Activating Cystic Fibrosis Transmembrane Conductance Regulator Channels with Pore Blocker Analogs

Cystic fibrosis (CF) is caused by mutations that disrupt the surface localization and/or gating of the CF transmembrane conductance regulator (CFTR) chloride channel. The most common CF mutant is ΔF508-CFTR, which inefficiently traffics to the surfaces of most cells. The ΔF508 mutation may also disrupt the opening of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear. Here, we describe potent activators of wild-type and ΔF508-CFTR channels that are structurally related to 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged pore blocker that we show to have mixed agonistic activity (channel activation plus voltage-dependent pore block). These CFTR agonists include 1) an uncharged NPPB analog that stimulates channel opening at submicromolar concentrations without blocking the pore and 2) curcumin, a dietary compound recently reported to augment ΔF508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and ΔF508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of ΔF508-CFTR channels in excised membrane patches by 10–15-fold under conditions in which wild-type channels were already maximally active. Our results support the emerging view that CFTR channel activity is substantially reduced by the ΔF508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface. Cystic fibrosis (CF) is caused by mutations that disrupt the surface localization and/or gating of the CF transmembrane conductance regulator (CFTR) chloride channel. The most common CF mutant is ΔF508-CFTR, which inefficiently traffics to the surfaces of most cells. The ΔF508 mutation may also disrupt the opening of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear. Here, we describe potent activators of wild-type and ΔF508-CFTR channels that are structurally related to 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged pore blocker that we show to have mixed agonistic activity (channel activation plus voltage-dependent pore block). These CFTR agonists include 1) an uncharged NPPB analog that stimulates channel opening at submicromolar concentrations without blocking the pore and 2) curcumin, a dietary compound recently reported to augment ΔF508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and ΔF508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of ΔF508-CFTR channels in excised membrane patches by 10–15-fold under conditions in which wild-type channels were already maximally active. Our results support the emerging view that CFTR channel activity is substantially reduced by the ΔF508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface. IntroductionCystic fibrosis (CF) 1The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; NBD, nucleotide-binding domain; PKA, protein kinase A; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoate; BHK, baby hamster kidney; HEK, human embryonic kidney; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; PKI, protein kinase A inhibitory peptide; NPPB-AM, 5-nitro-2-(3-phenylpropylamino)benzamide; NPPB-sulf, 5-nitro-2-(3-phenylpropylamino)benzenesulfonamide. is caused by inadequate CF transmembrane conductance regulator (CFTR) channel activity in the lung and intestines (1Welsh M.J. Smith A.E. Cell. 1993; 73: 1251-1254Abstract Full Text PDF PubMed Scopus (1216) Google Scholar). CFTR channels are normally activated by MgATP binding to the nucleotide-binding domains (NBDs) and phosphorylation of the regulatory domain by protein kinase A (PKA) (2Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (369) Google Scholar, 3Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (790) Google Scholar). Many different mutations in these domains can cause CF, but the most common CF mutation is deletion of Phe-508 from NBD1 (ΔF508) (4Zielenski J. Tsui L.C. Annu. Rev. Genet. 1995; 29: 777-807Crossref PubMed Scopus (512) Google Scholar). The consensus view is that this mutation disrupts the biosynthetic maturation and surface localization of CFTR (5Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1407) Google Scholar, 6Riordan J.R. Am. J. Hum. Genet. 1999; 64: 1499-1504Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). However, it has been argued that ΔF508-CFTR channels may reach the surfaces of a subset of cells in vivo (7Kälin N. Claaβ A. Sommer M. Puchelle E. Tümmler B. J. Clin. Investig. 