The cystic fibrosis mutation G551D alters the non-Michaelis-Menten behavior of the cystic fibrosis transmembrane conductance regulator (CFTR) channel and abolishes the inhibitory Genistein binding site

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) channel activity explains most of the manifestations of the cystic fibrosis (CF) disease. To understand the consequences of CF mutations on CFTR channel activity, we compared the pharmacological properties of wild-type (wt) and G551D-CFTR. Dose-dependent relationships of wt-CFTR activated by genistein follows a non-Michaelis-Menten behavior consistent with the presence of two binding sites. With phosphorylated CFTR, a high affinity site for genistein is the activator (K(s) approximately 3 microm), whereas a second site of low affinity (K(i) approximately 75 microm) is the inhibitor. With non-phosphorylated CFTR, K(s) was increased (K(s) approximately 12 microm), but K(i) was not affected (K(i) approximately 70 microm). In G551D-CFTR cells, channel activity was recovered by co-application of forskolin and genistein in a dose-dependent manner. A further stimulation of G551D-CFTR channel activity was measured at concentrations from 30 microm to 1 mm. The dose response is described by a classical Michaelis-Menten kinetics with only a single apparent site (K(m) approximately 11 microm). Our results suggest glycine 551 in NBD1 as an important location within the low affinity inhibitory site for genistein and offers new evidence for pharmacological alteration caused by an NBD1 mutation of CFTR. This study also reveals how a mutation of an ion channel converts a non-Michaelis-Menten behavior (two binding sites) into a classical Michaelis-Menten model (one binding site).     

G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects

Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) result in cystic fibrosis (CF). CFTR is a chloride channel that is regulated by phosphorylation and gated by ATP binding and hydrolysis at its nucleotide binding domains (NBDs). G551D-CFTR, the third most common CF-associated mutation, has been characterized as having a lower open probability (Po) than wild-type (WT) channels. Patients carrying the G551D mutation present a severe clinical phenotype. On the other hand, G1349D, also a mutant with gating dysfunction, is associated with a milder clinical phenotype. Residues G551 and G1349 are located at equivalent positions in the highly conserved signature sequence of each NBD. The physiological importance of these residues lies in the fact that the signature sequence of one NBD and the Walker A and B motifs from the other NBD form the ATP-binding pocket (ABP1 and ABP2, named after the location of the Walker A motif) once the two NBDs dimerize. Our studies show distinct gating characteristics for these mutants. The G551D mutation completely eliminates the ability of ATP to increase the channel activity, and the observed activity is approximately 100-fold smaller than WT-CFTR. G551D-CFTR does not respond to ADP, AMP-PNP, or changes in [Mg(2+)]. The low activity of G551D-CFTR likely represents the rare ATP-independent gating events seen with WT channels long after the removal of ATP. G1349D-CFTR maintains ATP dependence, albeit with a Po approximately 10-fold lower than WT. Interestingly, compared to WT results, the ATP dose-response relationship of G1349D-CFTR is less steep and shows a higher apparent affinity for ATP. G1349D data could be well described by a gating model that predicts that binding of ATP at ABP1 hinders channel opening. Thus, our data provide a quantitative explanation at the single-channel level for different phenotypes presented by patients carrying these two mutations. In addition, these results support the idea that CFTR's two ABPs play distinct functional roles in gating.     

The glycine residues G551 and G1349 within the ATP-binding cassette signature motifs play critical roles in the activation and inhibition of cystic fibrosis transmembrane conductance regulator channels by phloxine B

The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (Ks = 3.2 +/- 1.6 microM: , Ki = 38 +/- 1.4 microM: ) and delF508 CFTR (Ks = 3 +/- 1.8 microM: , Ki = 33 +/- 1 microM: ). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (Ks = 2 +/- 1.13 microM: ) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (Ki = 40 +/- 1.01 microM: ) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR.     

