ABCMdb

ABCC7 p.Ser660Ala

CF databases:	c.1978T>A , p.Ser660Thr (CFTR1) ? , The mutation was detected by DGGE analysis and characterized by direct sequencing. We have seen it only once, in over 1000 control chromosomes from Italian population.
Predicted by SNAP2:	A: N (82%), C: N (66%), D: D (53%), E: D (53%), F: D (59%), G: N (82%), H: N (61%), I: D (59%), K: D (53%), L: D (59%), M: N (57%), N: N (87%), P: N (72%), Q: N (61%), R: D (53%), T: N (87%), V: N (53%), W: D (63%), Y: D (59%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: D, Y: N,

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[hide] A functional R domain from cystic fibrosis transme... Proc Natl Acad Sci U S A. 2000 May 9;97(10):5657-62. Ostedgaard LS, Baldursson O, Vermeer DW, Welsh MJ, Robertson AD
A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution.
Proc Natl Acad Sci U S A. 2000 May 9;97(10):5657-62., 2000-05-09 [PMID:10792060]

Abstract [show]
Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel activity. To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708-831 of CFTR. Phosphorylated, but not unphosphorylated, R8 stimulated activity of CFTR channels lacking this domain, indicating that R8 is functional. Unexpectedly, this functional R8 was predominantly random coil, as revealed by CD and limited proteolysis. The CD spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer. The folding agent trimethylamine N-oxide induced only a small increase in the helical content of nonphosphorylated R8 and even less change in the helical content of phosphorylated R8. These data, indicating that the R domain is predominantly random coil, may explain the seemingly complex way in which phosphorylation regulates CFTR channel activity.

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26 An example is CFTR-⌬R͞ S660A, in which residues 708-835 are deleted and Ser-660 is mutated to alanine (12, 19, 23). Login to comment
46 10 ͉ 5657-5662 S660A in lipid bilayers (13, 14). Login to comment

[hide] Severed channels probe regulation of gating of cys... J Gen Physiol. 2000 Sep;116(3):477-500. Csanady L, Chan KW, Seto-Young D, Kopsco DC, Nairn AC, Gadsby DC
Severed channels probe regulation of gating of cystic fibrosis transmembrane conductance regulator by its cytoplasmic domains.
J Gen Physiol. 2000 Sep;116(3):477-500., [PMID:10962022]

Abstract [show]
Opening and closing of a CFTR Cl(-) channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634-836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1-633 and 837-1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them.

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332 Based on these findings, and the effect of phosphorylated R-domain peptide on ⌬R(708-835)-S660A channels, the phosphorylated R domain was proposed to stimulate channel activity by enhancing the affinity of CFTR for ATP (Winter and Welsh, 1997). Login to comment
357 However, in the absence of the phosphopeptide, the opening rate of ⌬R(708-835)-S660A, with or without PKA, KrCO KPo ( ) was only ‫%03ف‬ that of phosphorylated WT, and increased only to ‫%54ف‬ of the latter, even in the presence of the phosphopeptide (Winter and Welsh, 1997; compare Ma et al., 1997). Login to comment
363 This activation is intriguing, because the effect of PKA on CFTR has generally been attributed to phosphorylation of serine residues within the R domain, based on both biochemical and functional evidence: CNBr cleavage and peptide mapping experiments on CFTR protein prephosphorylated by PKA with ␥-32P ATP found no evidence for phosphorylation outside the R domain (Cheng et al., 1991; Picciotto et al., 1992; Seibert et al., 1995), and PKA no longer stimulated ⌬R(708-835) channels after mutation of serine 660 [⌬R(708-835)-S660A, Rich et al., 1993; compare Ma et al., 1997]. Login to comment
365 Also, lack of response of ⌬R(708-835)-S660A channels to PKA could reflect steric constraints introduced by linking residue 707 to 836. Login to comment
372 Interestingly, a likely ␣-helical section of CFTR`s NH2 terminus (within residues 46-60) was recently reported to bind to the R domain, and enhance channel activity (Naren et al., 1999): point mutations in that stretch of amino acids impaired the gating of WT (an effect apparently on channel closing rate), but not of ⌬R(708-835)-S660A, channels. Login to comment

[hide] Contribution of R domain phosphoserines to the fun... Am J Physiol Lung Cell Mol Physiol. 2000 Nov;279(5):L835-41. Baldursson O, Berger HA, Welsh MJ
Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia.
Am J Physiol Lung Cell Mol Physiol. 2000 Nov;279(5):L835-41., [PMID:11053017]

Abstract [show]
The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl(-) current. Mutation of four in vivo phosphorylation sites, Ser(660), Ser(737), Ser(795), and Ser(813) (S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser(660) or Ser(813) alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser(660) nor Ser(813) alone increased current to wild-type levels; both residues were required. Changing Ser(737) to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser(737) may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines.

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56 In each of the serine to alanine mutants, S660A, S737A, S795A, and S813A, alanine replaced serine at the designated residue. Login to comment
107 The S660A and S813A variants generated small but significant cAMP-stimulated Cl- currents. Login to comment
112 The S660A and S813A variants generated currents, but they were no greater than those obtained with SQuad-A. Login to comment
186 Mutation of either residue alone significantly decreased current; with maximal stimulation by cAMP agonists, neither the S660A nor the S813A mutant gave more current than the S-Quad-A Fig. 5. Login to comment
195 With submaximal cAMP agonists, S660A- and S813A-generated current was also reduced, but it was slightly greater than that obtained with S-Quad-A. Login to comment

[hide] A macromolecular complex of beta 2 adrenergic rece... Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):342-6. Epub 2002 Dec 26. Naren AP, Cobb B, Li C, Roy K, Nelson D, Heda GD, Liao J, Kirk KL, Sorscher EJ, Hanrahan J, Clancy JP
A macromolecular complex of beta 2 adrenergic receptor, CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA.
Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):342-6. Epub 2002 Dec 26., 2003-01-07 [PMID:12502786]

Abstract [show]
It has been demonstrated previously that both the cystic fibrosis transmembrane conductance regulator (CFTR) and beta(2) adrenergic receptor (beta(2)AR) can bind ezrinradixinmoesin-binding phosphoprotein 50 (EBP50, also referred to as NHERF) through their PDZ motifs. Here, we show that beta(2) is the major adrenergic receptor isoform expressed in airway epithelia and that it colocalizes with CFTR at the apical membrane. beta(2)AR stimulation increases CFTR activity, in airway epithelial cells, that is glybenclamide sensitive. Deletion of the PDZ motif from CFTR uncouples the channel from the receptor both physically and functionally. This uncoupling is specific to the beta(2)AR receptor and does not affect CFTR coupling to other receptors (e.g., adenosine receptor pathway). Biochemical studies demonstrate the existence of a macromolecular complex involving CFTR-EBP50-beta(2)AR through PDZ-based interactions. Assembly of the complex is regulated by PKA-dependent phosphorylation. Deleting the regulatory domain of CFTR abolishes PKA regulation of complex assembly. This report summarizes a macromolecular signaling complex involving CFTR, the implications of which may be relevant to CFTR-dysfunction diseases.

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No. Sentence Comment
198 Deleting most of the R domain of CFTR (S660A ⌬RCFTR; lacking amino acids 708-835; ref. 32) abolished the PKA sensitivity of CFTR binding in the complex (Fig. 4 E and F). Login to comment

[hide] Capsaicin potentiates wild-type and mutant cystic ... Mol Pharmacol. 2004 Jun;65(6):1415-26. Ai T, Bompadre SG, Wang X, Hu S, Li M, Hwang TC
Capsaicin potentiates wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride-channel currents.
Mol Pharmacol. 2004 Jun;65(6):1415-26., [PMID:15155835]

Abstract [show]
To examine the effects of capsaicin on cystic fibrosis transmembrane conductance regulator (CFTR), we recorded wild-type and mutant CFTR chloride-channel currents using patch-clamp methods. The effects of capsaicin were compared with those of genistein, a well-characterized CFTR activator. In whole-cell experiments, capsaicin potentiates cAMP-stimulated wild-type CFTR currents expressed in NIH 3T3 cells or Chinese hamster ovary cells in a dose-dependent manner with a maximal response approximately 60% of that with genistein and an apparent Kd of 48.4 +/- 6.8 microM. In cell-attached recordings, capsaicin alone fails to activate CFTR in cells that show negligible basal CFTR activity, indicating that capsaicin does not stimulate the cAMP cascade. The magnitude of potentiation with capsaicin depends on the channel activity before drug application; the lower the prestimulated Po, the higher the potentiation. Single-channel kinetic analysis shows that capsaicin potentiates CFTR by increasing the opening rate and decreasing the closing rate of the channel. Capsaicin may act as a partial agonist of genistein because the maximally enhanced wild-type CFTR currents with genistein are partially inhibited by capsaicin. Capsaicin increases DeltaR-CFTR, a protein kinase A (PKA)-independent, constitutively active channel, in cell-attached patches. In excised inside-out patches, capsaicin potentiates the PKA-phosphorylated, ATP-dependent CFTR activity. Both capsaicin and genistein potentiate the cAMP-stimulated G551D-CFTR, DeltaF508-CFTR, and 8SA mutant channel currents. The binding site for capsaicin is probably located at the cytoplasmic domain of CFTR, because pipette application of capsaicin fails to potentiate CFTR activity. In conclusion, capsaicin is a partial agonist of genistein in activation of the CFTR chloride channel. Both compounds affect ATP-dependent gating of CFTR.

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No. Sentence Comment
142 We further explored the action of capsaicin using CFTR mutants whose eight major PKA consensus serines are substituted with alanine (S660A, S686A, S700A, S712A, S737A, S768A, S795A, and S813A), so called S-oct-A or 8SA. Login to comment

[hide] Molecular dissection of the butyrate action reveal... Mol Pharmacol. 2004 Nov;66(5):1248-59. Epub 2004 Aug 10. Sugita M, Kongo H, Shiba Y
Molecular dissection of the butyrate action revealed the involvement of mitogen-activated protein kinase in cystic fibrosis transmembrane conductance regulator biogenesis.
Mol Pharmacol. 2004 Nov;66(5):1248-59. Epub 2004 Aug 10., [PMID:15304546]

Abstract [show]
Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which belongs to the superfamily of ATP-binding cassette transporters and uniquely possesses an additional large cytoplasmic domain [regulatory (R) domain]. CFTR inefficiently folds by means of co- and post-translational interactions with the cytosolic chaperones as well as luminal chaperones in the endoplasmic reticulum (ER). Aberrant folding and defective trafficking of the CFTR protein, which functions as an apical membrane Cl(-) channel, is the principal cause of cystic fibrosis. Recent data indicated that butyrate improves CFTR trafficking partly by regulating molecular chaperones; however, the precise mechanism of butyrate action remains elusive. In the present study, we examine the molecular aspect underlying the butyrate action in CFTR biogenesis by evaluating the expression and localization of the green fluorescent protein (GFP)-tagged CFTR transgenes in Cos7 cells. Our data show that butyrate significantly promoted stability of the ER-located form of GFP-wild-type (wt)-CFTR, followed by an increase in the amount of plasma membrane GFP-wt-CFTR. In contrast, the expression of the R domain deletion mutant GFP-DeltaR-CFTR was slightly increased by butyrate. The butyrate action on wt-CFTR expression was partially blocked by PD98059 (2'-amino-3'-methoxyflavone), a specific inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK), which is the upstream activator of extracellular-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK). Furthermore, activation of ERK/MAPK by the coexpression of constitutively active MAPKK/MEK predominantly augmented the expression of wt-CFTR, but not of DeltaR-CFTR, induced by butyrate. These data suggest that butyrate may facilitate the biogenesis and trafficking of wt-CFTR by requiring the presence of the R domain and further involving active ERK/MAPK in its biogenesis.

