Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation

Renaud Dérand1, Laurence Bulteau-Pignoux1, Yvette Mettey2, Olga Zegarra-Moran3, L. Daniel Howell4, Christoph Randak5, Luis J. V. Galietta3, Jonathan A. Cohn4, Caroline Norez1, Leila Romio3, Jean-Michel Vierfond2, Michel Joffre1, and Frédéric Becq1

1 Laboratoire de Physiologie des Régulations Cellulaires, Unité Mixte de Recherche 6558, 86022 Poitiers, and 2 Laboratoire de Chimie Organique, Faculté de Médecine et de Pharmacie, 86005 Poitiers, France; 3 Laboratorio di Genetica Molecolare, Istituto G. Gaslini, Genoa 16148, Italy; 4 Durham Veterans Affairs and Duke University Medical Center, Durham, North Carolina; and 5 Kinderklinik im Dr. von Haunerschen Kinderspital, Ludwig-Maximilians-Universität, Munich, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (EC50 = 85 µM) and activated CFTR in Calu-3 cells (EC50 = 47 µM). MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. The activation of CFTR by MPB-91 is independent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTR having 10 mutated protein kinase A sites (10SA-CFTR). The new pharmacological agent MPB-91 may be an important candidate drug to ameliorate the ion transport defect associated with CF and to point out a new pathway to modulate CFTR activity.

pharmacology; disease-causing mutation; cystic fibrosis; nucleotide binding domains; cystic fibrosis transmembrane conductance regulator


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CYSTIC FIBROSIS (CF), the most common lethal autosomal recessive genetic disease, is caused by mutations of the CF gene (25, 33), which normally encodes a multifunctional and integral apical membrane chloride channel: the CF transmembrane conductance regulator (CFTR) (25). CFTR mediates chloride transepithelial transport (7, 13, 29), and is activated by phosphorylation (4, 7, 31) and gated by ATP hydrolysis (for review, see Refs. 10 and 15). More than 900 CF mutations are known (www.genet.sickkids.on.ca), and a lot of them generate defective chloride transport and perturb the quantity and composition of epithelial fluids. This results in the manifestations of the disease, which include airway obstruction and infection, pancreatic failure, male infertility, and elevated levels of salt in sweat (reviewed in Ref. 12).

The main mutations are a deletion of the phenylalanine at position 508 (Delta F508, class II mutation), which represents ~66% of the mutated chromosomes, and a glycine-to-aspartic acid missense mutation at codon 551 (G551D, class III mutation) with a frequency of 2-5% (33). Both mutations are located in the first nucleotide binding domain (NBD1) (21), but, whereas Delta F508-CFTR is not correctly addressed toward the membrane (8, 13), the processing, maturation, and assigned apical location of G551D CFTR are not affected (30, 33). However, in cells expressing the G551D mutant, chloride transport cannot be activated by cAMP-elevating agents (1, 11, 33). Although the regulatory domain of CFTR could be normally phosphorylated (6), a decreased nucleotide binding (21) and ATPase activity at NBD1 (14, 19) have been demonstrated.

We have recently shown that benzo[c]quinolizinium compounds (MPB-07 and MPB-27) are activators of wild-type CFTR (2). We have further performed a structure-activity study and identified 5-butyl-6- hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91), a compound able to restore chloride secretion in G551D and wild-type CFTR-expressing cells. The role of ATPase activity and phosphorylation in the MPB-dependent activation of CFTR was investigated.


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METHODS
RESULTS
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MPB-91. The methods for chemical synthesis of MPB-91 were similar to those previously described (2). The starting material, 2-pentylpyridine, was obtained by condensation of 2-picolyllithium with 1-bromobutane. Then 4.48 g of 2-pentylpyridine (0.03 mol) in tetrahydrofuran (30 ml) were metalated at 0°C with lithium diisopropylamide (0.033 mol) and cooled to -40°C, and 3.08 g of methyl-2,3-dichlorobenzoate (0.015 mol) in tetrahydrofuran (15 ml) were added. The mixture was stirred for 1 h at -40°C and hydrolyzed at 20°C with 10 ml of water. The product was warmed to 200°C for 1 h. The residue was chromatographed to obtain a cream powder [melting point = 160°C, yield = 15%; analysis of C17H17N1O1Cl2: C, 63.37; H, 5.32; N, 4.35; found: C, 63.36; H, 5.27; N, 4.32; infrared (KBr): 3439, 3121, 2951, 2923, 2895, 2863, 2496, 2344, 1629, 1589, 1502, 1457, 1488, 1400; 1H-NMR (Me2SO-d6): sigma  9.25 (d, J = 7 Hz, 1H, H1), 8.5 (d, J = 7 Hz, 1 H), 8.1-6.9 (m, 5H + OH), 2.9 (t, J = 4 Hz, 2H, CH2), 1.5-1.3 (m, 4H, 2 CH2), 0.9 (t, 3H, CH3); mass spectrum (ion chromatography mass spectrometry): 286 (M-HCl)].