1999; 103: 1379-1389Crossref PubMed Scopus (228) Google Scholar). There is accumulating evidence that the ΔF508 mutation also affects the gating (opening and closing) of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear (8Li C. Ramjeesingh M. Reyes E. Chang X. Rommens J.M. Bear C.E. Nat. Genet. 1993; 3: 311-316Crossref PubMed Scopus (154) Google Scholar, 9Schultz B.D. Frizzell R.A. Bridges R.J. J. Membr. Biol. 1999; 170: 51-66Crossref PubMed Scopus (52) Google Scholar, 10Dalemans W. Barbry P. Champigny G. Jallet S. Dott K. Dreyer D. Crystal R.G. Pavironi A. Lecocq J.P. Lazdunski M. Nature. 1991; 354: 526-528Crossref PubMed Scopus (560) Google Scholar, 11Wang F.S. Zeltwanger S. Hu S. Hwang T-C. J. Physiol. (Lond.). 2000; 524: 637-648Crossref Scopus (87) Google Scholar). Determining the extent to which the ΔF508 mutation disrupts channel activity is central to the development of appropriate treatment strategies.CFTR gating is also modulated by reactive glutathione species, which inhibit channel opening in the presence of MgATP and PKA by glutathionylating a cysteine in NBD2 (12Wang W. Oliva C. Li G. Holmgen A. Lillig C.H. Kirk K.L. J. Gen. Physiol. 2005; 125: 127-141Crossref PubMed Scopus (75) Google Scholar). Here, we report that, while studying the effect of glutathionylation on CFTR gating, we observed that these modified channels could be activated by 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged compound previously shown to block the pore in a voltage-dependent manner (13Zhang Z-R. Zeltwanger S. McCarty N.A. J. Membr. Biol. 2000; 175: 35-52Crossref PubMed Scopus (84) Google Scholar). Based on this observation, we identified structurally related compounds that behave as pure CFTR agonists that stimulate channel opening without blocking the pore. By using these compounds as functional probes of mutant CFTR gating, we show that (i) the ΔF508 mutation profoundly decreases the opening rates of surface-localized channels and (ii) this defect can be corrected by such compounds. Our results support the view that effective CF therapies may require the use of CFTR channel openers (9Schultz B.D. Frizzell R.A. Bridges R.J. J. Membr. Biol. 1999; 170: 51-66Crossref PubMed Scopus (52) Google Scholar, 14Yang H. Shelat A.A. Guy R.K. Gopinath V.S. Ma T. Du K. Lukacs G.L. Taddei A. Folli C. Pedemonte N. Galietta L.J.V. Verkman A.S. J. Biol. Chem. 2003; 278: 35079-35085Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar).EXPERIMENTAL PROCEDURESCell Culture and Transfections—Baby hamster kidney (BHK) cells stably expressing human wild-type CFTR (BHK-CFTR cells) were provided by Dr. J. W. Hanrahan (McGill University). Human embryonic kidney (HEK) 293T cells were transiently transfected with wild-type or mutant CFTR cDNA using the Lipofectamine transfection kit (Invitrogen) following the manufacturer's recommendations. Cells were cultured in Dulbecco's modified Eagle's medium (Mediatech) supplemented with 5 or 10% fetal bovine serum and 1 mm penicillin/streptomycin. CFTR expression in the transfected HEK-293T cells was verified by immunoblotting. The growth medium the cells also mm to cells T. Chang J.R. Hanrahan J. Physiol. (Lond.). 2003; Scopus Google Scholar). cells that were stably transfected with ΔF508-CFTR were cultured as K. D. B. E. J.P. Am. J. Biol. PubMed Scopus Google Scholar). cells were on and used Cells expressing ΔF508-CFTR or is the regulatory were at these are that surface expression cells are cultured at and channel were in the excised were from to of or channel CFTR channels were activated following by of the of the to the of PKA and MgATP CFTR were in mm 1 mm and mm were using a from to with a were at channel were at from and channel were at and and were using rates were from by the of plus by the by the channel opening rates and channel open probabilities were that the of the of This was to patches which were open the the that this the to patches that are to activation by compounds. was using epithelial cells stably transfected with ΔF508-CFTR or transiently transfected with wild-type CFTR were cultured as and in as K. D. B. E. J.P. Am. J. Biol. PubMed Scopus Google Scholar). were at to the surface expression of this A to was by to block were to both at the A was to as a or CFTR the inhibitory effect of an of and on the by CFTR channels in an membrane This effect is to glutathionylation of a cysteine in which a in the channel opening (12Wang W. Oliva C. Li G. Holmgen A. Lillig C.H. Kirk K.L. J. Gen. Physiol. 