Direct interaction of a small-molecule modulator with G551D-CFTR, a cystic fibrosis-causing mutation associated with severe disease

CF (cystic fibrosis) is caused by mutations in CFTR (CF transmembrane conductance regulator), which cause its mistrafficking and/or dysfunction as a regulated chloride channel on the apical surface of epithelia. CFTR is a member of the ABC (ATP-binding-cassette) superfamily of membrane proteins and a disease-causing missense mutation within the ABC signature sequence; G551D-CFTR exhibits defective phosphorylation and ATP-dependent channel gating. Studies of the purified and reconstituted G551D-CFTR protein revealed that faulty gating is associated with defective ATP binding and ATPase activity, reflecting the key role of G551 in these functions. Recently, high-throughput screens of chemical libraries led to identification of modulators that enhance channel activity of G551D-CFTR. However, the molecular target(s) for these modulators and their mechanism of action remain unclear. In the present study, we evaluated the mechanism of action of one small-molecule modulator, VRT-532, identified as a specific modulator of CF-causing mutants. First, we confirmed that VRT-532 causes a significant increase in channel activity of G551D-CFTR using a novel assay of CFTR function in inside-out membrane vesicles. Biochemical studies of purified and reconstituted G551D-CFTR revealed that potentiation of the ATPase activity of VRT-532 is mediated by enhancing the affinity of the mutant for ATP. Interestingly, VRT-532 did not affect the ATPase activity of the Wt (wild-type) CFTR, supporting the idea that this compound corrects the specific molecular defect in this mutant. To summarize, these studies provide direct evidence that this compound binds to G551D-CFTR to rescue its specific defect in ATP binding and hydrolysis.     

Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel gated by ATP binding and hydrolysis at its nucleotide binding domains (NBD). The NBDs dimerize in a head-to-tail configuration, forming two ATP binding pockets (ABP) with the ATP molecules buried at the dimer interface. Previous studies have indicated that ABP2, formed by the Walker A and B motifs of NBD2 and the signature sequence of NBD1, is the site critical for the ATP-dependent opening of CFTR. The G551D mutation in ABP2, the third most common cystic fibrosis-associated mutation, abolishes ATP-dependent gating, resulting in an open probability that is approximately 100-fold lower than that of wild-type channels. Interestingly, we found that the ATP analog N6-(2-phenylethyl)-ATP (P-ATP) increases G551D currents mainly by increasing the open time of the channel. This effect is reduced when P-ATP is applied together with ATP, suggesting a competition between ATP and P-ATP for a common binding site. Introducing mutations that lower the nucleotide binding affinity at ABP2 did not alter significantly the effects of P-ATP on G551D-CFTR, whereas an equivalent mutation at ABP1 (consisting of the Walker A and B motifs of NBD1 and the signature sequence of NBD2) dramatically decreased the potency of P-ATP, indicating that ABP1 is the site where P-ATP binds to increase the activity of G551D-CFTR. These results substantiate the idea that nucleotide binding at ABP1 stabilizes the open channel conformation. Our observation that P-ATP enhances the G551D activity by binding at ABP1 implicates that ABP1 can potentially be a target for drugs to bind and increase the channel activity.     

The Glycine Residues G551 and G1349 within the ATP-Binding Cassette Signature Motifs Play Critical Roles in the Activation and Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Channels by Phloxine B

The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (K_s = 3.2 ± 1.6 μM, K_i = 38 ± 1.4 μM) and delF508 CFTR (K_s = 3 ± 1.8 μM, K_i = 33 ± 1 μM). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (K_s = 2 ± 1.13 μM) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (K_i = 40 ± 1.01 μM) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR.     

