ABCMdb

PMID: 14502435

Derand R, Bulteau-Pignoux L, Becq F
Comparative pharmacology of the activity of wild-type and G551D mutated CFTR chloride channel: effect of the benzimidazolone derivative NS004.
J Membr Biol. 2003 Jul 15;194(2):109-17., 2003-07-15 [PubMed]

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No. Mutations Sentence Comment

0 ABCC7 p.Gly551Asp Comparative Pharmacology of the Activity of Wild-type and G551D Mutated CFTR Chloride Channel: Effect of the Benzimidazolone Derivative NS004 R. De´ rand, L. Bulteau-Pignoux, F. Becq Laboratoire des Biomembranes et Signalisation Cellulaire, UMR 6558 CNRS, Universite´ de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers, France Received: 2 December 2002/Revised: 10 April 2003 Abstract. Login to comment 1 ABCC7 p.Gly551Asp 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. Login to comment 4 ABCC7 p.Gly551Asp In G551D-CFTR-expressing CHO cells, neither forskolin (from 0.1 to 10 lM) nor NS004 (from 0.1 to 200 lM) added separately were able to stimulate channel activity. Login to comment 5 ABCC7 p.Gly551Asp However, in the presence of 10 lM forskolin, NS004 stimulated G551D-CFTR activity in a dose-dependent manner with an EC50 » 1.5 lM. Login to comment 6 ABCC7 p.Gly551Asp We also determined the half-maximal effective concentration of forskolin (EC50 » 3.2 lM) required to stimulate G551D channel activity in presence of 1.5 lM NS004. Login to comment 7 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp No inhibitory effect was observed at high concentration of NS004 with both wtand G551D-CFTR. Login to comment 8 ABCC7 p.Gly551Asp 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. Login to comment 9 ABCC7 p.Gly551Asp The inhibitory profile of G551D-CFTR channel activity was similar to that of wild type, i.e., inhibition by glibenclamide (100 lM) and DPC (250 lM) but not by DIDS (200 lM) nor calixarene (100 nM). Login to comment 10 ABCC7 p.Gly551Asp 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. Login to comment 11 ABCC7 p.Gly551Asp Key words: Cystic Fibrosis - NS004 - G551D mutant - Pharmacology - Phosphorylation Introduction Cystic fibrosis (CF), the most common lethal autosomal recessive genetic disease among Caucasians, results from mutations of the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel that normally mediates ClÀ transepithelial transport in epithelia (Riordan et al., 1989; Tabcharani et al., 1991; Quinton, 1999). Login to comment 14 ABCC7 p.Gly551Asp The class III mutation glycine-to-aspartic acid at codon 551 (G551D) is located within the NBD1 domain (Cutting et al., 1990). Login to comment 16 ABCC7 p.Gly551Asp With a frequency of 2-5%, depending on the population of origin, G551D is one of the five most frequent CF mutations and is always associated with a severe CF phenotype (Cutting et al., 1990), pulmonary dysfunction and pancreatic insufficiency. Login to comment 17 ABCC7 p.Gly551Asp G551D-mutated protein is fully glycosylated, correctly located at the apical membrane (i.e., normal biosynthesis, trafficking and processing) (Smit et al., 1993; Welsh & Smith, 1993) and normally phosphorylated at the R J. Membrane Biol. 194, 109-117 (2003) DOI: 10.1007/s00232-003-2030-z Correspondence to: F. Becq; email: frederic.becq@univ-poitiers.fr domain by cAMP-dependent protein kinase (Chang et al., 1993). Login to comment 19 ABCC7 p.Gly551Asp G551D mutation confers a decreased nucleotide binding (Logan et al., 1994) and a reduced ATPase activity at NBD1 (Li et al., 1996; Howell, Borchardt & Cohn, 2000). Login to comment 20 ABCC7 p.Gly551Asp The pharmacological