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39 To construct the pEGFP-⌬R-CFTR mammalian expression vector, an AflII and SacI fragment from pTM1-CFTR⌬R-S660A containing the C-terminal region of CFTR was swapped with the wild-type cassette present in pEGFP-C1-CFTR. Login to comment
104 In contrast, the B form of the deletion mutant GFP-⌬R-CFTR, which lacks much of the R domain and replaces Ser660 with alanine (Rich et al., 1993), was detected as an approximately 15-kDa increase in electrophoresis mobility (ϳ158 kDa) (Fig. 1B). Login to comment
303 Acknowledgments We thank Dr. K. Foskett for providing pEGFP-CFTR, Dr. M. Welsh for CFTR⌬R-S660A, and Dr. E. Nishida for LA-SDSE-MAPKK and SASA-MAPKK. Login to comment

[hide] Functional roles of nonconserved structural segmen... J Gen Physiol. 2005 Jan;125(1):43-55. Epub 2004 Dec 13. Csanady L, Chan KW, Nairn AC, Gadsby DC
Functional roles of nonconserved structural segments in CFTR's NH2-terminal nucleotide binding domain.
J Gen Physiol. 2005 Jan;125(1):43-55. Epub 2004 Dec 13., [PMID:15596536]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the gene mutated in cystic fibrosis patients, belongs to the family of ATP-binding cassette (ABC) proteins, but, unlike other members, functions as a chloride channel. CFTR is activated by protein kinase A (PKA)-mediated phosphorylation of multiple sites in its regulatory domain, and gated by binding and hydrolysis of ATP at its two nucleotide binding domains (NBD1, NBD2). The recent crystal structure of NBD1 from mouse CFTR (Lewis, H.A., S.G. Buchanan, S.K. Burley, K. Conners, M. Dickey, M. Dorwart, R. Fowler, X. Gao, W.B. Guggino, W.A. Hendrickson, et al. 2004. EMBO J. 23:282-293) identified two regions absent from structures of all other NBDs determined so far, a "regulatory insertion" (residues 404-435) and a "regulatory extension" (residues 639-670), both positioned to impede formation of the putative NBD1-NBD2 dimer anticipated to occur during channel gating; as both segments appeared highly mobile and both contained consensus PKA sites (serine 422, and serines 660 and 670, respectively), it was suggested that their phosphorylation-linked conformational changes might underlie CFTR channel regulation. To test that suggestion, we coexpressed in Xenopus oocytes CFTR residues 1-414 with residues 433-1480, or residues 1-633 with 668-1480, to yield split CFTR channels (called 414+433 and 633+668) that lack most of the insertion, or extension, respectively. In excised patches, regulation of the resulting CFTR channels by PKA and by ATP was largely normal. Both 414+433 channels and 633+668 channels, as well as 633(S422A)+668 channels (lacking both the extension and the sole PKA consensus site in the insertion), were all shut during exposure to MgATP before addition of PKA, but activated like wild type (WT) upon phosphorylation; this indicates that inhibitory regulation of nonphosphorylated WT channels depends upon neither segment. Detailed kinetic analysis of 414+433 channels revealed intact ATP dependence of single-channel gating kinetics, but slightly shortened open bursts and faster closing from the locked-open state (elicited by ATP plus pyrophosphate or ATP plus AMPPNP). In contrast, 633+668 channel function was indistinguishable from WT at both macroscopic and microscopic levels. We conclude that neither nonconserved segment is an essential element of PKA- or nucleotide-dependent regulation.

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204 Indeed, the single point mutation S660A was found to have a negligible effect on the gating of phosphorylated CFTR channels in excised patches exposed to saturating [ATP] (Winter and Welsh, 1997). Login to comment
206 These results on gating of strongly phosphorylated CFTR channels in excised patches do not contradict the finding (Wilkinson et al., 1997) of a Յ2-fold reduction in sensitivity to activation by IBMX (hence presumably by PKA) of S660A or S670A CFTR channels in intact oocytes. Login to comment

[hide] Reversible silencing of CFTR chloride channels by ... J Gen Physiol. 2005 Feb;125(2):127-41. Epub 2005 Jan 18. Wang W, Oliva C, Li G, Holmgren A, Lillig CH, Kirk KL
Reversible silencing of CFTR chloride channels by glutathionylation.
J Gen Physiol. 2005 Feb;125(2):127-41. Epub 2005 Jan 18., [PMID:15657297]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and ATP-dependent chloride channel that modulates salt and water transport across lung and gut epithelia. The relationship between CFTR and oxidized forms of glutathione is of potential interest because reactive glutathione species are produced in inflamed epithelia where they may be modulators or substrates of CFTR. Here we show that CFTR channel activity in excised membrane patches is markedly inhibited by several oxidized forms of glutathione (i.e., GSSG, GSNO, and glutathione treated with diamide, a strong thiol oxidizer). Three lines of evidence indicate that the likely mechanism for this inhibitory effect is glutathionylation of a CFTR cysteine (i.e., formation of a mixed disulfide with glutathione): (a) channels could be protected from inhibition by pretreating the patch with NEM (a thiol alkylating agent) or by lowering the bath pH; (b) inhibited channels could be rescued by reducing agents (e.g., DTT) or by purified glutaredoxins (Grxs; thiol disulfide oxidoreductases) including a mutant Grx that specifically reduces mixed disulfides between glutathione and cysteines within proteins; and (c) reversible glutathionylation of CFTR polypeptides in microsomes could be detected biochemically under the same conditions. At the single channel level, the primary effect of reactive glutathione species was to markedly inhibit the opening rates of individual CFTR channels. CFTR channel inhibition was not obviously dependent on phosphorylation state but was markedly slowed when channels were first "locked open" by a poorly hydrolyzable ATP analogue (AMP-PNP). Consistent with the latter finding, we show that the major site of inhibition is cys-1344, a poorly conserved cysteine that lies proximal to the signature sequence in the second nucleotide binding domain (NBD2) of human CFTR. This region is predicted to participate in ATP-dependent channel opening and to be occluded in the nucleotide-bound state of the channel based on structural comparisons to related ATP binding cassette transporters. Our results demonstrate that human CFTR channels are reversibly inhibited by reactive glutathione species, and support an important role of the region proximal to the NBD2 signature sequence in ATP-dependent channel opening.

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43 The S660A/⌬R-CFTR mutant and all but one of the alanine-substituted cysteine mutants were provided by M. Welsh (University of Iowa, Iowa City, IA) (Rich et al., 1991; Cotton and Welsh, 1997) and were subcloned into the pCDNA3 expression vector (Invitrogen). Login to comment
51 HEK-293T cells that were transfected with S660A/⌬R-CFTR or with the cys-free constructs were grown overnight at 27ЊC because these mutants are temperature-sensitive ER processing mutants as determined by immunoblot analysis. Login to comment
203 Fig. 9 (A and B) shows that S660A/⌬R-CFTR, which was previously shown to have moderate channel activity in the absence of PKA (Rich et al., 1991), was substantially and reversibly inhibited by diamide/GSH in the absence of added kinase. Login to comment
205 Interestingly, Grx could rescue completely the currents mediated by S660A/⌬R-CFTR, which indicates that the Grx-insensitive oxidation previously observed for the WT channel (Fig. 4) requires an intact R domain. Login to comment
259 (A) Inhibitory effect of 20 ␮M diamide/ GSH on macroscopic current mediated by ⌬R-S660A-CFTR in patch excised from transfected HEK-293T cell. Login to comment
260 (B) Mean data for ⌬R-S660A-CFTR showing inhibition by diamide/GSH (20 ␮M) and recovery by E. coli Grx1 (4 ␮M) plus 1 mM GSH. Login to comment

[hide] Activating cystic fibrosis transmembrane conductan... J Biol Chem. 2005 Jun 24;280(25):23622-30. Epub 2005 Apr 27. Wang W, Li G, Clancy JP, Kirk KL
Activating cystic fibrosis transmembrane conductance regulator channels with pore blocker analogs.
J Biol Chem. 2005 Jun 24;280(25):23622-30. Epub 2005 Apr 27., 2005-06-24 [PMID:15857825]

Abstract [show]
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 deltaF508-CFTR, which inefficiently traffics to the surfaces of most cells. The deltaF508 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 deltaF508-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 deltaF508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and deltaF508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of deltaF508-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 deltaF508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface.