Cell culture. Chinese hamster ovary (CHO) cells stably transfected with pNUT vector alone (CHO pNUT) or containing wild-type CFTR [CFTR(+) CHO], G551D CFTR (G551D CHO), or 10SA CFTR (10SA CHO) were provided by J. R. Riordan and X.-B. Chang (Scottsdale, AZ) (6, 31). Cells cultured at 37°C in 5% CO2 were maintained in alpha MEM containing 7% fetal bovine serum, 0.5% L-glutamine, 0.5% antibiotics (50 IU/ml penicillin and 50 µg/ml streptomycin), and 20-100 µM methotrexate. For detailed procedures see elsewhere (2, 32). Calu-3 (American Type Culture Collection), a cell line of human pulmonary origin (9), was cultured at 37°C in 5% CO2 and maintained in DMEM Ham's F-12 Nutritif Mix (1:1) supplemented by 10% FCS and 1% antibiotics (50 IU/ml penicillin and 50 µg/ml streptomycin).

Iodide efflux experiments and patch-clamp experiments. CFTR chloride channel activity was assayed at 37°C as previously described (2, 5) by measuring the rate of iodide (125I) efflux (5, 32) from CHO and Calu-3 cells cultured in multiwell plates to perform parallel experiments and comparison analysis. Briefly, the fraction of initial intracellular 125I lost during each time point was determined, and time-dependent rates of 125I efflux were calculated from ln(125It1/125It2)/(t1 - t2), where 125It is the intracellular 125I at time t, and t1 and t2 are successive time points (32). Curves were constructed by plotting rate of 125I vs. time. All comparisons were based on maximal values for the time-dependent rates (peak rates), excluding the points used to establish the baseline (5). In Fig. 6 the relative rates (r) correspond to r peak/r basal. In experiments in which the chloride transport inhibitors DIDS, glibenclamide, DPC, and Calixarene or the protein kinase inhibitors N-(2-[p-bromocinnamylamino]ethyl)- 5-isoquinolinesulfonamide (H-89) and 1,2-dimethoxy-N-methyl-[1,3]-benzodioxolo[5,6-c]phenantridinium chloride (chelerythrine chloride) were used, these agents were present in the loading solution and in the efflux buffer as indicated in RESULTS.

Whole cell recordings were performed as previously described (5). Media generating a chloride gradient of external concentration 151 mM and internal concentration 28 mM were used. The pipette solution contained (in mM) 113 L-aspartic acid, 113 CsOH, 27 CsCl, 1 NaCl, 1 EGTA, and 10 TES (pH 7.2, 285 mosmol). Mg-ATP (3 mM) was added just before patch-clamp experiments. The external solution contained (in mM) 145 NaCl, 4 CsCl, 1 CaCl2, 5 glucose, and 10 TES (pH 7.4, 340 mosM).

Ussing chamber experiments in G551D Fisher rat thyroid cells. The plasmid vector for expression of human CFTR gene in Fisher rat thyroid (FRT) cells was constructed by cloning the CFTR cDNA encoding sequence in the expression vector pTracer-cytomegalovirus (CMV; Invitrogen). Expression of the CFTR gene is controlled by the CMV promoter, whereas synthesis of the green fluorescent protein-Zeocin fusion protein is controlled by the SV40 promoter. Site-directed mutagenesis was done using the GeneEditor kit (Promega). FRT cells were cultured on 60-mm petri dishes with Coon's modified F-12 medium containing 5% serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells were stably transfected using 10 µl Effectene (Qiagen) with 1 µg of the construct according to the manufacturer's instructions. Clones were selected and maintained in 800 or 600 µg/ml Zeocin, respectively.