Genistein improves regulatory interactions between G551D-cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) in addition to its well defined Cl(-) channel properties regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and non-epithelial cells, whereas the presence of ENaC increases CFTR functional expression. This interregulation is reproduced in Xenopus oocytes where both the open probability and surface expression of wild type CFTR Cl(-) channels are increased when CFTR is co-expressed with alphabetagamma-mouse ENaC (mENaC) and conversely when the activity of mENaC is inhibited after wild type CFTR activation. Using the Xenopus oocyte expression system, different functional regulatory interactions were observed between G551D-CFTR and alphabetagamma-mENaC. The co-expression of G551D-CFTR and alphabetagamma-mENaC resulted in a 5-fold increase in G551D-CFTR Cl(-) current compared with oocytes expressing G551D-CFTR alone. Oocytes co-injected with both G551D-CFTR and ENaC expressed an amiloride-sensitive whole cell current that was similar to that observed before and after G551D-CFTR activation with forskolin/isobutylmethylxanthine. Treatment with genistein both enhanced the functional expression of G551D-CFTR and improved regulatory interactions between G551D-CFTR and ENaC. These data suggest that genistein may be useful in patients with cystic fibrosis and the G551D-CFTR mutation.     

Proteomic identification of calumenin as a G551D-CFTR associated protein

Cystic fibrosis (CF) is the most common lethal autosomal recessive disease in the Caucasian population. It is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To date, over 1910 mutations have been identified in the CFTR gene. Among these mutations, the CF-causing missense mutation G551D-CFTR (approx. 5% of cases) encodes for a CFTR chloride channel with normal expression on the cell surface. Nevertheless, it is associated with severe disease due to its altered channel activation. The aim of the present study was to identify specific interacting proteins of G551D-CFTR. Co-immunoprecipitated proteins with G551D-CFTR were resolved by 2D-gel electrophoresis (2-DE). Mass Spectrometry revealed that calumenin was present in the protein complex linked to G551D-CFTR. Despite its basal expression was not modified in G551D-CFTR expressing cells when compared to Wt-CFTR expressing cells, it was more abundant in the G551D-CFTR complex detected by immunoprecipitation. The calumenin-CFTR interaction was also shown by Surface Plasmon Resonance and further confirmed by computational analysis of the predicted calumenin's partners. Because in our cellular model calumenin was found in the endoplasmic reticulum (ER) by immunofluorescence experiments, we suggest that calumenin is likely involved in the mutated CFTR's maturation. In conclusion, we showed for the first time that calumenin binds to CFTR and that it is increased in the G551D-CFTR complex. We suggest that it may be involved in the physiopathology of G551D-CFTR and that G551D-CFTR may follow a specific maturation and trafficking pathway. We also hypothesize that UPR may be triggered independently of the retention of G551D-CFTR in the ER because Grp78/Bip expression is increased in the cells. Finally, we propose here that Calumenin is a new CFTR chaperone.     

Potentiation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents by the chemical solvent tetrahydrofuran

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.     

Dual activity of aminoarylthiazoles on the trafficking and gating defects of the cystic fibrosis transmembrane conductance regulator chloride channel caused by cystic fibrosis mutations

A large fraction of mutations causing cystic fibrosis impair the function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel by causing reduced channel activity (gating defect) and/or impaired exit from the endoplasmic reticulum (trafficking defect). Such defects need to be treated with separate pharmacological compounds termed potentiators and correctors, respectively. Here, we report the characterization of aminoarylthiazoles (AATs) as compounds having dual activity. Cells expressing mutant CFTR were studied with functional assays (fluorescence-based halide transport and short circuit current measurements) to assess the effect of acute and chronic treatment with compounds. We found that AATs are effective on F508del, the most frequent cystic fibrosis mutation, which is associated with both a gating and a trafficking defect. AATs are also effective on mutations like G1349D and G551D, which cause only a gating defect. Evaluation of a panel of AAT analogs identified EN277I as the most effective compound. Incubation of cells expressing mutant CFTR with EN277I caused a strong stimulation of channel activity as demonstrated by single channel recordings. Compounds with dual activity such as AATs may be useful for the development of effective drugs for the treatment of cystic fibrosis.     