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27 Cells expressing ⌬F508-CFTR or ⌬R-S660A-CFTR (where "R" is the regulatory domain) were grown for 1-2 days at 27 °C (low temperature-corrected) because these mutants are temperature-sensitive endoplasmic reticulum processing mutants that exhibit low surface expression when cells are cultured at 37 °C. Login to comment
105 Second, NPPB-AM markedly stimulated the currents mediated by a regulatory domain deletion mutant (⌬R-S660A-CFTR) (Fig. 3A) that exhibits low constitutive activity in the absence of PKA (23). Login to comment
157 A, NPPB-AM and NPPB markedly stimulate ⌬R-S660A-CFTR currents minus PKA across an excised HEK-293T patch. Login to comment
203 NPPB-AM and NPPB are capable of stimulating CFTR opening without affecting channel phosphorylation by PKA, i.e. these compounds enhance wild-type or ⌬R-S660A-CFTR activity in the absence of active kinase. Login to comment
208 In this regard, the ⌬R-S660A-CFTR construct used here (23) has much lower channel activity than highly phosphorylated wild-type channels (Po estimated at Ͻ0.05) (29), although this activity is not dependent on PKA. Login to comment
234 Michael Welsh and John Wakefield for sharing the ⌬R-S660A-CFTR construct and the ⌬F508-CFTR-transfected CFBE41o-cells, respectively. Login to comment

[hide] Curcumin opens cystic fibrosis transmembrane condu... J Biol Chem. 2007 Feb 16;282(7):4533-44. Epub 2006 Dec 18. Wang W, Bernard K, Li G, Kirk KL
Curcumin opens cystic fibrosis transmembrane conductance regulator channels by a novel mechanism that requires neither ATP binding nor dimerization of the nucleotide-binding domains.
J Biol Chem. 2007 Feb 16;282(7):4533-44. Epub 2006 Dec 18., 2007-02-16 [PMID:17178710]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are essential mediators of salt transport across epithelia. Channel opening normally requires ATP binding to both nucleotide-binding domains (NBDs), probable dimerization of the two NBDs, and phosphorylation of the R domain. How phosphorylation controls channel gating is unknown. Loss-of-function mutations in the CFTR gene cause cystic fibrosis; thus, there is considerable interest in compounds that improve mutant CFTR function. Here we investigated the mechanism by which CFTR is activated by curcumin, a natural compound found in turmeric. Curcumin opened CFTR channels by a novel mechanism that required neither ATP nor the second nucleotide-binding domain (NBD2). Consequently, this compound potently activated CF mutant channels that are defective for the normal ATP-dependent mode of gating (e.g. G551D and W1282X), including channels that lack NBD2. The stimulation of NBD2 deletion mutants by curcumin was strongly inhibited by ATP binding to NBD1, which implicates NBD1 as a plausible activation site. Curcumin activation became irreversible during prolonged exposure to this compound following which persistently activated channels gated dynamically in the absence of any agonist. Although CFTR activation by curcumin required neither ATP binding nor heterodimerization of the two NBDs, it was strongly dependent on prior channel phosphorylation by protein kinase A. Curcumin is a useful functional probe of CFTR gating that opens mutant channels by circumventing the normal requirements for ATP binding and NBD heterodimerization. The phosphorylation dependence of curcumin activation indicates that the R domain can modulate channel opening without affecting ATP binding to the NBDs or their heterodimerization.

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50 The ⌬R/S660A-CFTR mutant was provided by M. Welsh (University of Iowa) (23), subcloned into the pCDNA3 vector, and truncated by PCR mutagenesis to make ⌬1198/⌬R/S660A-CFTR. Login to comment
204 D, curcumin activates ⌬1198/⌬R-S660A-CFTR channels in the absence of ATP and PKA. Login to comment
211 We also tested the effect of curcumin on channels that lack a large portion of the R domain in addition to NBD2 (⌬1198/⌬R/S660A), because previous work has shown that ⌬R/S660A-CFTR channels exhibit constitutive activity in the absence of added kinase (23). Login to comment
267 Acknowledgments-We thank J. P. Clancy for the HeLa cells stably transfected with W1282X-CFTR and M. J. Welsh for the ⌬R/S660A-CFTR construct. Login to comment

[hide] CFTR regulatory region interacts with NBD1 predomi... Nat Struct Mol Biol. 2007 Aug;14(8):738-45. Epub 2007 Jul 29. Baker JM, Hudson RP, Kanelis V, Choy WY, Thibodeau PH, Thomas PJ, Forman-Kay JD
CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices.
Nat Struct Mol Biol. 2007 Aug;14(8):738-45. Epub 2007 Jul 29., [PMID:17660831]

Abstract [show]
The regulatory (R) region of the cystic fibrosis transmembrane conductance regulator (CFTR) is intrinsically disordered and must be phosphorylated at multiple sites for full CFTR channel activity, with no one specific phosphorylation site required. In addition, nucleotide binding and hydrolysis at the nucleotide-binding domains (NBDs) of CFTR are required for channel gating. We report NMR studies in the absence and presence of NBD1 that provide structural details for the isolated R region and its interaction with NBD1 at residue-level resolution. Several sites in the R region with measured fractional helical propensity mediate interactions with NBD1. Phosphorylation reduces the helicity of many R-region sites and reduces their NBD1 interactions. This evidence for a dynamic complex with NBD1 that transiently engages different sites of the R region suggests a structural explanation for the dependence of CFTR activity on multiple PKA phosphorylation sites.

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19 The R region may have an additional stimulatory role, as shown by CFTR channels lacking much of the R region (D708À835/S660A), which gate independently of PKA yet are further stimulated by the addition in trans of phosphorylated R region (residues 645À835)15. Login to comment

[hide] Potentiation of cystic fibrosis transmembrane cond... Mol Membr Biol. 2008 Sep;25(6-7):528-38. Hughes LK, Ju M, Sheppard DN
Potentiation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents by the chemical solvent tetrahydrofuran.
Mol Membr Biol. 2008 Sep;25(6-7):528-38., [PMID:18989824]

Abstract [show]
The chemical solvent tetrahydrofuran (THF) increases short-circuit current (I(sc)) in renal epithelia endogenously expressing the cystic fibrosis transmembrane conductance regulator (CFTR). To understand how THF increases I(sc), we employed the Ussing chamber and patch-clamp techniques to study cells expressing recombinant human CFTR. THF increased I(sc) in Fischer rat thyroid (FRT) epithelia expressing wild-type CFTR with half-maximal effective concentration (K(D)) of 134 mM. This THF-induced increase in I(sc) was enhanced by forskolin (10 microM), inhibited by the PKA inhibitor H-89 (10 microM) and the thiazolidinone CFTR(inh)-172 (10 microM) and attenuated greatly in FRT epithelia expressing the cystic fibrosis mutants F508del- and G551D-CFTR. By contrast, THF (100 mM) was without effect on untransfected FRT epithelia, while other solvents failed to increase I(sc) in FRT epithelia expressing wild-type CFTR. In excised inside-out membrane patches, THF (100 mM) potentiated CFTR Cl(-) channels open in the presence of ATP (1 mM) alone by increasing the frequency of channel openings without altering their duration. However, following the phosphorylation of CFTR by PKA (75 nM), THF (100 mM) did not potentiate channel activity. Similar results were obtained with the triangle upR-S660A-CFTR Cl(-) channel that is not regulated by PKA-dependent phosphorylation and using 2'deoxy-ATP, which gates wild-type CFTR more effectively than ATP. Our data suggest that THF acts directly on CFTR to potentiate channel gating, but that its efficacy is weak and dependent on the phosphorylation status of CFTR.

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7 Similar results were obtained with the DR-S660A-CFTR Cl( channel that is not regulated by PKA-dependent phosphorylation and using 2?deoxy-ATP, which gates wild-type CFTR more effectively than ATP. Login to comment
28 Materials and methods Cells and cell culture For this study, we used Fischer rat thyroid (FRT) epithelial cells expressing wild-type, F508del- and G551D- human CFTR [8] and mouse mammary epithelial (C127) cells stably expressing either wild-type human CFTR or the variant DR-S660A-CFTR Figure 1. Login to comment
179 THF potentiates the DR-S660A-CFTR Cl( channel and wild-type CFTR gated by 2?-deoxy-ATP The lack of effect of THF on the CFTR Cl( channel after phosphorylation by PKA, suggests that the RD might be a target for the actions of THF. Login to comment
180 To test this hypothesis, we used the CFTR construct DR-S660A [9], performing the same experiments on this CFTR variant as those with wild-type CFTR. Login to comment
181 The CFTR mutant DR-S660A lacks much of the RD (residues 708Á835) and disables the dibasic phosphorylation site at serine 660. Login to comment
183 Supplementary Information (SI) Figure 1 (online version only) demonstrates that THF (100 mM) augments the activity of DR-S660A-CFTR Cl( channels by increasing the frequency of channel openings. Login to comment
227 Our observation that THF potentiates the DR-S660A-CFTR Cl( channel argues that the solvent interacts with a site distinct from the RD. Login to comment

[hide] Direct sensing of intracellular pH by the cystic f... J Biol Chem. 2009 Dec 18;284(51):35495-506. Epub . Chen JH, Cai Z, Sheppard DN
Direct sensing of intracellular pH by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel.
J Biol Chem. 2009 Dec 18;284(51):35495-506. Epub ., 2009-12-18 [PMID:19837660]

Abstract [show]
In cystic fibrosis (CF), dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel disrupts epithelial ion transport and perturbs the regulation of intracellular pH (pH(i)). CFTR modulates pH(i) through its role as an ion channel and by regulating transport proteins. However, it is unknown how CFTR senses pH(i). Here, we investigate the direct effects of pH(i) on recombinant CFTR using excised membrane patches. By altering channel gating, acidic pH(i) increased the open probability (P(o)) of wild-type CFTR, whereas alkaline pH(i) decreased P(o) and inhibited Cl(-) flow through the channel. Acidic pH(i) potentiated the MgATP dependence of wild-type CFTR by increasing MgATP affinity and enhancing channel activity, whereas alkaline pH(i) inhibited the MgATP dependence of wild-type CFTR by decreasing channel activity. Because these data suggest that pH(i) modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pH(i) dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Site 2 mutants, but not site 1 mutants, perturbed both potentiation by acidic pH(i) and inhibition by alkaline pH(i), suggesting that site 2 is a critical determinant of the pH(i) sensitivity of CFTR. The effects of pH(i) also suggest that site 2 might employ substrate-assisted catalysis to ensure that ATP hydrolysis follows NBD dimerization. We conclude that the CFTR Cl(-) channel senses directly pH(i). The direct regulation of CFTR by pH(i) has important implications for the regulation of epithelial ion transport.