2005; 125: 127-141Crossref PubMed Scopus (75) Google Scholar). NPPB was to block the CFTR we observed a in at at which pore block by the negatively charged compound is The that are by NPPB at are on (i) in membrane patches excised from cells and (ii) by of NPPB and or by CFTR blocker B.D. Frizzell R.A. Bridges R.J. Am. J. Physiol. Google Scholar). could the effect of NPPB This effect was in that CFTR were by voltage-dependent of the CFTR pore mm P. Hanrahan J. Gen. Physiol. PubMed Scopus Google and mm (13Zhang Z-R. Zeltwanger S. McCarty N.A. J. Membr. Biol. 2000; 175: 35-52Crossref PubMed Scopus (84) Google or by a of NPPB The by NPPB was to a effect of this compound on CFTR channels the in was from the the (12Wang W. Oliva C. Li G. Holmgen A. Lillig C.H. Kirk K.L. J. Gen. Physiol. 2005; 125: 127-141Crossref PubMed Scopus (75) Google the of CFTR channel opening without the phosphorylation and NPPB stimulate PKA an excised HEK-293T the at the The results were NPPB affects a excised from an HEK-293T of and on CFTR channel activity in a The was were activated by PKA, by that stimulates the opening rates of CFTR The conditions were as C. The are rates the of The of the channel opening rates and channel the and conditions that is the of were as and and and of NPPB stimulate this mutant at PKA concentrations in excised HEK-293T the at the in NPPB stimulates wild-type CFTR at in excised HEK-293T channels were activated with PKA The conditions were as E. are at from and the wild-type and were to the by NPPB at channel activation by an uncharged NPPB of compounds. stimulates in both wild-type channels in excised are of HEK-293T in a to the function The conditions were as and C. were to the at The was which increased the by The results are has a effect on CFTR also stimulates CFTR and stably The results are of on the effect of this pore blocker on we NPPB could stimulate the activities of CFTR channels that are at under conditions of by that NPPB also CFTR at channels were by the with a of PKA by PKA inhibitory to inhibit in both were increased by concentrations of at a voltage-dependent block was channels that were by to a PKA CFTR activation a voltage-dependent block by NPPB the of channel activity by NPPB is related to the of CFTR that NPPB as a mixed or CFTR this compound stimulates channels in to blocking the pore in a voltage-dependent NPPB CFTR by binding to the that a pore we on a CFTR pore mutant that is to block by NPPB X. J. 1999; PubMed Scopus Google Scholar). 1 and that NPPB the by at to a extent with wild-type CFTR at of phosphorylation PKA by the wild-type the were by NPPB at NPPB as a pure the pore which that this compound stimulates channel opening by binding to a that is from the the without the inhibitory effect of NPPB on CFTR in on the of this as by the of the this and to compounds that are pure CFTR we and related compounds on CFTR channel activity NPPB been in a A. A. G. PubMed Scopus Google a and a of these compounds is an effective of A. A. G. PubMed Scopus Google Scholar). However, we that is a potent of CFTR channels we observed that the by CFTR channels or channels in excised membrane at and was The effect of was and by the of and The by with the of CFTR phosphorylation at of as was observed the NPPB and However, the by was with evidence CFTR at CFTR activation by compound the presence of both MgATP and at a of PKA the effect on membrane patches excised from cells we observed in CFTR at concentrations as as with 1 and as a pure CFTR the to and NPPB stimulate ΔF508-CFTR of a ΔF508-CFTR an excised HEK-293T PKA, that stimulates ΔF508-CFTR wild-type in excised patches to a PKA the of to results were from wild-type or HEK-293T cells with the of the cells stably transfected with cells were as under ΔF508-CFTR channel activation by in an excised HEK-293T channels to conditions were as PKA mm and of on the opening rates and ΔF508-CFTR in excised membrane patches HEK-293T rates the of The of the channel opening rates and channel the and conditions that is the of were as and and and a excised HEK-293T the activation of ΔF508-CFTR channels by and the in channel under conditions this The conditions were as of by a is to that of a dietary compound that has in the CF in M. D. J. S. Du K. Lukacs G.L. M.J. PubMed Scopus Google reported that the biosynthetic maturation and functional of the ΔF508-CFTR mutant in cells and in that the this effect is and in activity and function in the the maturation of the ΔF508-CFTR protein in the is D. Pedemonte N. Galietta L.J.V. Verkman A.