Phloxine B interacts with the cystic fibrosis transmembrane conductance regulator at multiple sites to modulate channel activity

The fluorescein derivative phloxine B is a potent modulator of the cystic fibrosis transmembrane conductance regulator (CFTR). Low micromolar concentrations of phloxine B stimulate CFTR Cl(-) currents, whereas higher concentrations of the drug inhibit CFTR. In this study, we investigated the mechanism of action of phloxine B. Phloxine B (1 microm) stimulated wild-type CFTR and the most common cystic fibrosis mutation, DeltaF508, by increasing the open probability of phosphorylated CFTR Cl(-) channels. At each concentration of ATP tested, the drug slowed the rate of channel closure without altering the opening rate. Based on the effects of fluorescein derivatives on transport ATPases, these data suggest that phloxine B might stimulate CFTR by binding to the ATP-binding site of the second nucleotide-binding domain (NBD2) to slow the dissociation of ATP from NBD1. Channel block by phloxine B (40 microm) was voltage-dependent, enhanced when external Cl(-) concentration was reduced and unaffected by ATP (5 mm), suggesting that phloxine B inhibits CFTR by occluding the pore. We conclude that phloxine B interacts directly with CFTR at multiple sites to modulate channel activity. It or related agents might be of value in the development of new treatments for diseases caused by the malfunction of CFTR.     

A single amino acid substitution in CFTR converts ATP to an inhibitory ligand

Cystic fibrosis (CF), one of the most common lethal genetic diseases, is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel that, when phosphorylated, is gated by ATP. The third most common pathogenic mutation, a glycine-to-aspartate mutation at position 551 or G551D, shows a significantly decreased open probability (P_o) caused by failure of the mutant channel to respond to ATP. Recently, a CFTR-targeted drug, VX-770 (Ivacaftor), which potentiates G551D-CFTR function in vitro by boosting its P_o, has been approved by the FDA to treat CF patients carrying this mutation. Here, we show that, in the presence of VX-770, G551D-CFTR becomes responsive to ATP, albeit with an unusual time course. In marked contrast to wild-type channels, which are stimulated by ATP, sudden removal of ATP in excised inside-out patches elicits an initial increase in macroscopic G551D-CFTR current followed by a slow decrease. Furthermore, decreasing [ATP] from 2 mM to 20 µM resulted in a paradoxical increase in G551D-CFTR current. These results suggest that the two ATP-binding sites in the G551D mutant mediate opposite effects on channel gating. We introduced mutations that specifically alter ATP-binding affinity in either nucleotide-binding domain (NBD1 or NBD2) into the G551D background and determined that this disease-associated mutation converts site 2, formed by the head subdomain of NBD2 and the tail subdomain of NBD1, into an inhibitory site, whereas site 1 remains stimulatory. G551E, but not G551K or G551S, exhibits a similar phenotype, indicating that electrostatic repulsion between the negatively charged side chain of aspartate and the γ-phosphate of ATP accounts for the observed mutational effects. Understanding the molecular mechanism of this gating defect lays a foundation for rational drug design for the treatment of CF.     

Optimization of the Degenerated Interfacial ATP Binding Site Improves the Function of Disease-related Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated ΔF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed.     

Comparative Pharmacology of the Activity of Wild-type and G551D Mutated CFTR Chloride Channel: Effect of the Benzimidazolone Derivative NS004

The pharmacological activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel mutated at glycine 551 (G551D-CFTR) was studied in the presence of the benzimidazolone derivative NS004 and compared to that of wild-type (wt) CFTR. Using iodide (¹²⁵I) efflux and whole-cell patch-clamp techniques we found dose-dependent stimulation of phosphorylated wt-CFTR channels by NS004 with an EC ₅₀ 11 µM. With non-phosphorylated CFTR, the effect of NS004 was apparent only at concentration >100 µM. In G551D-CFTR-expressing CHO cells, neither forskolin (from 0.1 to 10 µM) nor NS004 (from 0.1 to 200 µM) added separately were able to stimulate channel activity. However, in the presence of 10 µM forskolin, NS004 stimulated G551D-CFTR activity in a dose-dependent manner with an EC ₅₀ 1.5 µM. We also determined the half-maximal effective concentration of forskolin (EC ₅₀ 3.2 µM) required to stimulate G551D channel activity in presence of 1.5 µM NS004. No inhibitory effect was observed at high concentration of NS004 with both wt- and G551D-CFTR. Whole-cell recordings of CFTR chloride currents from cells expressing wild-type or G551D-CFTR in the presence of NS004 were linear, time- and voltage-independent. The inhibitory profile of G551D-CFTR channel activity was similar to that of wild type, i.e., inhibition by glibenclamide (100 µM) and DPC (250 µM) but not by DIDS (200 µM) nor calixarene (100 nM). These results show that NS004 activates wt-CFTR channel and restores G551D-CFTR channel activity, the potency of which depends on both the concentration of NS004 and the phosphorylation status of CFTR.     

Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein

The patch-clamp technique was used to investigate the effects ofthe isoflavone genistein on disease-causing mutations (G551D andF508) of the cystic fibrosis transmembrane conductance regulator (CFTR). In HeLa cells recombinantly expressing thetrafficking-competent G551D-CFTR, the forskolin-stimulated Cl currentswere small, and average open probability of G551D-CFTR wasP_o = 0.047 ± 0.019. Addition of genistein activated Cl currents~10-fold, and the P_o of G551D-CFTRincreased to 0.49 ± 0.12, which is aP_o similar towild-type CFTR. In cystic fibrosis (CF) epithelial cells homozygous forthe trafficking-impaired F508 mutation, forskolin and genistein activated Cl currents only after 4-phenylbutyrate treatment. These datasuggested that genistein activated CFTR mutants that were present inthe cell membrane. Therefore, we tested the effects of genistein in CFpatients with the G551D mutation in nasal potential difference (PD)measurements in vivo. The perfusion of the nasal mucosa of G551D CFpatients with isoproterenol had no effect; however, genisteinstimulated Cl-dependent nasal PD by, on average, 2.4 ± 0.6 mV, which corresponds to 16.9% of the responses (to -adrenergicstimulation) found in healthy subjects.

     

Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia

Cystic fibrosis (CF) is caused by the functional expression defect of the CF transmembrane conductance regulator (CFTR) chloride channel at the apical plasma membrane. Impaired bacterial clearance and hyperactive innate immune response are hallmarks of the CF lung disease, yet the existence of and mechanism accounting for the innate immune defect that occurs before infection remain controversial. Inducible expression of either CFTR or the calcium-activated chloride channel TMEM16A attenuated the proinflammatory cytokines interleukin-6 (IL-6), IL-8, and CXCL1/2 in two human respiratory epithelial models under air–liquid but not liquid–liquid interface culture. Expression of wild-type but not the inactive G551D-CFTR indicates that secretion of the chemoattractant IL-8 is inversely proportional to CFTR channel activity in cftr^{∆F508/∆F508} immortalized and primary human bronchial epithelia. Similarly, direct but not P2Y receptor–mediated activation of TMEM16A attenuates IL-8 secretion in respiratory epithelia. Thus augmented proinflammatory cytokine secretion caused by defective anion transport at the apical membrane may contribute to the excessive and persistent lung inflammation in CF and perhaps in other respiratory diseases associated with documented down-regulation of CFTR (e.g., chronic obstructive pulmonary disease). Direct pharmacological activation of TMEM16A offers a potential therapeutic strategy to reduce the inflammation of CF airway epithelia.     

Benzoflavone activators of the cystic fibrosis transmembrane conductance regulator: towards a pharmacophore model for the nucleotide-binding domain

