ABCMdb

PMID: 15163550

Melin P, Thoreau V, Norez C, Bilan F, Kitzis A, Becq F
The cystic fibrosis mutation G1349D within the signature motif LSHGH of NBD2 abolishes the activation of CFTR chloride channels by genistein.
Biochem Pharmacol. 2004 Jun 15;67(12):2187-96., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Gly1349Asp The cystic fibrosis mutation G1349D within the signature motif LSHGH of NBD2 abolishes the activation of CFTR chloride channels by genistein Patricia Melin, Vincent Thoreau, Caroline Norez, Fre´de´ric Bilan, Alain Kitzis, Fre´de´ric Becq* Institut de Physiologie et Biologie Cellulaires CNRS UMR 6187, Universite´ de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers, France Received 27 October 2003; accepted 5 February 2004 Abstract Cystic fibrosis (CF) is a common lethal genetic disease caused by autosomal recessive mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that belongs to the ATP-Binding Cassette (ABC) family of transporters. Login to comment 1 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The class III CF mutations G551D and G1349D are located within the ''signature`` sequence LSGGQ and LSHGH of NBD1 and NBD2, respectively. Login to comment 2 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp We have constructed by site-directed mutagenesis vectors encoding green fluorescent protein (GFP)-tagged wild-type (wt) CFTR or CFTR containing delF508, G551D, G1349D and G551D/G1349D to study their pharmacology after transient expression in COS-7 cells. Login to comment 3 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp We show that IBMX and the benzo[c]quinolizinium derivative MPB-91 stimulates the activity of G1349D-, G551D- and G551D/G1349D-CFTR only in the presence of cAMP-promoting agents like forskolin or cpt-cAMP. Login to comment 4 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Similar half-maximal effective concentrations (EC50) of MPB-91 (22-36 mM) have been determined for wt-, G551D-, G1349D- and G551D/G1349D-CFTR. Login to comment 6 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp By contrast, the response of G1349D- and G551D-CFTR to genistein is dramatically altered. Login to comment 7 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp First, genistein is not able to stimulate G1349D- and G551D/G1349D-CFTR. Login to comment 8 ABCC7 p.Gly551Asp Second, genistein stimulates G551D-CFTR without any inhibition at high concentration. Login to comment 12 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Keywords: CFTR; Signature sequences; Genistein; G1349D; G551D; delF508 1. Login to comment 27 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The glycine-to-aspartic acid missense mutations G551D and G1349D are class III mutations located within the signature sequence LSGGQ in NBD1 and LSHGH in NBD2, respectively [13-15]. Login to comment 28 ABCC7 p.Gly551Asp G551D is one of the five most frequent CF mutation with a frequency of 2-5% depending of the population of origin. Login to comment 42 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The delF508, G551D and G1349D mutations were created using the sense oligonucleotides 50 -CAT- TAAAGAAAATATCATTGGTGTTTCCTATGATG-30 , 50 -GGAATCACACTGAGTGGAGATCAACGAGCAA- GAATTTCTT-30 and 50 -GGCTGTGTCCTAAGCCAT- GACCACAAGCAGTTGATGTGC-30 , respectively, and the corresponding antisense oligonucletotides. Login to comment 43 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Double mutant G551D/G1349D was derived from G551D. Login to comment 49 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp COS-7 cells were either transfected with empty pEGFP-C1 vector (mock) or with expression vector encoding wild-type GFP-tagged CFTR (wt-CFTR), or mutant forms of GFP-CFTR: delF508, G551D, G1349 or the double mutant G551D/G1349D (named 2GD-CFTR). Login to comment 79 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Results To study the pharmacology of CFTR, we have introduced green fluorescent protein (GFP)-tagged CFTR proteins into COS-7 cells, i.e. wt-CFTR and four CFTR mutants i.e. delF508, G551D, G1349D and the double mutant G551D/G1349D (named 2GD). Login to comment 85 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp GFP-G1349D (lane 5), GFP-G551D/G1349D (lane 6) and GFP-G551D (lane 7) appeared with a similar pattern like the non-mutated form of CFTR (lane 3). Login to comment 90 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The two mutated channels G1349D- and 2GD-CFTR, like G551D-CFTR, did not respond to forskolin stimulation (n ¼ 4 for each construct, Fig. 2A), as expected from class III CF mutations [13,19]. Login to comment 91 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp However, when a cAMP cocktail composed of forskolin, IBMX and cpt-cAMP was used instead of forskolin alone, the chloride channel activity of G1349D and 2GD mutants was stimulated, indicating that both proteins have functional cAMP-dependent chloride channel activity (n ¼ 4, Fig. 2B and C) like