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, of to NPPB-AM, we were by the that could have a effect on CFTR gating, which of the functional that was reported by M. D. J. S. Du K. Lukacs G.L. M.J. PubMed Scopus Google Scholar). that also the by wild-type CFTR in excised membrane This effect was it was that observed show that also the activities of ΔF508-CFTR the that the most potent CFTR activity by the channel opening of effect on CFTR that CFTR channel activity in excised membrane patches which channel is without CFTR this compound and the compound CFTR following PKA or the of 1 and the by a regulatory domain deletion mutant that activity in the of PKA Gregory R.J. P. Smith A.E. Welsh M.J. 1991; PubMed Scopus (193) Google Scholar). effect on the MgATP of CFTR activity as in MgATP in the presence and of this compound However, the opening rates and channel CFTR channels in patches channels and that this compound stimulates the opening of CFTR channels at a of the of ΔF508-CFTR under in the of NPPB and on the most common CF and is a gating mutant R.J. S.H. Souza D.W. Paul S. P. Welsh M.J. Smith A.E. Cell. Biol. 1991; PubMed Scopus Google that, ΔF508-CFTR, is to the cell surface with to that of wild-type The mutation to a in NBD1 that a in MgATP binding or the binding and the opening of the pore the transmembrane domains Annu. Rev. Biol. PubMed Scopus Google Scholar). the activity of this mutant was by a that maximally the wild-type channel However, activity was by of the charged compound (NPPB), that were to the uncharged NPPB we observed an concentrations of NPPB activation with wild-type channel activation This that the mutation in NBD1 the of NPPB of activation that ΔF508-CFTR channels were by NPPB, NPPB-AM, and under conditions that maximally wild-type HEK-293T cells transiently transfected with ΔF508-CFTR or CF epithelial cells stably transfected with ΔF508-CFTR cells) K. D. B. E. J.P. Am. J. Biol. PubMed Scopus Google were cultured at to the surface expression of this mutant Marshall J. Smith A.E. Welsh M.J. Nature. PubMed Scopus Google Scholar). was the an with a PKA to the that ΔF508-CFTR channels phosphorylation to the F.S. Zeltwanger S. Hu S. Hwang T-C. J. Physiol. (Lond.). 2000; 524: 637-648Crossref Scopus (87) Google Scholar). a 10–15-fold in ΔF508-CFTR in excised patches in the presence of normally concentrations of MgATP and PKA A and observed wild-type channel ΔF508-CFTR channel activity in the of kinase we observed that the of ΔF508-CFTR channel activity by was to a in the channel opening and that this is patches which the of channel the of the 10–15-fold of ΔF508-CFTR by under conditions in which the wild-type channel was maximally the of compound the activity of the wild-type channel by in the of This that the ΔF508-CFTR channels were at an of under these conditions channel is in this a that is with the view that ΔF508-CFTR channels activity they reach the membrane (8Li C. Ramjeesingh M. Reyes E. Chang X. Rommens J.M. Bear C.E. Nat. Genet. 1993; 3: 311-316Crossref PubMed Scopus (154) Google Scholar). The the extent to which the ΔF508 mutation affects CFTR channel activity may be in to a the in the channel and opening rates channels the of channels in the is unclear. this a which we to this as the the of we a channel and opening of and However, following the of NPPB-AM, as as were observed a of it was that the of channels in this been and that, the channel and opening rates been the that ΔF508-CFTR channel opening is under these conditions and that this gating defect is normally to of in the of channels in a membrane CFTR in 5 that also wild-type and ΔF508-CFTR channels in intact epithelial cell monolayers. of epithelial cells from a and cells transfected with ΔF508-CFTR or wild-type CFTR were in K. D. B. E. J.P. Am. J. Biol. PubMed Scopus Google Scholar). on the chloride wild-type or monolayers. this compound the by wild-type or ΔF508-CFTR channels without the of a and This effect of CFTR phosphorylation by it could be by treatment with a kinase the to on the which of ΔF508-CFTR protein of at compound and 1 the effect of a of of on the by ΔF508-CFTR a dietary compound previously reported to activate CFTR channels Zeltwanger S. Nairn A.C. Hwang T-C. J. Gen. Physiol. PubMed Scopus Google to the at