Our previous screen of flavones and related heterocycles for the ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel indicated that UCCF-029, a 7,8-benzoflavone, was a potent activator. In the present study, we describe the synthesis and evaluation, using cell-based assays, of a series of benzoflavone analogues to examine structure-activity relationships and to identify compounds having greater potency for activation of both wild type CFTR and a mutant CFTR (G551D-CFTR) that causes cystic fibrosis in some human subjects. Using UCCF-029 as a structural guide, a panel of 77 flavonoid analogues was prepared. Analysis of the panel in FRT cells indicated that benzannulation of the flavone A-ring at the 7,8-position greatly improved compound activity and potency for several flavonoids. Incorporation of a B-ring pyridyl nitrogen either at the 3- or 4-position also elevated CFTR activity, but the influence of this structural modification was not as uniform as the influence of benzannulation. The most potent new analogue, UCCF-339, activated wild-type CFTR with a K(d) of 1.7 microM, which is more active than the previous most potent flavonoid activator of CFTR, apigenin. Several compounds in the benzoflavone panel also activated G551D-CFTR, but none were as active as apigenin. Pharmacophore modeling suggests a common binding mode for the flavones and other known CFTR activators at one of the nucleotide-binding sites, allowing for the rational development of more potent flavone analogues.     

F508del-CFTR increases intracellular Ca(2+) signaling that causes enhanced calcium-dependent Cl(-) conductance in cystic fibrosis

In many cells, increase in intracellular calcium ([Ca(2+)](i)) activates a Ca(2+)-dependent chloride (Cl(-)) conductance (CaCC). CaCC is enhanced in cystic fibrosis (CF) epithelial cells lacking Cl(-) transport by the CF transmembrane conductance regulator (CFTR). Here, we show that in freshly isolated nasal epithelial cells of F508del-homozygous CF patients, expression of TMEM16A and bestrophin 1 was unchanged. However, calcium signaling was strongly enhanced after induction of expression of F508del-CFTR, which is unable to exit the endoplasmic reticulum (ER). Since receptor-mediated [Ca(2+)](i) increase is Cl(-) dependent, we suggested that F508del-CFTR may function as an ER chloride counter-ion channel for Ca(2+). This was confirmed by expression of the double mutant F508del/G551D-CFTR, which remained in the ER but had no effects on [Ca(2+)](i). Moreover, F508del-CFTR could serve as a scavenger for inositol-1,4,5-trisphosphate [IP3] receptor binding protein released with IP(3) (IRBIT). Our data may explain how ER-localized F508del-CFTR controls intracellular Ca(2+) signaling.     

High-affinity activators of cystic fibrosis transmembrane conductance regulator (CFTR) chloride conductance identified by high-throughput screening

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein that reduce cAMP-stimulated Cl(-) conductance in airway and other epithelia. The purpose of this investigation was to identify new classes of potent CFTR activators. A collection of 60,000 diverse drug-like compounds was screened at 10 microm together with a low concentration of forskolin (0.5 microm) in Fisher rat thyroid epithelial cells co-expressing human CFTR and a green fluorescent protein-based Cl(-) sensor. Primary screening yielded 57 strong activators (greater activity than reference compound apigenin), most of which were unrelated in chemical structure to known CFTR activators, and 284 weaker activators. Secondary analysis of the strong activators included analysis of CFTR specificity, forskolin requirement, transepithelial short-circuit current, activation kinetics, dose response, toxicity, and activation mechanism. Three compounds, the most potent being a dihydroisoquinoline, activated CFTR by elevating cellular cAMP, probably by phosphodiesterase inhibition. Fourteen compounds activated CFTR without cAMP elevation or phosphatase inhibition, suggesting direct CFTR interaction. The most potent compounds had tetrahydrocarbazol, hydroxycoumarin, and thiazolidine core structures. These compounds induced CFTR Cl(-) currents rapidly (<5 min) with K(d) down to 200 nm and were CFTR-selective, reversible, and nontoxic. Several compounds, the most potent being a trifluoromethylphenylbenzamine, activated the CF-causing mutant G551D, but with much weaker affinity (K(d) > 10 microm). When added for 10 min, none of the compounds activated DeltaPhe(508)-CFTR in transfected cells grown at 37 degrees C (with DeltaPhe(508)-CFTR trapped in the endoplasmic reticulum). However, after correction of trafficking by 48 h of growth at 27 degrees C, tetrahydrocarbazol and N-phenyltriazine derivatives strongly stimulated Cl(-) conductance with K(d) < 1 microm. The new activators identified here may be useful in defining molecular mechanisms of CFTR activation and as lead compounds in CF drug development.