For Ussing chamber experiments, cells were seeded at high density (5 × 105 cells/cm2) on Snapwell inserts (Costar, Corning), maintained at 37°C, and gassed with 5% CO2-95% air. The medium was replaced every 48 h. Transepithelial resistance was measured daily with an epithelial voltohmeter (Millipore-ERS, Millipore) using chopstick-like electrodes. After 4-7 days, FRT monolayers developed a transepithelial resistance in the range of 2-4 kOmega /cm2. Experiments were done at days 8-9 after seeding. Snapwell filters were mounted into an Ussing-like vertical diffusion chamber (Costar). To detect CFTR activity, the basolateral membrane of FRT epithelia was permeabilized with 250 µg/ml amphotericin B, and a transepithelial chloride gradient was applied as previously reported (28). The apical chamber was bathed with a low- chloride solution containing (in mM) 140 sodium gluconate, 1 MgSO4, 2 CaCl2, 1 HCl, 10 glucose, and 24 NaHCO3. The basolateral chamber was instead bathed with a high-chloride solution containing (in mM) 126 NaCl, 24 NaHCO3, 0.4 KH2PO4, 2.1 KH2PO4, 1 MgSO4, 1 CaCl2, and 10 glucose. Experiments were done at 37°C, and solutions were bubbled with 5% CO2-95% air. The transepithelial potential difference was short circuited at 0 mV with a voltage clamp (Bioengineering, The University of Iowa) connected to the chambers through Ag-AgCl electrodes and agar bridges. To study the activity of CFTR, all experiments were done in the presence of 10 µM amiloride on the apical chamber.

ATPase assays of fusion protein. Production of the fusion protein NBD1/R/GST and corresponding enzyme assays were performed as previously described (14, 22, 27). A second fusion protein (MBP/NBD2) of the maltose-binding protein (MBP) and the human CFTR protein sequence from Gly-1208 to Leu-1399 (numbering according to Ref. 25), enclosing the whole predicted second nucleotide binding domain of CFTR (NBD2), was expressed, purified, and characterized in Escherichia coli (23, 24); all enzymatic assays were performed as described in Refs. 23 and 24.

Chemicals. Forskolin, 1,9-dideoxyforskolin, 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate (CPT-cAMP), glibenclamide, DPC, DIDS, H-89, and chelerythrine chloride were from Sigma Chemical (St. Louis, MO). 5,11,17,23-Tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]arene (TS-TM calix[4]arene) was a generous gift of Drs. Singh and Bridges (University of Pittsburgh, Pittsburgh). All other products were from Sigma, except alpha MEM and DMEM/Ham Nutritif Mix F-12, which were from Fisher PAA and GIBCO BRL.

Statistics. Results are expressed as means ± SE of n observations. To compare sets of data, we used either an ANOVA or Student's t-test. Differences were considered statistically significant when P < 0.05 [nonsignificant (NS) difference was P > 0.05]. All tests were performed using GraphPad Prism version 3.0 for Windows (GraphPad Software, San Diego, CA).


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Activation of wild-type CFTR channels by MPB-91 in Calu-3 and CFTR(+) CHO cells. We previously found that several substitutions within the benzo[c]quinolizinium (MPB) skeleton are crucial for CFTR activation (2). These are the hydroxyl at position 6 and the chlorine atom at positions 7 (MPB-27) or 10 (MPB-07). Here we performed chemical modifications of the MPB-07 structure by keeping the 10-chlorine and 6-hydroxyl substituents and changing the substituent at position 5. Through a screening process using a cell-based iodide efflux assay, we identified the 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium derivative named MPB-91 (Fig. 1A) and found that it activates wild-type CFTR in CFTR(+) CHO cells and in the human airway epithelial Calu-3 cell lines (Fig. 1, B, C, and F). Relative to control rates in the absence of agonists (i.e., in the presence of DMSO alone), rates of MPB-91-dependent iodide efflux were stimulated in CFTR(+) CHO cells 1.93 ± 0.15-fold (n = 8, P < 0.01) and in Calu-3 cells 1.91 ± 0.12-fold (n = 20, P < 0.001). An EC50 of 46.7 ± 1.3 µM (n = 3) was determined in iodide efflux experiments on Calu-3 cells. We found it necessary to study the additive stimulatory effect of forskolin and MPB-91 in CFTR(+) CHO cells, since the effect on G551D CFTR had been tested in the same cell line. In CFTR(+) CHO cells stimulated by low concentrations of forskolin (500 nM, peak rate 0.20 ± 0.02 min-1, n = 4, vs. control without forskolin: 0.01 ± 0.01 min-1, n = 4, P < 0.01), 250 µM MPB-91 further stimulated CFTR (peak rates: MPB-91 + forskolin, 0.33 ± 0.01 min-1, n = 4, P < 0.01; Fig. 1E). Similar results were observed in Calu-3 cells (not shown). Additional control experiments have been performed on cells that do not express CFTR (CHO pNUT; Fig. 1D). MPB-91 at 250 µM failed to stimulate any significant iodide efflux (relative rate: 1.11 ± 0.14 min-1, n = 8), even in the presence of forskolin (not shown). Figure 1F presents peak rates of MPB-91-induced iodide efflux in each cell line vs. control condition.