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46 These included (i) mouse mammary epithelial cells (C127 cells) expressing wild-type human CFTR, the CFTR variant ⌬R-S660A (13) or the CF mutant G1349D (14), (ii) Fischer rat thyroid epithelial cells expressing the CF mutant G551D (15), and (iii) NIH-3T3 cells expressing the CFTR construct K1250M (16). Login to comment
158 To test this hypothesis, we adopted two strategies; first, we employed the CFTR construct ⌬R-S660A that deletes a large part of the RD and is not regulated by PKA-dependent phosphorylation (13) to explore separately how pHi influences the function of the RD and NBDs. Login to comment
161 Fig. 5A shows representative recordings of a single ⌬R-S660A-CFTR Cl-channel at different pHi, whereas Fig. 5, B-D quantifies the effects of pHi on channel gating. Consistent with previous studies (13), at pHi 7.3 the Po of ⌬R-S660A-CFTR was attenuated markedly compared with that of wild-type CFTR FIGURE 4. Login to comment
176 Acidic pHi potentiates the activity of ⌬R-S660A-CFTR. Login to comment
177 A, representative recordings show the effects of pHi on the activity of a single ⌬R-S660A-CFTR Cl-channel. Login to comment
178 Dotted lines indicate where the channel is closed, and downward deflections correspond to channel openings. B, C, and D, effects of pHi on the Po, MBD, and IBI of ⌬R-S660A (columns) and wild-type CFTR (circles). Login to comment
179 Data are means Ϯ S.E. (B, n ϭ 6 for all data; C and D, ⌬R-S660A-CFTR, n ϭ 3; wild-type-CFTR, n Ն 6); asterisks indicate ⌬R-S660A-CFTR values that are significantly different from the pHi 7.3 control (p Ͻ 0.05). Login to comment
181 Acidifying to pHi 6.3 potentiated the Po of wild-type CFTR 0.4-fold, but that of ⌬R-S660A-CFTR 3.3-fold (Fig. 5B). Login to comment
182 Fig. 5, C and D, demonstrates that the marked potentiation of ⌬R-S660A-CFTR Po at pHi 6.3 was primarily caused by a 0.9-fold decrease in IBI, but it was also enhanced by a 1.5-fold increase of MBD. Login to comment
183 Alkalinizing to pHi 8.3 decreased the Po of wild-type CFTR 0.3-fold, but was without effect on the Po of ⌬R-S660A-CFTR (Fig. 5B). Login to comment
184 An explanation for this striking difference between wild-type and ⌬R-S660A-CFTR is shown in Fig. 5D. Login to comment
185 Between pHi 7.3 and 8.3, the IBI of wild-type CFTR increased 0.3-fold, whereas that of ⌬R-S660A-CFTR decreased 0.5-fold (Fig. 5D). Login to comment
186 Fig. 5D also reveals that the relationship between IBI and pHi is linear for wild-type CFTR, but bell-shaped for ⌬R-S660A-CFTR over the pHi range 6.3-8.3. Login to comment
187 We interpret the failure of pHi 8.3 to attenuate ⌬R-S660A-CFTR channel gating to suggest that alkaline pHi might inhibit CFTR channel gating, at least in part, by modulating RD function. Login to comment
188 However, the robust potentiation of ⌬R-S660A-CFTR channel gating at pHi 6.3 argues that acidic pHi likely enhances CFTR channel gating by acting on sites distinct from the RD. Login to comment
254 Second, studies of the CFTR construct ⌬R-S660A suggest that OH- ions might inhibit CFTR channel gating, at least in part, by modulating RD function. Login to comment
283 In contrast to the effects of strong alkaline pHi on wild-type CFTR, our studies of the ⌬R-S660A-CFTR Cl-channel at pHi 8.3 argue that alkaline pHi inhibits CFTR channel gating, at FIGURE 8. Login to comment
290 The reason why ⌬R-S660A-CFTR is not inhibited at pHi 8.3 is that its prolonged IBI is attenuated, not extended, at this pHi value. Login to comment
292 However, it is interesting to note that for both ⌬R-S660A-CFTR and the ATP-binding site 1 mutant D572N-CFTR, the relationship between pHi and IBI is bell-shaped, not linear, between pHi 6.3 and 8.3 (Figs. 5D and 8B). Login to comment

[hide] ATP-independent CFTR channel gating and allosteric... Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3888-93. Epub 2010 Feb 3. Wang W, Wu J, Bernard K, Li G, Wang G, Bevensee MO, Kirk KL
ATP-independent CFTR channel gating and allosteric modulation by phosphorylation.
Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3888-93. Epub 2010 Feb 3., 2010-02-23 [PMID:20133716]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. The extent to which CFTR gating conforms to classic allosteric schemes of ligand activation is unclear. Here, we describe point mutations in the CFTR cytosolic loops that markedly increase ATP-independent (constitutive) channel activity. This finding is consistent with an allosteric gating mechanism in which ligand shifts the equilibrium between inactive and active states but is not essential for channel opening. Constitutive mutations mapped to the putative symmetry axis of CFTR based on the crystal structures of related ABC transporters, a common theme for activating mutations in ligand-gated channels. Furthermore, the ATP sensitivity of channel activation was strongly enhanced by these constitutive mutations, as predicted for an allosteric mechanism (reciprocity between protein activation and ligand occupancy). Introducing constitutive mutations into CFTR channels that cannot open in response to ATP (i.e., the G551D CF mutant and an NBD2-deletion mutant) substantially rescued their activities. Importantly, constitutive mutants that opened without ATP or NBD2 still required R domain phosphorylation for optimal activity. Our results confirm that (i) CFTR gating exhibits features of protein allostery that are shared with conventional ligand-gated channels and (ii) the R domain modulates CFTR activity independent of ATP-induced NBD dimerization.

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No. Sentence Comment
213 ΔR-S660A-CFTR (29) was provided by Michael Welsh (University of Iowa, Iowa City, IA).Cells transfected with cysteine-free or ΔR constructs were cultured at 26-28 °C for 24-48 h before patch clamping to increase expression of these mutants, which mature less efficiently than WT-CFTR (16, 17, 30). Login to comment

[hide] State-dependent regulation of cystic fibrosis tran... J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15. Wang G
State-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gating by a high affinity Fe3+ bridge between the regulatory domain and cytoplasmic loop 3.
J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15., 2010-12-24 [PMID:20952391]

Abstract [show]
The unique regulatory (R) domain differentiates the human CFTR channel from other ATP-binding cassette transporters and exerts multiple effects on channel function. However, the underlying mechanisms are unclear. Here, an intracellular high affinity (2.3 x 10(-19) M) Fe(3+) bridge is reported as a novel approach to regulating channel gating. It inhibited CFTR activity by primarily reducing an open probability and an opening rate, and inhibition was reversed by EDTA and phenanthroline. His-950, His-954, Cys-832, His-775, and Asp-836 were found essential for inhibition and phosphorylated Ser-768 may enhance Fe(3+) binding. More importantly, inhibition by Fe(3+) was state-dependent. Sensitivity to Fe(3+) was reduced when the channel was locked in an open state by AMP-PNP. Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe(3+) no matter whether NBD2 was present or not. Therefore, although ATP binding-induced dimerization of NBD1-NBD2 is required for channel gating, regulation of CFTR activity by Fe(3+) may involve an interaction between the R domain and CL3. These findings may support proximity of the R domain to the cytoplasmic loops. They also suggest that Fe(3+) homeostasis may play a critical role in regulating pathophysiological CFTR activity because dysregulation of this protein causes cystic fibrosis, secretary diarrhea, and infertility.

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45 In addition, removal of a segment 760-783 or 817-838 (NEG2) or much of the R domain (⌬708-835/ S660A) from CFTR eliminates the PKA dependence of channel activity (26-28). Login to comment

[hide] The inhibition mechanism of non-phosphorylated Ser... J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8. Wang G
The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8., 2011-01-21 [PMID:21059651]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporters but serves as a chloride channel dysfunctional in cystic fibrosis. The activity of CFTR is tightly controlled not only by ATP-driven dimerization of its nucleotide-binding domains but also by phosphorylation of a unique regulatory (R) domain by protein kinase A (PKA). The R domain has multiple excitatory phosphorylation sites, but Ser(737) and Ser(768) are inhibitory. The underlying mechanism is unclear. Here, sulfhydryl-specific cross-linking strategy was employed to demonstrate that Ser(768) or Ser(737) could interact with outwardly facing hydrophilic residues of cytoplasmic loop 3 regulating channel gating. Furthermore, mutation of these residues to alanines promoted channel opening by curcumin in an ATP-dependent manner even in the absence of PKA. However, mutation of Ser(768) and His(950) with different hydrogen bond donors or acceptors clearly changed ATP- and PKA-dependent channel activity no matter whether curcumin was present or not. More importantly, significant activation of a double mutant H950R/S768R needed only ATP. Finally, in vitro and in vivo single channel recordings suggest that Ser(768) may form a putative hydrogen bond with His(950) of cytoplasmic loop 3 to prevent channel opening by ATP in the non-phosphorylated state and by subsequent cAMP-dependent phosphorylation. These observations support an electron cryomicroscopy-based structural model on which the R domain is closed to cytoplasmic loops regulating channel gating.

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42 In addition, removal of residues 760-783 or 817-838 (NEG2) or much of the R domain (⌬708-835/S660A) from CFTR eliminates PKA dependence of channel activity (26-28). Login to comment
62 ⌬R-S660A-CFTR was provided by Michael Welsh (University of Iowa) (31). Login to comment

[hide] Pharmacological therapy for cystic fibrosis: from ... J Cyst Fibros. 2011 Jun;10 Suppl 2:S129-45. Becq F, Mall MA, Sheppard DN, Conese M, Zegarra-Moran O
Pharmacological therapy for cystic fibrosis: from bench to bedside.
J Cyst Fibros. 2011 Jun;10 Suppl 2:S129-45., [PMID:21658632]

Abstract [show]
With knowledge of the molecular behaviour of the cystic fibrosis transmembrane conductance regulator (CFTR), its physiological role and dysfunction in cystic fibrosis (CF), therapeutic strategies are now being developed that target the root cause of CF rather than disease symptoms. Here, we review progress towards the development of rational new therapies for CF. We highlight the discovery of small molecules that rescue the cell surface expression and defective channel gating of CF mutants, termed CFTR correctors and CFTR potentiators, respectively. We draw attention to alternative approaches to restore epithelial ion transport to CF epithelia, including inhibitors of the epithelial Na(+) channel (ENaC) and activators of the Ca(2+)-activated Cl(-) channel TMEM16A. The expertise required to translate small molecules identified in the laboratory to drugs for CF patients depends on our ability to coordinate drug development at an international level and our ability to provide pertinent biological information using suitable disease models.

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244 5-nitro-2-(3-phenyl propylamino)benzoate (NPPB) analogues [142] BHK, HEK293, CFBE41o- transfected WT, F508del, R-S660A-CFTR Electrophysiology NPPB analogues stimulate WT and F508del-CFTR channel activity at submicromolar concentrations without blocking the channel pore. Login to comment

[hide] Regulation of murine cystic fibrosis transmembrane... J Physiol. 1998 Nov 1;512 ( Pt 3):751-64. Lansdell KA, Kidd JF, Delaney SJ, Wainwright BJ, Sheppard DN
Regulation of murine cystic fibrosis transmembrane conductance regulator Cl- channels expressed in Chinese hamster ovary cells.
J Physiol. 1998 Nov 1;512 ( Pt 3):751-64., 1998-11-01 [PMID:9769419]

Abstract [show]
1. We investigated the effect of protein kinases and phosphatases on murine cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, expressed in Chinese hamster ovary (CHO) cells, using iodide efflux and the excised inside-out configuration of the patch-clamp technique. 2. The protein kinase C (PKC) activator, phorbol dibutyrate, enhanced cAMP-stimulated iodide efflux. However, PKC did not augment the single-channel activity of either human or murine CFTR Cl- channels that had previously been activated by protein kinase A. 3. Fluoride, a non-specific inhibitor of protein phosphatases, stimulated both human and murine CFTR Cl- channels. However, calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, did not enhance cAMP-stimulated iodide efflux. 4. The alkaline phosphatase inhibitor, (-)-bromotetramisole augmented cAMP-stimulated iodide efflux and, by itself, stimulated a larger efflux than that evoked by cAMP agonists. However, (+)-bromotetramisole, the inactive enantiomer, had the same effect. For murine CFTR, neither enantiomer enhanced single-channel activity. In contrast, both enantiomers increased the open probability (Po) of human CFTR, suggesting that bromotetramisole may promote the opening of human CFTR. 5. As murine CFTR had a low Po and was refractory to stimulation by activators of human CFTR, we investigated whether murine CFTR may open to a subconductance state. When single-channel records were filtered at 50 Hz, a very small subconductance state of murine CFTR was observed that had a Po greater than that of human CFTR. The occupancy of this subconductance state may explain the differences in channel regulation observed between human and murine CFTR.

