. Fig.…”

Section: Site-directed Editase Can Specifically Correct a Premature Tmentioning

confidence: 99%

Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing

Montiel-González

Vallecillo-Viejo

Yudowski

et al. 2013

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

198

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Adenosine deaminases that act on RNA are a conserved family of enzymes that catalyze a natural process of site-directed mutagenesis. Biochemically, they convert adenosine to inosine, a nucleotide that is read as guanosine during translation; thus when editing occurs in mRNAs, codons can be recoded and the changes can alter protein function. By removing the endogenous targeting domains from human adenosine deaminase that acts on RNA 2 and replacing them with an antisense RNA oligonucleotide, we have engineered a recombinant enzyme that can be directed to edit anywhere along the RNA registry. Here we demonstrate that this enzyme can efficiently and selectively edit a single adenosine. As proof of principle in vitro, we correct a premature termination codon in mRNAs encoding the cystic fibrosis transmembrane conductance regulator anion channel. In Xenopus oocytes, we show that a genetically encoded version of our editase can correct cystic fibrosis transmembrane conductance regulator mRNA, restore fulllength protein, and reestablish functional chloride currents across the plasma membrane. Finally, in a human cell line, we show that a genetically encoded version of our editase and guide RNA can correct a nonfunctional version of enhanced green fluorescent protein, which contains a premature termination codon. This technology should spearhead powerful approaches to correcting a wide variety of genetic mutations and fine-tuning protein function through targeted nucleotide deamination.

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“…Most ABC proteins are presumed to hydrolyze ATP in transporting solutes across the cell membrane as has been demonstrated for the periplasmic permeases (Hyde et al, 1990;Ames and Lecar, 1992). However, CFTR is a chloride channel which requires both PKAdependent phosphorylation to initiate activity and the continued presence of ATP for steady state channel gating (Anderson, Berger, Rich, Gregory, Smith, and Welsh, 1991;Nagel, Hwang, Nastiuk, Nairn, and Gadsby, 1992;Venglarik, Schultz, Frizzell, and Bridges, 1994; see reviews by Anderson, Sheppard, Berger, and Welsh, 1992;Collins, 1992). It has been suggested that CFTR hydrolyzes ATP as a necessary step in channel gating.…”

Section: Introductionmentioning

confidence: 99%

“…More complex schemes have been proposed which include nucleotide binding at both putative NBDs with hydrolysis occurring at the first NBD , the second NBD , or both NBDs, but with one hydrolytic event leading to channel opening and the second hydrolytic event leading to channel closure (Baukrowitz, Hwang, Nairn, and Gadsby, 1994;Hwang, Nagel, Nairn, and Gadsby, 1994). We (Venglarik et al, 1994) proposed a scheme to account for details of the gating properties seen at 37~ that is an extension of that proposed by Winter, Sheppard, Carson, and Welsh (1994). The kinetic scheme presented as Scheme I, requires the presence of ATP for channel gating to occur, but neither requires nor precludes ATP hydrolysis as a step in this gating.…”

Section: Introductionmentioning

confidence: 99%

Regulation of CFTR Cl- channel gating by ADP and ATP analogues.

Schultz

Venglarik

Bridges

et al. 1995

The Journal of General Physiology

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A B S T R A C T The cystic fibrosis gene product (CFTR) isNucleotide triphosphates initiate channel activity, while nucleotide diphosphates and nonhydrolyzable ATP analogues do not. To further characterize the role of these compounds on CFTR channel activity we examined their effects on chloride channel currents in excised inside-out membrane patches from CFFR transfected mouse L cells. ADP competitively inhibited ATP-dependent CFTR channel gating with a Ki of 16 -+ 9 ~M. AMP neither initiated CVFR channel gating nor inhibited ATP-dependent CFFR channel gating. Similarly, ATP analogues with substitutions in the phosphate chain, including AMPCPP, AMPPCP, AMPPNP, and ATP~S failed to support CFTR channel activity when present at the cytoplasmic face of the membrane and none of these analogues, when present at three to 10-fold excess of ATP, detectably altered ATP-dependent CFFR channel gating. These data suggest that none of these ATP analogues interact with the ATP regulatory site of CFFR which we previously characterized and, therefore, no inference regarding a requirement for ATP hydrolysis in CFFR channel gating can be made from their failure to support channel activity. Furthermore, the data indicate that this nucleotide regulatory site is exquisitely sensitive to alterations in the phosphate chain of the nucleotide; only a nonsubstituted nucleotide di-or triphosphate interacts with this regulatory site. Alternative recording conditions, such as the presence of kinase and a reduction in temperature to 25~ result in a previously uncharacterized kinetic state of CFTR which may exhibit distinctly different nucleotide dependencies.

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ATP alters current fluctuations of cystic fibrosis transmembrane conductance regulator: evidence for a three-state activation mechanism.

Abstract: The cystic fibrosis gene product cystic fibrosis transmembrane conductance regulator (CFTR) is a low conductance, cAMP-regulated C1-channel. Removal of cytosolic ATP causes a cessation of cAMP-dependent kinase-phosphorylated CFTR channel activity that resumes upon ATP addition.

Cited by 78 publications

References 66 publications

Ibuprofen inhibits cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion.

Ibuprofen inhibits cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion.

Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing

Regulation of CFTR Cl- channel gating by ADP and ATP analogues.

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