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Fig. 1.   Average MPB-91-mediated iodide efflux in cystic fibrosis transmembrane conductance regulator (CFTR)(+) Chinese hamster ovary (CHO) (B), Calu-3 (C), and CFTR(-) CHO (D) cells. A: structure of 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91). Carbon atoms are numbered from 1 to 10. B-D: , basal (vehicle alone); , MPB-91 (250 µM). E: MPB-91 (250 µM) significantly potentiates forskolin (, 500 nM, n = 4; , forskolin + MPB-91, n = 4)-induced iodide efflux in CFTR(+) CHO cells. F: bar graphs comparing peak rates of iodide efflux on each cell line. Arrows indicate times the drugs were added. Error bars are SE. NS, nonsignificant difference (***P < 0.001).

Whole cell patch-clamp experiments were performed on the three cell lines. In CHO pNUT, neither forskolin (10 µM), MPB-91 (250 µM), nor forskolin + MPB-91 were able to stimulate any conductance, confirming that no endogenous MPB-91-activated current was present (data not shown). Figure 2 shows representative whole cell recordings from Calu-3 cells. In resting conditions only a small current was recorded (Fig. 2A), indicating a minimal basal channel activity. After addition of 250 µM MPB-91 into the bath (Fig. 2B), a significant time-independent and nonrectifying current was induced (6.56 ± 1.62 pA/pF, n = 7, vs. 1.53 ± 0.28 pA/pF, n = 7, when measured at +40 mV; Fig. 2C). This current was approximately one-half of that obtained with a high concentration (5 µM) of forskolin (current density 13.51 ± 3.08 pA/pF, n = 5, when measured at +40 mV). Figure 2D shows a typical time course of CFTR activation and inhibition. The current induced by 250 µM MPB-91 reached a maximal value within 2 min and was fully inhibited by either 100 µM glibenclamide (Fig. 2D) or 250 µM DPC (not shown) within 4 min. In iodide efflux experiments, we further determined the inhibitory profile of MPB-91-dependent CFTR activity and found 68%, 80%, and 12% inhibition with, respectively, 100 µM glibenclamide (P < 0.001, n = 8), 250 µM DPC (P < 0.001, n = 8), and 200 µM DIDS (NS, n = 8). On the basis of these observations, we concluded that MPB-91 activates specifically CFTR.


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Fig. 2.   Whole cell current activated by MPB-91 in Calu-3 cells. A and B: representative whole cell currents in basal condition (A) and after MPB-91 exposure (B, 250 µM). Currents are elicited by stepping from a holding potential of -40 mV to a series of test potentials from -100 to +100 mV in 20-mV increments. Cell capacitance was 60 pF. C: corresponding current (I)-voltage (V) relationships. D: time course of activation and inhibition of CFTR current at +40 mV (glib, glibenclamide at 100 µM). Errors bars are SE.

MPB-91 but not MPB-07 is an activator of G551D CFTR. Because little is known about the pharmacology of G551D CFTR, we determined the effects of MPB-07 and MPB-91 on the activity of G551D expressed in CHO cells using iodide efflux and whole cell patch-clamp methods. Control experiments confirmed that forskolin indeed failed to stimulate iodide efflux from G551D-CFTR-expressing cells (peak rate-to-basal: 0.10 ± 0.01 min-1, n = 14; 10 µM forskolin: 0.10 ± 0.01 min-1, n = 14). We found that MPB-07 (250 µM) either alone (peak rate: 0.09 ± 0.01 min-1, n = 4) or in combination with 10 µM forskolin (peak rate: 0.09 ± 0.01 min-1, n = 4) failed to stimulate the iodide efflux in G551D cells. In contrast, the substituted compound MPB-91 (250 µM) dramatically stimulated iodide efflux in the presence of forskolin (peak rate with 250 µM MPB-91: 0.21 ± 0.01 min-1, n = 52; peak rate without MPB-91: 0.09 ± 0.01 min-1, n = 14, P < 0.001). This efflux was inhibited by 61% and 68% with, respectively, 100 µM glibenclamide (P < 0.001, n = 8) or 250 µM DPC (P < 0.001, n = 8), but was not affected by 200 µM DIDS (NS, n = 8). When added simultaneously with the inactive form of forskolin, 1,9-dideoxyforskolin (10 µM, n = 4), MPB-91 did not stimulate iodide efflux, whereas in the presence of CPT-cAMP (200 µM, n = 4), iodide efflux was recovered (not shown).