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248 However, recent data suggest that the effect of fluoride may be independent of phosphatase inhibition, because fluoride stimulated the human CFTR mutant CFTRÄR-S660A (Berger et al. 1998). Login to comment

[hide] Structure and function of the CFTR chloride channe... Physiol Rev. 1999 Jan;79(1 Suppl):S23-45. Sheppard DN, Welsh MJ
Structure and function of the CFTR chloride channel.
Physiol Rev. 1999 Jan;79(1 Suppl):S23-45., [PMID:9922375]

Abstract [show]
Structure and Function of the CFTR Chloride Channel. Physiol. Rev. 79, Suppl.: S23-S45, 1999. - The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ABC transporter family that forms a novel Cl- channel. It is located predominantly in the apical membrane of epithelia where it mediates transepithelial salt and liquid movement. Dysfunction of CFTR causes the genetic disease cystic fibrosis. The CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. Here we review the structure and function of this unique channel, with a focus on how the various domains contribute to channel function. The MSDs form the channel pore, phosphorylation of the R domain determines channel activity, and ATP hydrolysis by the NBDs controls channel gating. Current knowledge of CFTR structure and function may help us understand better its mechanism of action, its role in electrolyte transport, its dysfunction in cystic fibrosis, and its relationship to other ABC transporters.

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No. Sentence Comment
521 When S660 in CFTRDR was mutated to alanine dan (42) proposed a two-domain model of the R domain: RD1 (amino acids 587-672) and RD2 (amino acids 679-(CFTRDR-S660A), PKA failed to stimulate channel activity. Login to comment
526 In addition, deletion studies of portions of the R domain suggest thatout that PKA cannot interact with CFTRDR-S660A, even though it does interact with CFTRDR. there are two halves to the R domain with the first conserved in other ABC transporters and the second uniqueLike CFTRDR, the amino-terminal portion of CFTR (D836X, which contains MSD1, NBD1, and the R domain) to CFTR (102, 103). Login to comment
533 Alternatively, the PKA-independent activity of D836X could be due to As described above, phosphorylation by PKA, deletion of FIG. 11 Effect of a recombinant R domain (R1, residues 645-834) on CFTRDR/S660A channel in presence of PKA. Login to comment
535 B and C: effect of R1 on rates of entry into and exit from burst for CFTR-DR/S660A channels in presence of PKA. Login to comment

[hide] Control of CFTR channel gating by phosphorylation ... Physiol Rev. 1999 Jan;79(1 Suppl):S77-S107. Gadsby DC, Nairn AC
Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis.
Physiol Rev. 1999 Jan;79(1 Suppl):S77-S107., [PMID:9922377]

Abstract [show]
Control of CTFR Channel Gating by Phosphorylation and Nucleotide Hydrolysis. Physiol. Rev. 79, Suppl.: S77-S107, 1999. - The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is the protein product of the gene defective in cystic fibrosis, the most common lethal genetic disease among Caucasians. Unlike any other known ion channel, CFTR belongs to the ATP-binding cassette superfamily of transporters and, like all other family members, CFTR includes two cytoplasmic nucleotide-binding domains (NBDs), both of which bind and hydrolyze ATP. It appears that in a single open-close gating cycle, an individual CFTR channel hydrolyzes one ATP molecule at the NH2-terminal NBD to open the channel, and then binds and hydrolyzes a second ATP molecule at the COOH-terminal NBD to close the channel. This complex coordinated behavior of the two NBDs is orchestrated by multiple protein kinase A-dependent phosphorylation events, at least some of which occur within the third large cytoplasmic domain, called the regulatory domain. Two or more kinds of protein phosphatases selectively dephosphorylate distinct sites. Under appropriately controlled conditions of progressive phosphorylation or dephosphorylation, three functionally different phosphoforms of a single CFTR channel can be distinguished on the basis of channel opening and closing kinetics. Recording single CFTR channel currents affords an unprecedented opportunity to reproducibly examine, and manipulate, individual ATP hydrolysis cycles in a single molecule, in its natural environment, in real time.

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No. Sentence Comment
133 Along the same lines, coimmunoprecipitation experiments suggested that phosphorylation of functional decrements attending incorporation of addi- P33-8/ 9j0e$$ja10 01-13-99 16:19:50 prsa APS-Phys Rev tional mutations at Ser-422 to yield a 10SA mutant (31), single-channel currents in mutant CFTR missing R-domain residues 708-835 (CFTRDR), with (163) or withoutat Ser-753 to give the 11SA mutant (178), and at four remaining R-domain serines and threonines (S670, T690, (122, but cf. Ref. 164) additional mutation of Ser-660 to alanine (CFTRDR-S660A), does not constitute proof thatT787, and S790) mutated together to yield a (11/4)SA mutant (177). Login to comment

[hide] Regulation of Activation and Processing of the Cys... J Biol Chem. 2012 Oct 11. Wang G, Duan DD
Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3.
J Biol Chem. 2012 Oct 11., [PMID:23060444]

Abstract [show]
NEG2, a short C-terminal segment (817-838) of the unique regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, has been reported to regulate CFTR gating in response to cAMP-dependent R domain phosphorylation. The underlying mechanism, however, is unclear. Here, K946 of cytoplasmic loop 3 (CL3) is proposed as counter-ion of D835, D836 or E838 of NEG2 to prevent channel activation by PKA. R764 or R766 of the S768 phosphorylation site of the R domain is proposed to promote channel activation possibly by weakening the putative CL3-NEG2 electrostatic attraction. First, not only D835A, D836A and E838A but also K946A reduced the PKA dependent CFTR activation. Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Third, R764A and R766A mutants enhanced the PKA-dependent activation. On the other hand, it is very exciting that D835R/D836R/E838R and K946D/H950D and H950R exhibited normal channel processing and activity while D835R/D836R/E838R/K946D/H950D was misprocessed and silent in response to forskolin. Further, D836R and E838R played a critical role in the asymmetric electrostatic regulation of CFTR processing and S768 phosphorylation may not be involved. Thus, a complex interfacial interaction among CL3, NEG2 and the S768 phosphorylation site may be responsible for the asymmetric electrostatic regulation of CFTR activation and processing.

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37 R-S660A-CFTR was provided by Michael Welsh (University of Iowa, Iowa City, IA). Login to comment
41 èc;R-S660A-CFTR was provided by Michael Welsh (University of Iowa, Iowa City, IA). Login to comment

[hide] Fluoride stimulates cystic fibrosis transmembrane ... Am J Physiol. 1998 Mar;274(3 Pt 1):L305-12. Berger HA, Travis SM, Welsh MJ
Fluoride stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity.
Am J Physiol. 1998 Mar;274(3 Pt 1):L305-12., [PMID:9530164]

Abstract [show]
While studying the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR), we found that addition of F- to the cytosolic surface of excised, inside-out membrane patches reversibly increased Cl- current in a dose-dependent manner. Stimulation required prior phosphorylation and the presence of ATP. F- increased current even in the presence of deferoxamine, which chelates Al3+, suggesting that stimulation was not due to AlF4-. F- also stimulated current in a CFTR variant that lacked a large part of the R domain, suggesting that the effect was not mediated via this domain. Studies of single channels showed that F- increased the open-state probability by slowing channel closure from bursts of activity; the mean closed time between bursts and single-channel conductance was not altered. These results suggested that F- influenced regulation by the cytosolic domains, most likely the nucleotide-binding domains (NBDs). Consistent with this, we found that mutation of a conserved Walker lysine in NBD2 changed the relative stimulatory effect of F- compared with wild-type CFTR, whereas mutation of the Walker lysine in NBD1 had no effect. Based on these and previous data, we speculate that F- interacts with CFTR, possibly via NBD2, and slows the rate of channel closure.

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134 Effect of Fon CFTR⌬R-S660A current. Because CFTR requires phosphorylation for activation and because Fis known to inhibit several protein phosphatases (34), we asked whether F- might stimulate CFTR activity by inhibiting a phosphatase. Login to comment
135 To test this possibility, we studied a CFTR variant (CFTR⌬R-S660A) in which part of the R domain (amino acids 708-835) is deleted and a remaining phosphorylation site (serine 660) is mutated to alanine (30). Login to comment
137 Figure 5 shows that addition of F- increased the current in CFTR⌬R-S660A (n ϭ 3), suggesting that F- did not stimulate the channel by inhibiting a phosphatase. Login to comment
157 Effect of Fon Cl- current in CFTR⌬R-S660A. Login to comment
158 Data show time course of current in an excised, inside-out membrane patch from NIH/3T3 cells expressing CFTR⌬R-S660A. Login to comment
182 The effect of F- was likely independent of phosphatase inhibition, since F- increased current in CFTR⌬R-S660A. Login to comment
201 It seemed unlikely that F- stimu- lated through an interaction with the R domain because F- stimulated CFTR⌬R-S660A. Login to comment
127 Effect of F2 on CFTRDR-S660A current. Because CFTR requires phosphorylation for activation and because F2 is known to inhibit several protein phosphatases (34), we asked whether F2 might stimulate CFTR activity by inhibiting a phosphatase. Login to comment
128 To test this possibility, we studied a CFTR variant (CFTRDR-S660A) in which part of the R domain (amino acids 708-835) is deleted and a remaining phosphorylation site (serine 660) is mutated to alanine (30). Login to comment
130 Figure 5 shows that addition of F2 increased the current in CFTRDR-S660A (n 5 3), suggesting that F2 did not stimulate the channel by inhibiting a phosphatase. Login to comment
150 Effect of F2 on Cl2 current in CFTRDR-S660A. Login to comment
151 Data show time course of current in an excised, inside-out membrane patch from NIH/3T3 cells expressing CFTRDR-S660A. Login to comment
175 The effect of F2 was likely independent of phosphatase inhibition, since F2 increased current in CFTRDR-S660A. Login to comment
193 It seemed unlikely that F2 stimulated through an interaction with the R domain because F2 stimulated CFTRDR-S660A. Login to comment