modulation of G551D-CFTR chloride-channel activity has not been clearly and systematically characterized and compared to that of wild-type CFTR channel. Login to comment 21 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The ability of high doses of IBMX to activate mutated CFTR, including G551D, has, however, been reported (Drumm et al., 1991; Becq et al., 1994) and the phenylimidazothiazoles bromotetramisole and levamisole (Becq et al., 1994; 1996) as well as genistein (Illek et al., 1999) have been shown to activate the G551D-CFTR channel. Login to comment 22 ABCC7 p.Gly551Asp Recently, we demonstrated that benzoquinolizinium derivatives activate wt-, G551D-CFTR chloride channels and modulate the trafficking of delF508-CFTR (Becq et al., 1999, De´ rand et al., 2001, Dormer et al., 2001). Login to comment 23 ABCC7 p.Gly551Asp In this report, we have investigated and compared the effect of the benzimidazolone NS004 on the activation of both wild-type (wt) and G551D-CFTR chloride channels stably expressed in CHO cells, using iodide (125 I) efflux and whole-cell patch-clamp techniques. Login to comment 24 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Materials and Methods CELL CULTURE Chinese Hamster Ovary (CHO) cells stably transfected with pNUT vector alone (pNUT CHO) or containing wild-type CFTR (CFTR(+) CHO) or G551D (G551D CHO) mutation were provided by J.R. Riordan and X.-B. Chang, Scottsdale, AZ, USA (Tabcharani et al., 1991; Chang et al., 1993). Login to comment 25 ABCC7 p.Gly551Asp Cells cultured at 37°C in 5% CO2 were maintained in aMEM containing 7% fetal bovine serum, 0.5% antibiotics (50 IU/ml penicillin and 50 lg/ml streptomycin) and 100 lM or 20 lM methotrexate for CFTR(+), G551D and pNUT CHO cells, respectively. Login to comment 94 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp STIMULATION OF G551D-CFTR-MEDIATED 125 I EFFLUX BY NS004 The pharmacological properties of CFTR chloride channel having the glycine-to-aspartate mutation at codon 551 (G551D) were then studied. Login to comment 96 ABCC7 p.Gly551Asp In G551D-CFTR cells, in the absence of Fsk, the peak rate of 125 I efflux was 0.09 ± 0.01 minÀ1 (n = 14, not shown) and did not differ significantly from experiments in which 10 lM Fsk was added (0.10 ± 0.01 minÀ1 , n = 14, Fig. 3A). Login to comment 97 ABCC7 p.Gly551Asp We then studied the effect of NS004 on G551D-CFTR activity in the presence of 10 lM Fsk. Login to comment 99 ABCC7 p.Gly551Asp In G551D-CFTR cells, 125 I efflux rates were 1.35 ± 0.17 (n = 12), 1.13 ± 0.04 (n = 8), and 3.41 ± 0.15 (n = 28) with 10 lM Fsk, 20 lM NS004 and 10 lM Fsk + 20 lM NS004, respectively. Login to comment 100 ABCC7 p.Gly551Asp Figure 3A also shows that the addition of 20 lM NS004, without Fsk, failed to activate any significant 125 I efflux in G551D-CFTR cells. Login to comment 102 ABCC7 p.Gly551Asp In contrast, in the presence of 10 lM Fsk, the stimulation of G551D-CFTR activity by NS004 increased dose-dependently with an EC50 = 1.47 ± 0.07 lM (Fig. 3B). Login to comment 103 ABCC7 p.Gly551Asp We also searched for the minimal concentration of forskolin required to stimulate G551D in the presence of a low concentration of NS004. Login to comment 104 ABCC7 p.Gly551Asp For this protocol, G551D-CFTR cells were simultaneously exposed to 1.5 lM NS004 and to increasing concentrations of Fsk. Login to comment 106 ABCC7 p.Gly551Asp The half-maximal effective concentration of forskolin needed to stimulate G551D-CFTR with 1.5 lM NS004 was EC50 = 3.15 ± 0.2 lM (n = 4, Fig. 4B). Login to comment 107 ABCC7 p.Gly551Asp These data indicate a