Figure 3C shows that addition of 250 µM MPB-91 in the presence of 10 µM forskolin stimulated a whole cell chloride current that was recorded neither under control conditions (noted basal, Fig. 3A) nor with forskolin alone (10 µM, Fig. 3B). The current density of the MPB-91-activated conductance (6.12 ± 0.82 pA/pF, n = 7, when measured at +40 mV; Fig. 3D) was statistically different (P < 0.001) from currents measured in the presence or absence of forskolin (respectively, 1.49 ± 0.19 pA/pF, n = 8, and 1.53 ± 0.82 pA/pF, n = 8, at +40 mV; Fig. 3D). These results indicate that the MPB-91-stimulated iodide efflux and whole cell chloride current in G551D cells are due to CFTR activation.


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Fig. 3.   Whole cell CFTR current activated by MPB-91 in CHO G551D cells. A-C: representative whole cell recordings in basal condition (A), after addition of forskolin (Fsk; 10 µM; B), and after addition of Fsk + MPB-91 (250 µM; C). Currents were elicited by stepping from a holding potential of -40 mV to a series of test potentials from -100 to +100 mV in 20-mV increments. Cell capacitance was 17 pS. D: corresponding I-V relationships. Errors bars are SE.

Figure 4 shows short-circuit current measurements on FRT cells transfected with G551D CFTR. Pretreatment with a maximal concentration of CPT-cAMP (500 µM) elicited a small effect. However, subsequent stimulation with MPB-91 produced a dose-dependent increase of transepithelial conductance in G551D CFTR cells (Fig. 4). For instance, in the presence of 200 µM MPB-91, the conductance increased to 376.4 ± 32.3 µS/cm2 (n = 4, P < 0.005). Lower concentrations of MPB-91, e.g., 50 and 100 µM, also evoked effects that were significantly larger than CPT-cAMP alone (P < 0.05 and P < 0.005, respectively). This increase of chloride conductance was totally abolished by glibenclamide (100 µM) exposure (Fig. 4A) or 1 mM DPC (not shown). MPB-91 and CPT-cAMP were completely ineffective on nontransfected parental FRT cells. The activation of G551D CFTR by MPB-91 occurred with an EC50 of 84.8 ± 9.3 µM (n = 3).


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Fig. 4.   Transepithelial conductance (G) response to MPB-91 on G551D CFTR-transfected FRT cells. A: response of a G551D CFTR monolayer to 500 µM 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate (CPT-cAMP) followed by increasing concentrations of MPB-91 (in µM) in the apical solution. B: conductance increase as a function of MPB-91 concentration. Each point is the mean of 4 experiments. Errors bars are SE.

Role of ATPase activities at both NBD1 and NBD2 in the activation of CFTR by MPB-91. Because NBD1 and NBD2 can bind and hydrolyze ATP to gate the CFTR channel (10), we investigated the effect of MPB-91 on both NBD1 and NBD2 ATPase activities. First, wild-type and G551D NBD1/R/GST ATPase activities were measured at their respective Michaelis-Menten constant (Km) values (70 and 250 µM; Ref. 14) and at 1 mM ATP. G551D NBD1 has a significantly reduced ATPase activity compared with wild-type NBD1 (Fig. 5A) as expected from previous studies (14, 19). MPB-91 does not affect the ATPase activity of wild-type or G551D NBD1 at any ATP and MPB-91 concentrations tested. Figure 5A shows results obtained with MPB-91 at 1 mM. We also determined the enzymatic activities of a recombinant NBD2 polypeptide fused to maltose-binding protein (23, 24). MPB-07, MPB-27, and MPB-91 (at 200 µM) did not influence ATPase activity, as shown Fig. 5B. A similar lack of effect was determined for GTPase and adenylate kinase activities of MBP/NBD2 (Table 1). Concerning MBP/NBD2 ATPase activity, they neither affected the Km for ATP (Km values: control, 783 ± 28 µM; MPB-07, 756 ± 16 µM; MPB-27, 738 ± 86; MPB-91, 740 ± 40 µM; n >=  2 for each condition), which, under the assumption of an equilibrium-binding model, mainly reflects the affinity of MBP/NBD2 for ATP, nor the catalytic rates (Vmax: control, 0.95 ± 0.01; MPB-07, 0.94 ± 0.01; MPB-27, 0.93 ± 0.03; MPB-91, 0.92 ± 0.01 µmol ATP · min-1 · µmol MPB-1; n >=  2 for each condition).