[hide] CFTR Cl- channel and CFTR-associated ATP channel: ... EMBO J. 1998 Feb 16;17(4):898-908. Sugita M, Yue Y, Foskett JK
CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates.
EMBO J. 1998 Feb 16;17(4):898-908., [PMID:9463368]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is regulated by phosphorylation of the R domain and ATP hydrolysis at two nucleotide-binding domains (NBDs). It is controversial whether CFTR conducts ATP or whether CFTR might be closely associated with a separate ATP conductance. To characterize ATP channels associated with CFTR, we analyzed Cl- and ATP single channel-currents in excised inside-out membrane patches from MDCK epithelial cells transiently expressing CFTR. With 100 mM ATP in the pipette and 140 mM Cl- in the bath, ATP channels were associated with CFTR Cl- channels in two-thirds of patches that included CFTR. CFTR Cl- channels and CFTR-associated ATP channels had slope conductances of 7.4 pS and 5.2 pS, respectively, and had distinct reversal potentials and sensitivities to channel blockers. CFTR-associated ATP channels exhibited slow gating kinetics that depended on the presence of protein kinase A and cytoplasmic ATP, similar to CFTR Cl- channels. Gating kinetics of the ATP channels as well as the CFTR Cl- channels were similarly affected by non-hydrolyzable ATP analogues and mutations in the CFTR R domain and NBDs. Our results indicate that phosphorylation- and nucleotide-hydrolysis-dependent gating of CFTR is directly involved in gating of an associated ATP channel. However, the permeation pathways for Cl- and ATP are distinct and the ATP conduction pathway is not obligatorily associated with the expression of CFTR.

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128 We first examined the deletion mutant CFTR∆R-S660A, Fig. 8. Login to comment
131 The residues altered in CFTR∆R-S660A, CFTR S-oct-D, K464A and K1250A mutants are shown. Login to comment
132 (B) Current traces from a MDCK cell expressing CFTR∆R-S660A in the presence or absence of PKA at various membrane potentials (representative of five independently observed channels). Login to comment
137 which lacks much of the R domain and replaces Ser-660 with alanine (Figure 8A). Login to comment
139 The CFTR∆R-S660A Cl- channels were active in MDCK cells (Po ϭ 0.05 Ϯ 0.003, nϭ3) requiring the presence of 903 cytosolic ATP alone (Figure 8B), and the gating activity did not increase following addition of PKA, in agreement with the previous results. Login to comment
140 However, the CFTR∆R-S660A mutant eliminated CFTR-associated ATP channel activities, even in the presence of both PKA and ATP (nϭ5; Figure 8B). Login to comment
272 Acknowledgements We thank M.Welsh for providing the CFTR∆R-S660A and CFTR S-oct-D mutants, R.Kopito for providing the K1250A and K464A mutants, J.Engelhardt for providing the R347E mutant, U.Patel for her precious technical help and D.Mak for helpful discussions. Login to comment
135 We first examined the deletion mutant CFTRƊR-S660A, Fig. 8. Login to comment
138 The residues altered in CFTRƊR-S660A, CFTR S-oct-D, K464A and K1250A mutants are shown. Login to comment
145 which lacks much of the R domain and replaces Ser-660 with alanine (Figure 8A). Login to comment
147 The CFTRƊR-S660A Cl-channels were active in MDCK cells (Po afd; 0.05 afe; 0.003, nafd;3) requiring the presence of 903 cytosolic ATP alone (Figure 8B), and the gating activity did not increase following addition of PKA, in agreement with the previous results. Login to comment
148 However, the CFTRƊR-S660A mutant eliminated CFTR-associated ATP channel activities, even in the presence of both PKA and ATP (nafd;5; Figure 8B). Login to comment
282 Acknowledgements We thank M.Welsh for providing the CFTRƊR-S660A and CFTR S-oct-D mutants, R.Kopito for providing the K1250A and K464A mutants, J.Engelhardt for providing the R347E mutant, U.Patel for her precious technical help and D.Mak for helpful discussions. Login to comment

[hide] Stimulation of CFTR activity by its phosphorylated... Nature. 1997 Sep 18;389(6648):294-6. Winter MC, Welsh MJ
Stimulation of CFTR activity by its phosphorylated R domain.
Nature. 1997 Sep 18;389(6648):294-6., [PMID:9305845]

Abstract [show]
Phosphorylation controls the activity of ion channels in many tissues. In epithelia, the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by phosphorylation of serine residues in its regulatory (R) domain and then gated by binding and hydrolysis of ATP by the nucleotide-binding domains. Current models propose that the unphosphorylated R domain serves as an inhibitory particle that occludes the pore, much like the inhibitory 'ball' in Shaker K+ channels; presumably, phosphorylation relieves this inhibition. Here we test this by adding an R-domain peptide to a CFTR variant in which much of the R domain had been deleted (CFTR-deltaR/S660A): in contrast to predictions, we found that adding an unphosphorylated R domain to CFTR-deltaR/S660A did not inhibit activity, whereas a phosphorylated R-domain peptide stimulated activity. To investigate how phosphorylation controls activity, we studied channel gating and found that phosphorylation of the R domain increases the rate of channel opening by enhancing the sensitivity to ATP. Our results indicate that CFTR is regulated by a new mechanism in which phosphorylation of one domain stimulates the interaction of ATP with another domain, thereby increasing activity.

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124 Here we test this by adding an R-domain peptide to a CFTR variant in which much of the R domain had been deleted (CFTR-DR/S660A): in contrast to predictions, we found that adding an unphosphorylated R domain to CFTR-DR/S660A did not inhibit activity, whereas a phosphorylated R-domain peptide stimulated activity. Login to comment
125 To investigate how phosphorylation controls activity, we studied channel gating and found that phosphorylation of the R domain increases the rate of channel opening by enhancing the sensitivity to ATP. Our results indicate that CFTR is regulated by a new mechanism in which phosphorylation of one domain stimulates the interaction of ATP with another domain, thereby increasing activity. We previously studied a CFTR variant, CFTR-DR/S660A, in which much of the R domain (residues 708-835) was deleted and the phosphorylation site was removed at Ser 660 by mutation to alanine4 . Login to comment
128 This model predicts that the activity of CFTR-DR/S660A should be similar to that of phosphorylated wild-type CFTR. Login to comment
129 However, when we examined the single-channel properties of CFTR-DR/ S660A, we found that Po was 0:13 Ϯ 0:01 (1 mM ATP, n ¼ 7), much lower than that of phosphorylated wild-type CFTR (Po ¼ 0:46 Ϯ 0:02; 1 mM ATP, n ¼ 17, P Ͻ 0:001). Login to comment
131 But deletion of the R domain in CFTR-DR/S660A could still have additional nonspecific effects on channel structure which might reduce activity. We considered, therefore, that the phosphorylated R domain might stimulate the channel and that the defective functioning of CFTR-DR/S660A is due to its lacking a phosphorylated R domain. Login to comment
132 To test this idea, we added a recombinant R domain (R1, residues 645-834, similar to a peptide reported previously10 ) to the cytosolic surface of CFTR-DR/S660A and measured activity by using the excised, inside-out patch-clamp technique. Login to comment
136 Additional evidence that the effect of phosphorylation was on R1 rather than on CFTR-DR/S660A came from our finding that addition of phosphorylated R1 in the presence of protein-kinase-inhibitor peptide under conditions that block the effect of PKA11 gave similar results (43:2 Ϯ 3:4% increase; n ¼ 3). Login to comment
141 Our results also suggest that the low Po found for CFTR-DR/S660A results at least in part from a lack of stimulation by a phosphorylated R domain, rather than from nonspecific structural changes. Login to comment
142 The fact that R1 did not increase the Po of CFTR-DR/S660A to wild-type CFTR values may be because the endogenous R domain is constrained in the correct orientation by the site of interaction; the interaction of exogenously applied R1 with CFTR-DR/S660A is probably less efficient. Login to comment
143 Our results contrast with a report that addition of an in vitro translation mixture containing a different unphosphorylated R domain (residues letters to nature 294 NATURE |VOL 389 |18 SEPTEMBER 1997 a -20 -10 0 I(pA) 5 min R1 PKA b-30 -20 -10 0 5 min R1 ∆R/S660A Wild type 0 10 20 30 40 50 - + - + c * PKA I(pA)ChangewithR1addition(%) Figure 1 Effect of R1 on CFTR-DR/S660A and wild-type CFTR. Login to comment
144 a, b, Time course of Cl- current in excised, inside-out patches containing many CFTR-DR/S660A channels during addition of PKA and R1 (50 nM), as indicated by bars. Login to comment
148 c, Effect of R1 in the presence and absence of PKA on current measured from CFTR-DR/S660A and wild-type CFTR. Login to comment
152 For CFTR-DR/S660A, n is 10 and 9; for wild-type CFTR, n is 4 and 5, in the absence or presence of PKA, respectively. Login to comment
156 To find out how the phosphorylated R domain stimulates CFTR-DR/S660A, we investigated channel gating. Login to comment
158 Figure 2 shows that phosphorylated R1 increases the rate at which CFTR-DR/S660A opened into bursts, without affecting the rate of closure from bursts. Login to comment
166 Thus addition of the phosphorylated R domain to CFTR-DR/ S660A and mutation of phosphorylation sites in wild-type CFTR had opposite effects on the same kinetic step in channel gating, the rate of channel opening. Login to comment
170 Figure 4 shows that the mutations reduced Po at low concentrations of ATP; Po was letters to nature NATURE |VOL 389 |18 SEPTEMBER 1997 295 Figure 2 Effect of R1 on CFTR-DR/S660A channels in the presence of PKA. Login to comment
172 b, c, Effect of R1 on the rates of entry into and exit from a burst for CFTR-DR/S660A channels in the presence of PKA. Login to comment
185 Number of experiments for b/c and d were: 17/6 for wild-type (WT), 8 for wild-type (-PKA), 8/7 for S660A, 5/5 for S737A, 8/6 for S795A, 9/7 for S813A, and 11/4 for S-Quad-A. Login to comment
188 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Po 0 0.2 0.4 0.6 0.8 1 10 [ATP] (mM) S813A S795A S737A S660A WT (-PKA) WT Figure 4 Effect of ATP concentration on Po of CFTR containing phosphorylation site mutations. Login to comment
193 This might also be true for CFTR-DR/S660A. Login to comment
200 When phosphorylation modifies the R domain, it might have two effects: the first could be permissive, releasing a steric inhibition, consistent with our finding that deletion of the R domain produces a channel that is partially active; the second effect might be stimulatory, facilitating interaction of the NBDs with ATP, which would be consistent with results we obtained when a phosphorylated R domain was added to CFTR-DR/S660A and with the effects of CFTR-containing mutations in the endogenous R domain. Login to comment
126 To investigate how phosphorylation controls activity, we studied channel gating and found that phosphorylation of the R domain increases the rate of channel opening by enhancing the sensitivity to ATP. Our results indicate that CFTR is regulated by a new mechanism in which phosphorylation of one domain stimulates the interaction of ATP with another domain, thereby increasing activity. We previously studied a CFTR variant, CFTR-DR/S660A, in which much of the R domain (residues 708-835) was deleted and the phosphorylation site was removed at Ser 660 by mutation to alanine4 . Login to comment
130 However, when we examined the single-channel properties of CFTR-DR/ S660A, we found that Po was 0:13 6 0:01 (1 mM ATP, n &#bc; 7), much lower than that of phosphorylated wild-type CFTR (Po &#bc; 0:46 6 0:02; 1 mM ATP, n &#bc; 17, P , 0:001). Login to comment
133 To test this idea, we added a recombinant R domain (R1, residues 645-834, similar to a peptide reported previously10 ) to the cytosolic surface of CFTR-DR/S660A and measured activity by using the excised, inside-out patch-clamp technique. Login to comment
137 Additional evidence that the effect of phosphorylation was on R1 rather than on CFTR-DR/S660A came from our finding that addition of phosphorylated R1 in the presence of protein-kinase-inhibitor peptide under conditions that block the effect of PKA11 gave similar results (43:2 6 3:4% increase; n &#bc; 3). Login to comment