this These results that is a potent of wild-type and ΔF508-CFTR channels in intact epithelial cell monolayers. The effect of this compound on the a effect of this compound on of channels in these stimulates ΔF508-CFTR intact epithelial effect of on the a of epithelial cells stably transfected with ΔF508-CFTR and at The channel blocker was at the also the transfected with wild-type CFTR, and this effect was by with a kinase at ΔF508-CFTR activation by in monolayers. were to the have identified potent CFTR agonists on the that a pore blocker has mixed agonistic The uncharged NPPB as a pure CFTR that affects CFTR gating by the channel opening has that it a compound (i) (ii) cell and to activate ΔF508-CFTR we have that stimulates CFTR by the the effect of this dietary compound on ΔF508-CFTR channel activity in previously reported effect in ΔF508-CFTR mice M. D. J. S. Du K. Lukacs G.L. M.J. PubMed Scopus Google Scholar). This to be in with the results of D.W. Welsh M.J. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus (87) Google recently reported that can activate CFTR channels in membrane patches excised from transfected and NPPB are of CFTR opening without channel phosphorylation by PKA, these compounds wild-type or activity in the of However, the of activation is related to the of phosphorylation of This the effect of NPPB was NPPB a voltage-dependent block of CFTR channels under activation conditions PKA The activation by these compounds and the of phosphorylation may be to the that wild-type channels are maximally already channel (2Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (369) Google Scholar, 3Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (790) Google Scholar, E. J. M. Kirk K.L. 1999; PubMed Scopus Google Scholar). this the used Gregory R.J. P. Smith A.E. Welsh M.J. 1991; PubMed Scopus (193) Google has channel activity wild-type channels at E. J. M. Kirk K.L. 1999; PubMed Scopus Google this activity is on this the of channel activity by NPPB and observed in this that the ΔF508 mutation has a effect on CFTR channel activity The on this is with of gating defect ΔF508-CFTR (8Li C. Ramjeesingh M. Reyes E. Chang X. Rommens J.M. Bear C.E. Nat. Genet. 1993; 3: 311-316Crossref PubMed Scopus (154) Google a gating defect at a PKA (9Schultz B.D. Frizzell R.A. Bridges R.J. J. Membr. Biol. 1999; 170: 51-66Crossref PubMed Scopus (52) Google Scholar, 11Wang F.S. Zeltwanger S. Hu S. Hwang T-C. J. Physiol. (Lond.). 2000; 524: 637-648Crossref Scopus (87) Google and a gating defect W. Barbry P. Champigny G. Jallet S. Dott K. Dreyer D. Crystal R.G. Pavironi A. Lecocq J.P. Lazdunski M. Nature. 1991; 354: 526-528Crossref PubMed Scopus (560) Google Scholar). F.S. Zeltwanger S. Hu S. Hwang T-C. J. Physiol. (Lond.). 2000; 524: 637-648Crossref Scopus (87) Google reported that ΔF508-CFTR channels in membrane patches excised from cells activate wild-type channels to PKA, but that the gating of these channels is activation is we used a PKA and to activation to compounds. Our results that, under these ΔF508-CFTR channels have at activity in to reduced opening with wild-type channels at normally MgATP and PKA This defect ΔF508-CFTR channel opening was by NPPB-AM, it that the of ΔF508-CFTR channels under conditions been substantially results CFTR channels that were in HEK-293T cells or in epithelial this wild-type and ΔF508-CFTR channel activities to be to cell The the reduced channel activity of the ΔF508 mutant could be related to phosphorylation as argued by F.S. Zeltwanger S. Hu S. Hwang T-C. J. Physiol. (Lond.). 2000; 524: 637-648Crossref Scopus (87) Google Scholar). were under conditions in which the channels were to normally concentrations of PKA it is that ΔF508-CFTR channels are with wild-type channels under these the reduced ΔF508-CFTR channel activity could be to a defect in gating that is to a defect that has been to of the in the of mutant channels in membrane defect in ΔF508-CFTR channel activity has (i) This could the of the ΔF508 these mutant channels can reach the surfaces of a subset of epithelial cell as argued by (7Kälin N. Claaβ A. Sommer M. Puchelle E. Tümmler B. J. Clin. Investig. 