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Fig. 5.   Effect of MPB on the ATPase activity of nucleotide binding domains (NBD). A: ATPase activity of NBD1-containing fusion protein. Wild-type (WT) NBD1/R/GST (50 ng) and G551D NBD1/R/GST (50 ng) were tested for ATPase activity at their respective Km values (70 and 250 µM) and at 1 mM ATP. MPB-91 was tested at 1 mM. Errors bars are SE values for at least 5 separate experiments. B: ATPase activities of MBP/NBD2 are plotted as a function of substrate (ATP) concentration in the absence () or presence of 200 µM MPB-07 (open circle ), MPB-27 (triangle ), or MPB-91 (down-triangle). Solid lines are the result of fitting the depicted set of data obtained in the absence or presence of an individual MPB compound to the equation v = Vmax · [S]/(Km + [S]), which describes the dependence of the reaction velocity (v) on the concentration of the substrate (S), which is ATP. All data points depicted are representative of at least 2 determinations.


                              
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Table 1.   Effect of MPB-07, MPB-27, and MPB-91 on the enzymatic properties of a recombinant second nucleotide binding domain of CFTR fused to maltose-binding protein

Effect of protein kinase A and protein kinase C inhibitors. To determine whether wild-type and G551D CFTR activation by MPB-91 involved phosphorylation of the channel, the effects of two kinase inhibitors were investigated. H-89 (10 µM) and chelerythrine chloride (1 µM) were chosen as potent inhibitors of protein kinase A (PKA) and protein kinase C (PKC), respectively. Because our multiwell assay for CFTR activity allowed us to perform parallel experiments, the effect of kinase inhibitors was conducted in paired experiments including both forskolin and MPB-91 as agonists. We first investigated the effect of kinase inhibitors on CFTR activity in Calu-3 and CFTR(+) CHO cells. As expected, inhibition of forskolin-induced CFTR-mediated efflux was observed in the presence of H-89 in both CFTR(+) CHO and Calu-3 cells (85% and 90%, respectively, P < 0.001, n = 8 for each experiment; Fig. 6, A and B). We also observed inhibition with chelerythrine chloride, which was found more efficiently on CFTR(+) CHO cells than on Calu-3 cells [54% inhibition (P < 0.01) vs. 20% inhibition (P < 0.01), n = 8 for each condition, Fig. 6, A and B]. In contrast, results presented in Fig. 6C showed the lack of significant effect of both kinase inhibitors on MPB-91-stimulated iodide efflux in Calu-3 cells (P > 0.05, for both inhibitors). Similar results were observed in CFTR(+) CHO cells (not shown). Finally, experiments were performed on G551D CFTR cells exposed to forskolin (10 µM) and MPB-91 (250 µM). We observed <1% inhibition using H-89, which was not different from control without H-89 (P > 0.05, n = 8). Chelerythrine chloride affected 22% of efflux stimulated by forskolin + MPB-91 (P < 0.01, n = 8, Fig. 6D). MPB-91 was also tested on CHO cells having 10 PKA consensus sites converted into alanine (10SA CHO cells). Chang et al. (6) reported that, with the 10SA-CFTR mutant, no [32P]orthophosphate could be incorporated in the presence of PKA, although a moderate CFTR channel activity could still be recorded. Accordingly, we observed an attenuated iodide efflux in the presence of a high dose (10 µM) of forskolin (peak rate: 0.21 ± 0.02 min-1, n = 8, vs. with 5 µM forskolin on wild-type CFTR: 0.46 ± 0.03 min-1, n = 8, P < 0.01). However, when 250 µM MPB-91 was added to the 10SA-CFTR cell preparation, a significant stimulation of the iodide efflux was still observed in eight of eight experiments (Fig. 7) with a peak rate value (0.195 ± 0.01 min-1, n = 8) similar to that of MPB-91 on wild-type CFTR (compare with Fig. 1F).