[hide] CFTR activation: additive effects of stimulatory a... Am J Physiol. 1997 Jul;273(1 Pt 1):L127-33. Wilkinson DJ, Strong TV, Mansoura MK, Wood DL, Smith SS, Collins FS, Dawson DC
CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain.
Am J Physiol. 1997 Jul;273(1 Pt 1):L127-33., [PMID:9252549]

Abstract [show]
To investigate the functional significance of individual consensus phosphorylation sites within the R domain of cystic fibrosis transmembrane conductance regulator (CFTR), serines were eliminated by substituting them with alanine. Included in this analysis were serine-660, -670, -686, -700, -712, -737, -768, -795, and -813, which lie within protein kinase A consensus sequences, and serine-641, which does not. Elimination of single potential phosphorylation sites altered the sensitivity of CFTR (expressed in Xenopus oocytes) to activating conditions in a manner that was highly site dependent. Substitution at serine-660, -670, -700, -795, or -813 significantly increased the half-maximal activation constant (KA) for activation by 3-isobutyl-1-methylxanthine, which is consistent with the hypothesis that phosphorylation at any of these sites promotes CFTR activation. The effect of substitution at serine-813 was significantly greater than at the other sites. In contrast, alanine substitution at serine-737 or -768 actually decreased the KA for activation, suggesting that phosphorylation at either of these sites is inhibitory. Substitution at serine-641, -686, and -712 had no significant effect on activation sensitivity. The effects of multiple serine to alanine substitutions were consistent with the notion that phosphorylation at individual sites produced roughly additive effects, suggesting that the effect produced by phosphorylation of any one serine was not dependent on the phosphorylation state of other serines. These results are consistent with the notion that, although none of the phosphorylation sites studied here are absolutely necessary for activation of CFTR, individual sites contribute differently to the gating of the channel.

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No. Sentence Comment
85 Activation constants for wild-type and single serine-to-alanine mutant CFTRs PICA Phosphorylation CFTR K4, n-m n Class In vivo In vitro Wild type 0.65 t 0.08 26 S64lA 0.6520.12 14 N No concensus S660A 1.21+ 0.19* 11 S ++ ++ S670A 0.99t0.12* 12 s ? Login to comment
107 Symbols show averaged IBMX dose-response data for wild-type CFTR and several single-site serine-to-alanine mutants (S660A, S737A, S768A, S795A, and S813A). Login to comment
189 Gadsby and Nairn (10) called attention to the fact that the open probability of constructs like CFTRAR-S660A is well below that of wild-type channels. Login to comment

[hide] Phosphorylation-dependent block of cystic fibrosis... J Biol Chem. 1996 Mar 29;271(13):7351-6. Ma J, Tasch JE, Tao T, Zhao J, Xie J, Drumm ML, Davis PB
Phosphorylation-dependent block of cystic fibrosis transmembrane conductance regulator chloride channel by exogenous R domain protein.
J Biol Chem. 1996 Mar 29;271(13):7351-6., [PMID:8631756]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) constitutes a linear conductance chloride channel, which is regulated by cAMP-dependent protein kinase phosphorylation at multiple sites located in the intracellular regulatory (R) domain. Studies in a lipid bilayer system, reported here, provide evidence for the control of CFTR chloride channel by its R domain. The exogenous R domain protein (encoded by exon 13 plus 85 base pairs of exon 14) interacted specifically with the CFTR molecule and inhibited the chloride conductance in a phosphorylation-dependent manner. Only the unphosphorylated R domain protein blocked the CFTR channel. Such functional interaction suggests that the putative gating particle of the CFTR chloride channel resides in the R domain.

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No. Sentence Comment
159 However, CFTR⌬R-S660A, a molecule with amino acids 708-835 deleted (to render it constitutively open) with the remaining consensus PKA site mutated, is insensitive to activation by PKA (9), suggesting that activation by phosphorylation takes place in the R domain. Login to comment

[hide] Phosphate stimulates CFTR Cl- channels. Biophys J. 1994 Nov;67(5):1867-75. Carson MR, Travis SM, Winter MC, Sheppard DN, Welsh MJ
Phosphate stimulates CFTR Cl- channels.
Biophys J. 1994 Nov;67(5):1867-75., [PMID:7532021]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels appear to be regulated by hydrolysis of ATP and are inhibited by a product of hydrolysis, ADP. We assessed the effect of the other product of hydrolysis, inorganic phosphate (P(i)), on CFTR Cl- channel activity using the excised inside-out configuration of the patch-clamp technique. Millimolar concentrations of P(i) caused a dose-dependent stimulation of CFTR Cl- channel activity. Single-channel analysis demonstrated that the increase in macroscopic current was due to an increase in single-channel open-state probability (po) and not single-channel conductance. Kinetic modeling of the effect of P(i) using a linear three-state model indicated that the effect on po was predominantly the result of an increase in the rate at which the channel passed from the long closed state to the bursting state. P(i) also potentiated activity of channels studied in the presence of 10 mM ATP and stimulated Cl- currents in CFTR mutants lacking much of the R domain. Binding studies with a photoactivatable ATP analog indicated that Pi decreased the amount of bound nucleotide. These results suggest that P(i) increased CFTR Cl- channel activity by stimulating a rate-limiting step in channel opening that may occur by an interaction of P(i) at one or both nucleotide-binding domains.

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No. Sentence Comment
187 To determine whether the R domain is necessary for Pi stimulation of CFITR Cl- current, we examined two mutant CFTRs; CFTRAR and CFTRAR-S660A. Login to comment
189 In CFTRAR-S660A, amino acids 708-835 are deleted and serine 660 (a PKA phosphorylation site) is mutated to alanine. Login to comment
191 Stimulation was not dependent on the presence of serine 660, because we observed similar stimulation in the mutant CFTRAR-S660A (n = 6, not shown). Login to comment
186 To determine whether the R domain is necessary for Pi stimulation of CFITR Cl-current, we examined two mutant CFTRs; CFTRAR and CFTRAR-S660A. Login to comment
188 In CFTRAR-S660A, amino acids 708-835 are deleted and serine 660 (a PKA phosphorylation site) is mutated to alanine. Login to comment
190 Stimulation was not dependent on the presence of serine 660, because we observed similar stimulation in the mutant CFTRAR-S660A (n = 6, not shown). Login to comment

[hide] Regulation of the cystic fibrosis transmembrane co... J Biol Chem. 1993 Sep 25;268(27):20259-67. Rich DP, Berger HA, Cheng SH, Travis SM, Saxena M, Smith AE, Welsh MJ
Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain.
J Biol Chem. 1993 Sep 25;268(27):20259-67., [PMID:7690753]

Abstract [show]
Phosphorylation by cAMP-dependent protein kinase (PKA) regulates the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We previously showed that in vivo PKA phosphorylated 4 serines (Ser-660, Ser-737, Ser-795, and Ser-813) within the R domain. Here we show that a mutant CFTR lacking all 4 serines can still be phosphorylated by PKA to yield an activated Cl- channel, but channel open-state probability was substantially reduced. We also observed phosphorylation and Cl- channel activity in another mutant lacking all 8 consensus PKA serines in the R domain. We were unable to identify the residual phosphorylation sites by tryptic phosphopeptide mapping. These data suggest two possible interpretations: (a) additional, as yet unidentified, phosphorylation sites within CFTR may also open the channel, or (b) the 4 serines, previously identified as in vivo PKA phosphorylation sites, are the primary regulatory sites within CFTR, but in their absence, other sites can be phosphorylated to open the channel. The additional sites are likely located within the R domain: CFTR delta R-S660A, which lacks much of the R domain (residues 708-835) and replaces Ser-660 with an alanine, was no longer regulated by PKA. Substitution of aspartate for consensus PKA phosphorylation sites in the R domain mimicked the effect of phosphorylation. Mutants containing six or more serine-to-aspartate substitutions generated Cl- channels that opened without PKA phosphorylation. These results suggest that the R domain keeps the channel closed and that phosphorylation of the R domain or insertion of the negatively charged aspartate opens the channel, perhaps by electrostatic interactions.