synergistic effect of NS004 and forskolin, with the level of stimulation of G551D-CFTR activity being dependent on both NS004 and channel phosphorylation. Login to comment 108 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ACTIVATION OF G551D-CFTR CHLORIDE CURRENT BY NS004 Whole-cell patch-clamp experiments were next performed, using G551D-CFTR cells. Login to comment 110 ABCC7 p.Gly551Asp As expected from the iodide efflux experiments, G551D-CFTR cells are not responsive to up to 10 lM Fsk in whole-cell patch-clamp experiments, as previously reported (De´ rand et al., 2001; Illek et al., 1999). Login to comment 114 ABCC7 p.Gly551Asp This lack of responsiveness of G551D-CFTR to cAMP agonists constitutes a hallmark of the mutant and a major difference with wild-type CFTR. Login to comment 115 ABCC7 p.Gly551Asp To determine the effect of NS004 on G551D-CFTR current, cells were first exposed to 10 lM Fsk and then to 20 lM NS004. Login to comment 121 ABCC7 p.Gly551Asp Iodide efflux experiments in G551D-CFTR CHO cells. Login to comment 124 ABCC7 p.Gly551Asp (B) Dose-response relationship of the effect of NS004 on G551D-CFTR in the presence of 10 lM forskolin. Login to comment 129 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp INHIBITORY PROFILE OF G551D-CFTR CHLORIDE CHANNEL ACTIVITY STIMULATED BY NS004 Several inhibitors were then used to further characterize G551D-CFTR channel activated by NS004. Login to comment 131 ABCC7 p.Gly551Asp G551D-CFTR currents were first activated by Fsk+NS004 (Fig. 5E) and then inhibited in the presence of 100 lM glibenclamide (Figs. Login to comment 136 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp From these observations, we concluded that the inhibitory profile of G551D-CFTR activated by NS004 did not differ from that of wild-type CFTR, and that G551D mutation did not affect these pharmacological properties of CFTR. Login to comment 137 ABCC7 p.Gly551Asp Discussion The present work reports the characteristics of activation of G551D-CFTR channels by the benzimidazolone derivative NS004 and offers a comparison with wt-CFTR channel activity. Login to comment 139 ABCC7 p.Gly551Asp First, NS004 acts on phosphorylated wt and G551D-CFTR. Login to comment 141 ABCC7 p.Gly551Asp Third, the inhibitory profile of G551D-CFTR channel activity appears similar to that of wt-CFTR. Login to comment 149 ABCC7 p.Gly551Asp Indeed, one major difference concerning the phosphorylation process is that higher concentrations of forskolin are required to achieve stimulation of the G551D-CFTR channel activity by NS004. Login to comment 150 ABCC7 p.Gly551Asp Based on these observations, we could then speculate that NS004 activates G551D- Fig. 4. Login to comment 151 ABCC7 p.Gly551Asp Minimum concentration of forskolin to stimulate G551D-CFTR activity. Login to comment 154 ABCC7 p.Gly551Asp (B) Dose-response relationship of forskolin on G551D-CFTR in the presence of 1.5 lM NS004. Login to comment 160 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp In this study, we found that NS004 was able to stimulate forskolin-dependent phosphorylated G551D-CFTR activity. Login to comment 161 ABCC7 p.Gly551Asp Interestingly, we also showed that, although up to 10 lM forskolin did not activate G551D-CFTR, only 1 lM was sufficient when 1.5 lM NS004 was also present. Login to comment 162 ABCC7 p.Gly551Asp The maximum level of stimulation of G551D-CFTR was, however, achieved with higher concentrations of both agonists (see Fig. 3). Login to comment 165 ABCC7 p.Gly551Asp The glycine-to-aspartic acid missense mutation at codon 551 (G551D) is a class