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Fig. 6.   Effect of kinase inhibitors on iodide efflux. Inhibition of forskolin-mediated iodide efflux in CFTR(+) CHO (A) and Calu-3 (B) cells. , Forskolin (5 µM); diamond , H-89 (10 µM); triangle , chelerythrine chloride (1 µM). C and D: MPB-91-mediated iodide efflux in Calu-3 (C) and in G551D CHO cells (D). , MPB-91 (250 µM); diamond , H-89 (10 µM); triangle , chelerythrine chloride (1 µM). All results are means ± SE of 8 experiments for each protocol.



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Fig. 7.   MPB-91 stimulates iodide efflux in 10SA CHO. Average iodide efflux curves presenting 10SA CFTR activation by forskolin (10 µM, n = 4; triangle ) and by MPB-91 (250 µM, n = 8; ). All results are means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The development of specific CFTR modulators (2, 3, 5, 17, 26) is of key importance for our understanding of the role of chloride channels in the physiology of epithelia and in other tissues including heart and nephron. It is also crucial for the physiopathology of CF, chronic obstructive pulmonary disease, and other diseases of electrolyte transport.

Activation of CFTR chloride channel by MPB-91. Our study first illustrated that investigation of the structure-activity relationship (SAR) of CFTR modulators may lead to the discovery of more potent compounds. Indeed, we found that MPB-07, which is able to activate wild-type CFTR (2), is not effective as a G551D CFTR activator. Modification of the chemical structure using SAR identified an important position within the MPB skeleton, leading to MPB-91, a compound that kept its ability to activate wild-type CFTR but more importantly acquired the property to activate G551D CFTR. We report here that this compound modulates chloride secretion in airway epithelial cells and, in a recombinant system, activates the CFTR chloride channel. The chloride transport observed in Calu-3 cells by iodide efflux and by patch-clamp experiments presents all the characteristics of CFTR (i.e., kinetics properties and pharmacological profile). We demonstrated that MPB-91 restores CFTR-mediated chloride secretion in cells expressing G551D CFTR. The inhibitory profile of G551D CFTR channel activity is similar to that described for wild-type CFTR (13, 26).

Role of NBD1 domains in MPB-91-mediated CFTR activation. It is generally accepted that ATP binding and hydrolysis are crucial steps leading to CFTR chloride channel activation (10). Electrophysiological data indicates that hydrolysis at NBD1 is a prerequisite to channel opening, whereas ATP hydrolysis at NBD2 is implicated in channel closure (10). However, it has been recently reported that ATP binding alone could be sufficient to promote CFTR opening (15). Because ATP hydrolysis at NBD1 has been linked to CFTR activation, we initially hypothesized that MPB-91 could modulate wild-type ATPase and/or could restore normal G551D ATPase function (e.g., by reducing the Km value for ATP). To study this, we first measured the ATPase activity of wild-type NBD1 in the presence of MPB-91. Our results clearly demonstrated that MPB-91 did not compete with ATP binding, did not modulate ATPase activity, and had no significant effect on reduced ATPase activity of G551D NBD1. We then concluded that the mutation G551D interferes with the mechanism of channel opening that is dependent of ATP hydrolysis at NBD1. Because the compound MPB-91 was found to be able to activate wild-type CFTR and G551D CFTR without directly affecting NBD1 ATPase activity, these observations strongly suggest that an alternative pathway of activation exists that bypasses the defective ATPase activity of G551D CFTR proteins.

Role of NBD2 domains in MPB-91-mediated CFTR activation. Recent studies have proposed that genistein could activate CFTR (16) by a direct binding to NBD2 associated to inhibition of ATPase activity, which in turn prolongs the open time of the channel (23). We performed an analysis of ATPase activity at NBD2 and showed that MPB-91 (and other MPB derivatives) affected neither Vmax nor Km of the reaction. This clearly indicated that our compounds did not activate CFTR by interfering with NBD2 (and NBD1) ATPase activity, although we cannot exclude that these compounds act on NBD by interactions that could change the conformation of the channel.