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No. Sentence Comment
8 The additional sites are likelylocated within the R domain: CFTRAR-S660A, which lacksmuch of the R domain (residues 708-835)and replaces Ser-660 withan alanine,was no longer regulated byPKA. Login to comment
66 In vivo phosphorylation ofwild-typeand mutant CFTR CFTR S-Quad-A (S66OA,S737A,S795A,S813A), or CFTR S-Oct-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) weretran- siently expressed in HeLa cells. Login to comment
94 Single-channelanalysis of gle-channel traces from an excised, in-cell expressing CFTR S-Quad-A side-out membrane patch from a HeLa (S660A,S737A,S795A,S813A).Each trace is about 14s long. Login to comment
109 RESULTS Serine-to-Alanine Substitutionsin the R Domain Do NotAbolish CFTR Cl- Channel Activity-& a firststep to address the possibility that additional PKA phosphorylation sites might regulate CFTR, we asked whether the S-Quad-A (S660A,S737A,S795A,S813A) mutant could still bephosphorylated in vivofollowing CAMPstimulation. Login to comment
124 CFTR S-Oct-A (S660A,S686A,S700A, CFTR A 795 737 4+B5 -813 B," -02 -B3 sion was high, we expected the mutant channels to be less active than wild-type CFTR C1-channels. Login to comment
139 CFTR (A) or CFTR S-Quad-A (S660A,S737A,S795A,S813A)( B )were transiently expressed inCOS-7 cells. Login to comment
161 CFTRAR-S660A is similar toCFTRAR except that Ser-660, the sole remaining in vivo PKA phosphorylation site (13), is mutatedto alanine. Login to comment
166 CFTRAR-S660A C1-channels had biophysical properties similar towild-type CFTR C1-channels: single-channel slope conductance was 9.2 *0.4 pS (n = 3).The poof CFTRARS660ACl- channels was0.15 *0.04 (n= 6) in the presence ofATPalone and did not increasefollowingaddition of PKA (n = 4) (Fig. lOB). Login to comment
174 Fig. 1lA shows that simultaneous substitutionof the four in vivo PKA sites with aspartates (in the S-Quad-D mutant) did not generate constitutively active CFTR C1-channels as assessed by the SPQ fluorescence assay: the mutant channels opened only after stimulation by CAMP.We observed similar results withCFTR S-Quint-D which contained substitutionsof A MockS-Hept-A C S-OCt-A transfected (A) or were transfected with pMT-CFTR S-Hept-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A) (B)or pMT-CFl`R S-Oct-A FIG.9. Login to comment
176 COS-7 cells were mock- (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A)(C). Login to comment
232 CFTRAR-S660A contains only one "nonclassic" (R-X-X-Sor R-X-S) PKA phosphorylation site (Ser-753)in thedeleted segment; its consensus sequence is R-I-S.Alternatively, if PKAphosphorylated "nonclassic"sites within other partsof CFTR which interacted with theR domain, then deletion of part of the R domainwould disrupt those interactions whether or not the additional sites were phosphorylated. Login to comment
234 Finally, we cannot rule out thepossi- bility that PKA simply cannot interact with CFTRAR-S660A, even though it does interact withCFTRAR (14). Login to comment
7 The additional sites are likelylocated within the R domain: CFTRAR-S660A, which lacksmuch of the R domain (residues 708-835)and replaces Ser-660 with an alanine,was no longer regulated byPKA. Login to comment
65 In vivo phosphorylation ofwild-typeand mutant CFTR CFTR S-Quad-A (S66OA,S737A,S795A,S813A), or CFTR S-Oct-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) were transiently expressed in HeLa cells. Login to comment
95 Single-channelanalysis of gle-channel traces from an excised, in-cell expressing CFTR S-Quad-A side-out membrane patch from a HeLa (S660A,S737A,S795A,S813A).Each trace is about 14s long. Login to comment
111 RESULTS Serine-to-Alanine Substitutions in the R Domain Do NotAbolish CFTR Cl-Channel Activity-& a firststep to address the possibility that additional PKA phosphorylation sites might regulate CFTR, we asked whether the S-Quad-A (S660A,S737A,S795A,S813A) mutant could still be phosphorylated in vivofollowing CAMPstimulation. Login to comment
126 CFTR S-Oct-A (S660A,S686A,S700A, CFTR A 795 737 4+B5 - 8 1 3 B," -02 -B3 sion was high, we expected the mutant channels to be less active than wild-type CFTR C1-channels. Login to comment
142 CFTR (A) or CFTR S-Quad-A (S660A,S737A,S795A,S813A) ( B )were transiently expressed inCOS-7 cells. Login to comment
165 CFTRAR-S660A is similar toCFTRAR except that Ser-660, the sole remaining in vivo PKA phosphorylation site (13), is mutatedto alanine. Login to comment
170 CFTRAR-S660A C1-channels had biophysical properties similar towild-type CFTR C1-channels: single-channel slope conductance was 9.2 *0.4 pS (n = 3).The poof CFTRARS660ACl- channels was 0.15 *0.04 (n= 6) in the presence ofATPalone and did not increasefollowingaddition of PKA (n = 4) (Fig. lOB). Login to comment
178 Fig. 1lA shows that simultaneous substitution of the four in vivo PKA sites with aspartates (in the S-Quad-D mutant) did not generate constitutively active CFTR C1-channels as assessed by the SPQ fluorescence assay: the mutant channels opened only after stimulation by CAMP.We observed similar results withCFTR S-Quint-D which contained substitutionsof A Mock S-Hept-A C S-OCt-A transfected (A) or were transfected with pMT-CFTR S-Hept-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A) (B) or pMT-CFl`R S-Oct-A FIG. 9. Login to comment
180 COS-7 cells were mock- (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) (C). Login to comment
235 CFTRAR-S660A contains only one "nonclassic" (R-X-X-Sor R-X-S) PKA phosphorylation site (Ser-753)in thedeleted segment; its consensus sequence is R-I-S.Alternatively, if PKAphosphorylated "nonclassic"sites within other parts of CFTR which interacted with theR domain, then deletion of part of the R domainwould disrupt those interactions whether or not the additional sites were phosphorylated. Login to comment
237 Finally, we cannot rule out thepossibility that PKA simply cannot interact with CFTRAR-S660A, even though it does interact withCFTRAR (14). Login to comment

[hide] Protein kinase A (PKA) still activates CFTR chlori... J Biol Chem. 1993 May 25;268(15):11304-11. Chang XB, Tabcharani JA, Hou YX, Jensen TJ, Kartner N, Alon N, Hanrahan JW, Riordan JR
Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites.
J Biol Chem. 1993 May 25;268(15):11304-11., [PMID:7684377]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in transepithelial ion transport by acting as a tightly regulated apical chloride channel. Regulation is achieved by the concerted action of ATP at conserved nucleotide binding folds and serine phosphorylation at multiple sites by protein kinases A (PKA) and C (PKC). A previous investigation concluded that activation by PKA is critically dependent on phosphorylation at four of the nine predicted PKA sites in the R domain (S660A, S737A, S795A, S813A), because a "Quad" mutant lacking these sites could not be activated. We show in the present work that not only can this mutant be phosphorylated and activated, but a mutant in which all 10 predicted PKA sites have been altered still retains significant PKA-activated function. Potentiation of the PKA response by PKC is also preserved in this mutant. Thus CFTR may be regulated by cryptic PKA sites which also mediate interactions between different kinases. Such hierarchical phosphorylation of CFTR by obvious and cryptic PKA sites could provide a metered response to secretagogues.

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No. Sentence Comment
7 A previous investigationconcluded thatactivationby PKA is crit- icallydependent on phosphorylation at four of the nine predicted PKA sites in the R domain (S660A, S737A, S795A,SS13A), becausea "Quad"mutant lacking these sites couldnotbeactivated. Login to comment
37 The following mutations were introduced into CFTR, S422A (TCT to GCT), S660A (TCA to GCA), S686A (TCT to GCT), S700A (TCT toGCT), S712A (TCC to GCC), S737A (TCC to GCC), S768A (TCT toGCT), T788A (ACAto GCA),S795A (TCA to GCA), S813A (TCA to GCA), S660E (TCA to GAA),S737E (TCC to GAG), S795E (TCA to GAA), and S813E (TCA to GAA). Login to comment
39 The counterparts of CFTR cDNA in pUCF2.5 were replaced by PCR- mutated versions by interchange of the following fragments, S660A, DraIIIIEcoRI fragment, S737A, EcoRIIHpaIfragment, S795A and S813A,StyIIStyI fragment(Fig. 1A). Login to comment
40 A mutant containing S660/737/ 795/813A (pUCF2.5/4SA) was assembled by replacing the counterparts of a plasmid containing S795/813A with the DraIII/EcoRI fragment (S660A) and EcoRI/HpaI fragment (S737A). Login to comment
69 For example, S660A means that serine residue 660 was changed to alanine. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Neuron. 1992 May;8(5):821-9. Welsh MJ, Anderson MP, Rich DP, Berger HA, Denning GM, Ostedgaard LS, Sheppard DN, Cheng SH, Gregory RJ, Smith AE
Cystic fibrosis transmembrane conductance regulator: a chloride channel with novel regulation.
Neuron. 1992 May;8(5):821-9., [PMID:1375035]

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136 This point is illustrated by consideration of two mutants: in S737/795/813A, phosphorylation of Ser-660 ap- Review: The CFTR Cl-Channel pears to be sufficient to open the channel; yet in S660A, which lacks Ser-660 but retains Ser-737, -795, and -813, CAMP activates the channel. Login to comment
143 However, CFTRAR retains Ser-660, and when a combined mutant, CFTRAR-S660A, was created, only constitutive channel activity was found; CAMP no longer caused a further increase in activity (Cheng et al., 1991). Login to comment
206 Similar results were obtained with CFTRAR-S660A channels (unpublished data). Login to comment

[hide] Role of tyrosine phosphorylation in the muscarinic... J Biol Chem. 2013 Jul 26;288(30):21815-23. doi: 10.1074/jbc.M113.479360. Epub 2013 Jun 11. Billet A, Luo Y, Balghi H, Hanrahan JW
Role of tyrosine phosphorylation in the muscarinic activation of the cystic fibrosis transmembrane conductance regulator (CFTR).
J Biol Chem. 2013 Jul 26;288(30):21815-23. doi: 10.1074/jbc.M113.479360. Epub 2013 Jun 11., [PMID:23760269]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl(-)) channel, which plays an important role in physiological anion and fluid secretion, and is defective in several diseases. Although its activation by PKA and PKC has been studied extensively, its regulation by receptors is less well understood. To study signaling involved in CFTR activation, we measured whole-cell Cl(-) currents in BHK cells cotransfected with GPCRs and CFTR. In cells expressing the M3 muscarinic acetylcholine receptor, the agonist carbachol (Cch) caused strong activation of CFTR through two pathways; the canonical PKA-dependent mechanism and a second mechanism that involves tyrosine phosphorylation. The role of PKA was suggested by partial inhibition of cholinergic stimulation by the specific PKA inhibitor Rp-cAMPS. The role of tyrosine kinases was suggested by Cch stimulation of 15SA-CFTR and 9CA-CFTR, mutants that lack 15 PKA or 9 PKC consensus sequences and are unresponsive to PKA or PKC stimulation, respectively. Moreover the residual Cch response was sensitive to inhibitors of the Pyk2 and Src tyrosine kinase family. Our results suggest that tyrosine phosphorylation acts on CFTR directly and through inhibition of the phosphatase PP2A. Results suggest that PKA and tyrosine kinases contribute to CFTR regulation by GPCRs that are expressed at the apical membrane of intestinal and airway epithelia.

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102 Carbachol Stimulates CFTR through PKA and Non-PKA Signaling Pathways-To explore PKA-independent regulation of CFTR without using inhibitors that might have confounding effects on other pathways, we studied the activation of 15SA-CFTR (S422A/S660A/S670A/S686A/T690A/S700A/S712A/ S737A/S753A/S768A/T787A/T788A/S790A/S795A/S813A). Login to comment