III mutation located within NBD1, which disrupts activation and regulation of CFTR at the plasma membrane (Cutting et al., 1990). Login to comment 166 ABCC7 p.Gly551Asp The G551D mutated protein is, however, fully glycosylated and correctly located at the apical membrane. Login to comment 167 ABCC7 p.Gly551Asp Importantly, G551D proteins have a decreased nucleotide binding and a reduced ATPase activity at NBD1 (Logan et al., 1994; Li et al., 1996; Howell Fig. 5. Login to comment 168 ABCC7 p.Gly551Asp Electrophysiological characteristics of G551D-CFTR chloride current. Login to comment 169 ABCC7 p.Gly551Asp Whole-cell recordings using G551D-CFTR cells stimulated by 10 lM Forskolin (Fsk) and 20 lM NS004. Login to comment 172 ABCC7 p.Gly551Asp (A-C) Representative currents showing the stimulation of G551D-CFTR chloride currents only with forskolin +NS004. Login to comment 173 ABCC7 p.Gly551Asp Cell capacitance was 17 pF. (D-F) Effect of 100 lM glibenclamide on NS004-activated chloride conductance in G551D-CHO cells. Login to comment 176 ABCC7 p.Gly551Asp Despite this apparent normal property, the channel activity of G551D mutant can not be stimulated by the classical pathway (Gregory et al., 1991). Login to comment 177 ABCC7 p.Gly551Asp The importance of phosphorylation in the activation process of G551D-CFTR channel came from various studies. Login to comment 178 ABCC7 p.Gly551Asp For example, it was found that phosphatase inhibitors activated G551D-CFTR as well as wt-CFTR (Becq et al., 1994). Login to comment 180 ABCC7 p.Gly551Asp Thus, phosphorylation of the G551D-CFTR protein appears to be essential before opening the channel by other means. Login to comment 181 ABCC7 p.Gly551Asp Since the ATPase activity at NBD1 is abnormal for G551D, it can be hypothesized that both the altered ATPase activity and the unresponsiveness to PKA stimulation are linked. Login to comment 190 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Genistein has been proposed as an activator of the trafficking-competent G551D mutant after forskolin exposure (Illek et al., 1999; Bulteau-Pignoux et al., 2002; De´ - rand et al., 2002). Login to comment 191 ABCC7 p.Gly551Asp Previous work from our laboratory showed that benzo[c]quinolizinium derivatives (MPB-91 in particular) are also potent activators of G551D-CFTR (Derand et al., 2001). Login to comment 194 ABCC7 p.Gly551Asp Current-voltage relationships of G551D-CFTR chloride currents. Login to comment 200 ABCC7 p.Gly551Asp Effect of calixarene and glibenclamide on NS004-activated chloride conductance in G551D-CHO cells. Login to comment 201 ABCC7 p.Gly551Asp Representative experiments showing the time-dependent activation of G551D-CFTR chloride current in the presence of 10 lM forskolin and 20 lM NS004. Login to comment 204 ABCC7 p.Pro574His ABCC7 p.Lys1250Ala Further studies have also demonstrated that NS004 is a modulator of P574H (Champigny et al., 1995), K1250A (Al-Nakkash et al., 2001) and delF508 (Gribkoff et al., 1994; He et al., 1998; Al-Nakkash et al., 2001). Login to comment 209 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp When compared to wt-CFTR, the inhibitory profile of G551D was not altered, suggesting that the G551D mutation did not interfere with the pharmacological properties of the channel as we previously observed with genistein (Bulteau-Pignoux et al., 2002; De´ rand et al., 2002). Login to comment 213 ABCC7 p.Gly551Asp We provided evidence in this report that NS004 and phosphorylation have a synergistic effect, which allows the stimulation of G551D-CFTR chloride channel activity. Login to comment