1999; 103: 1379-1389Crossref PubMed Scopus (228) Google and (ii) therapies that the biosynthetic defect of this mutant may be Our results support the emerging view H. Shelat A.A. Guy R.K. Gopinath V.S. Ma T. Du K. Lukacs G.L. Taddei A. Folli C. Pedemonte N. Galietta L.J.V. Verkman A.S. J. Biol. Chem. 2003; 278: 35079-35085Abstract Full Text Full Text PDF PubMed Scopus (193) Google that the treatment of most CF may require therapies that include CFTR channel IntroductionCystic fibrosis (CF) 1The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; NBD, nucleotide-binding domain; PKA, protein kinase A; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoate; BHK, baby hamster kidney; HEK, human embryonic kidney; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; PKI, protein kinase A inhibitory peptide; NPPB-AM, 5-nitro-2-(3-phenylpropylamino)benzamide; NPPB-sulf, 5-nitro-2-(3-phenylpropylamino)benzenesulfonamide. is caused by inadequate CF transmembrane conductance regulator (CFTR) channel activity in the lung and intestines (1Welsh M.J. Smith A.E. Cell. 1993; 73: 1251-1254Abstract Full Text PDF PubMed Scopus (1216) Google Scholar). CFTR channels are normally activated by MgATP binding to the nucleotide-binding domains (NBDs) and phosphorylation of the regulatory domain by protein kinase A (PKA) (2Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (369) Google Scholar, 3Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (790) Google Scholar). Many different mutations in these domains can cause CF, but the most common CF mutation is deletion of Phe-508 from NBD1 (ΔF508) (4Zielenski J. Tsui L.C. Annu. Rev. Genet. 1995; 29: 777-807Crossref PubMed Scopus (512) Google Scholar). The consensus view is that this mutation disrupts the biosynthetic maturation and surface localization of CFTR (5Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1407) Google Scholar, 6Riordan J.R. Am. J. Hum. Genet. 1999; 64: 1499-1504Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). However, it has been argued that ΔF508-CFTR channels may reach the surfaces of a subset of cells in vivo (7Kälin N. Claaβ A. Sommer M. Puchelle E. Tümmler B. J. Clin. Investig. 1999; 103: 1379-1389Crossref PubMed Scopus (228) Google Scholar). There is accumulating evidence that the ΔF508 mutation also affects the gating (opening and closing) of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear (8Li C. Ramjeesingh M. Reyes E. Chang X. Rommens J.M. Bear C.E. Nat. Genet. 1993; 3: 311-316Crossref PubMed Scopus (154) Google Scholar, 9Schultz B.D. Frizzell R.A. Bridges R.J. J. Membr. Biol. 1999; 170: 51-66Crossref PubMed Scopus (52) Google Scholar, 10Dalemans W. Barbry P. Champigny G. Jallet S. Dott K. Dreyer D. Crystal R.G. Pavironi A. Lecocq J.P. Lazdunski M. Nature. 1991; 354: 526-528Crossref PubMed Scopus (560) Google Scholar, 11Wang F.S. Zeltwanger S. Hu S. Hwang T-C. J. Physiol. (Lond.). 2000; 524: 637-648Crossref Scopus (87) Google Scholar). Determining the extent to which the ΔF508 mutation disrupts channel activity is central to the development of appropriate treatment strategies.CFTR gating is also modulated by reactive glutathione species, which inhibit channel opening in the presence of MgATP and PKA by glutathionylating a cysteine in NBD2 (12Wang W. Oliva C. Li G. Holmgen A. Lillig C.H. Kirk K.L. J. Gen. Physiol. 2005; 125: 127-141Crossref PubMed Scopus (75) Google Scholar). Here, we report that, while studying the effect of glutathionylation on CFTR gating, we observed that these modified channels could be activated by 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged compound previously shown to block the pore in a voltage-dependent manner (13Zhang Z-R. Zeltwanger S. McCarty N.A. J. Membr. Biol. 2000; 175: 35-52Crossref PubMed Scopus (84) Google Scholar). Based on this observation, we identified structurally related compounds that behave as pure CFTR agonists that stimulate channel opening without blocking the pore. By using these compounds as functional probes of mutant CFTR gating, we show that (i) the ΔF508 mutation profoundly decreases the opening rates of surface-localized channels and (ii) this defect can be corrected by such compounds. Our results support the view that effective CF therapies may require the use of CFTR channel openers (9Schultz B.D. Frizzell R.A. Bridges R.J. J. Membr. Biol. 1999; 170: 51-66Crossref PubMed Scopus (52) Google Scholar, 14Yang H. Shelat A.A. Guy R.K. Gopinath V.S. Ma T. Du K. Lukacs G.L. Taddei A. Folli C. Pedemonte N. Galietta L.J.V. Verkman A.S. J. Biol. Chem. 2003; 278: 35079-35085Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar).

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