Our results also demonstrated an important difference between MPB-91 and genistein. For example, these two drugs are both able to activate wild-type and G551D CFTR, but probably through two distinct mechanisms. At least two different classes of activators of CFTR began to emerge: those (like genistein) opening CFTR (16) and altering its ATPase activity at NBD2 (23) but not at NBD1 (14) and another class of compounds (like MPB) opening CFTR without affecting its enzymatic activity (this report). It is interesting to note that none of the modulators of CFTR studied so far interacted with ATPase activity at NBD1 (14). It may be speculated that compounds interfering with the ATPase activity of NBD1 would be inhibitors of CFTR channel activity.

Role of phosphorylation in the mechanism of activation of CFTR by MPB-91. One major difference currently observed between wild-type and G551D CFTR is the role of phosphorylation. In wild-type CFTR cells, stimulation of the cAMP pathway is sufficient to open CFTR chloride channels (10, 29). Although G551D CFTR could be normally phosphorylated (11) and despite the fact that phosphorylation by itself is not sufficient to activate G551D CFTR (11, 33), it is required for CFTR to be opened by MPB-91 and other compounds like genistein (17). Thus one hypothesis is that MPB-91 directly interacts with the phosphorylated protein itself rather than with associated kinases or phosphatases. Indeed, we have shown that MPB compounds have no effect on various phosphatases or kinases implicated in CFTR activation (2). In addition, MPB-91 does not alter the cAMP levels in both Calu-3 and G551D CHO cells (T. Métayé, R. Dérand, L. Bulteau, and F. Becq, unpublished data). Although activity of CFTR is critically dependent of its phosphorylation status (18, 20, 31), MPB-91 activates CFTR in low phosphorylation conditions (low concentrations of forskolin), in variants having 10 mutated PKA sites, and even in the presence of kinase inhibitors. In the presence of H-89 or chelerythrine chloride (to prevent CFTR phosphorylation), MPB-91 still activates CFTR channels, probably through a phosphorylation-independent pathway.

In G551D CHO cells we found, however, that MPB-91 alone is not sufficient to stimulate the chloride current. A similar effect was observed using genistein (17). However, once phosphorylated, G551D CFTR can be activated by MPB-91. Surprisingly, when H-89 or chelerythrine chloride were added, we never observed an inhibition of the CFTR activity. These results suggest that phosphorylation of G551D CFTR may act only as a switch, making the mutated protein responsive or not to MPB-91. On the basis of all our results, we believe that within the CFTR architecture, a binding site for MPB-91 is unmasked following phosphorylation. When MPB-91 occupies the putative site, inhibiting the kinases (PKA or PKC) has no effect. Further experiments are needed to clear this point.

In conclusion, in this report we have presented the potential interest of MPB-91 as a putative drug in CF, since it not only modulates wild-type CFTR but also activates CFTR having the disease-causing mutation G551D. We previously predicted that the benzo[c] quinolizinium compounds were good candidates for pharmacological CFTR activation in airways (2). By designing new derivatives based on the MPB-07 skeleton, we have identified an important site on the MPB skeleton (position 5) and have obtained a new compound that is more potent on CFTR channel activity (normal and mutated). The pathway to activate CFTR with MPB drugs appears to be different (no direct effect on NBD domains) from that involved with genistein. In addition, studying ATPase dependence of CFTR activation with a given modulator may thus reveal novel mechanisms to control the channel opening, which may help to better understand the underlying molecular mechanism leading to activation of normal and mutated CFTR.


    ACKNOWLEDGEMENTS

We thank Dr. P. Fanen from Institut National de la Santé et de la Recherche Médicale U468, Créteil, France, for plasmid construction. CHO cells were a generous gift of J. R. Riordan and X.-B. Chang, Scottsdale, AZ.


    FOOTNOTES

This work was supported by an Association Française de Lutte contre la Mucoviscidose Grant to R. Dérand and L. Bulteau-Pignoux, a National Institutes of Health Grant to J. A. Cohn, a Cystic Fibrosis Foundation Grant to L. D. Howell, and a Deutsche Forschungsgemeinschaft Grant to C. Randak.

Address for reprint requests and other correspondence: F. Becq, Laboratoire de Physiologie des Régulations Cellulaires, UMR 6558, 40 Ave. du Recteur Pineau, 86022 Poitiers, France (E-mail: frederic.becq{at}univ-poitiers.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 19 April 2001; accepted in final form 16 July 2001.


    REFERENCES
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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