G551D-CFTR (data not shown and [19]). Login to comment 95 ABCC7 p.Gly1349Asp Only glibenclamide inhibited the chloride channel activity of G1349D- and 2GD-CFTR (Fig. 2B and C). Login to comment 96 ABCC7 p.Gly1349Asp Note that the vehicle DMSO has no apparent effect on the iodide efflux (Fig. 2D) A summary of the data obtained with glibenclamide and DIDS is presented Fig. 2E for wt-, G1349D- and 2GD-CFTR stimulated by cAMP cocktail. Login to comment 97 ABCC7 p.Gly551Asp In a second set of experiments, we used the benzoquinolizinium drug MPB-91, a CFTR pharmacological agent already shown to activate wt and G551D-CFTR [22]. Login to comment 99 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp We found that MPB-91 stimulated the iodide efflux with similar EC50 of 29 Æ 1:4, 22 Æ 1:4, 22 Æ 1:3 and 36 Æ 1:1 mM for wt-, G551D-, G1349D and 2GD-CFTR, respectively (n ¼ 4 for each concentration). Login to comment 100 ABCC7 p.Gly1349Asp We also found that G1349D and the double mutant 2GD could be stimulated by forskolin and 100 mM IBMX (not shown) as previously reported by others [23,24]. Login to comment 109 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Lane 1: non transfected COS-7 cells; lane 2: mock-transfected cells; lane 3: GFP-wt-CFTR, lane 4: GFP-delF508-CFTR; lane 5: GFP-G1349D-CFTR; lane 6: GFP-2GD-CFTR; lane 7: GFP-G551D-CFTR. Login to comment 110 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp (C) confocal imaging showing plasma membrane localization of wt-CFTR, G551D-, G1349D- and 2GD-CFTR. Login to comment 118 ABCC7 p.Gly551Asp Fig. 4B shows that the G551D mutation altered the non-Michaelis-Menten behaviour of GFP-wt-CFTR in such a way that the inhibitory effect of genistein at high concentration was abolished (Fig. 4B, right traces). Login to comment 120 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp The calculated half-maximal effective concentration EC50 was 13 Æ 1:25 mM (n ¼ 4), in perfect agreement with the value of 11 mM determined in our previous study using CHO cells stably expressing 0 2 4 6 8 0.0 0.1 0.2 0.3 cAMP agents +glibenclamide +DIDS time (min) k(min -1 ) G1349D-CFTR 2GD-CFTR 0 2 4 6 8 0.0 0.1 0.2 0.3 cAMP agents + glibenclamide +DIDS time (min) k(min -1 ) 0 2 4 6 8 0.00 0.05 0.10 0.15 0.20 mock Fsk 10µM G1349D G551D 2GD wt-CFTR time (min) k(min -1 ) 0 2 4 6 8 0.0 0.1 0.2 0.3 0.4 delF508 2GD G551D G1349D DMSO 1% wt time (min) k(min -1 ) cAMP agents + DIDS + glib 0 50 100 wt-CFTR 2GD G1349D %ofmaximalactivation *** ns (A) (B) (C) (D) (E) Fig. 2. Login to comment 121 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Functional expression and pharmacology of GFP-tagged wt-, G551D-, G1349D- and 2GD-CFTR. Login to comment 123 ABCC7 p.Gly1349Asp B and C, analysis of the expression of G1349D-CFTR (B) and 2GD-CFTR (C) mutants in COS-7 cells. Login to comment 127 ABCC7 p.Gly1349Asp (E) summary of the % of maximal activation in the presence of glibenclamide and DIDS for wt-, G1349D and 2GD-CFTR, as indicated. Login to comment 131 ABCC7 p.Gly551Asp G551D-CFTR [19]. Login to comment 132 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp On the contrary, analysing the effect of genistein on the channel activity of the LSHGH mutant G1349D and of the double mutant 2GD demonstrated their inability to be stimulated even in the presence of 10 mM forskolin and 300 mM genistein. Login to comment 133 ABCC7 p.Gly1349Asp On the contrary, analysing the effect of genistein on the channel activity of the LSHGH mutant G1349D and of the double mutant 2GD demonstrated their inability to be stimulated even in the presence of 10 mM forskolin and 300 mM genistein. Login to comment 134 ABCC7 p.Gly1349Asp Lack of response given by G1349D-expressing cells was observed in the presence of six different concentrations of genistein (with 10 mM forskolin) and was not statistically different from the control, i.e. with forskolin but without genistein. Login to comment 135 ABCC7 p.Gly1349Asp Lack of response given by G1349D-expressing cells was observed in the presence of six different concentrations of genistein (with 10 mM forskolin) and was not statistically different from the control, i.e. with forskolin but without genistein. Login to comment 136 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The corresponding concentration-dependent activation relationships for wt, G551D and G1349D are superimposed on the same graph in Fig. 4E. Login to comment 137 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The corresponding concentration-dependent activation relationships for wt, G551D and G1349D are superimposed on the same graph in Fig. 4E. Login to comment 138 ABCC7 p.Gly1349Asp Since there is no shift to the right of the curve for G1349D, we concluded that the mutant CFTR was fully refractory to genistein stimulation. Login to comment 139 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Finally, similar experiments were conducted with CFTR chloride channels having the class II CF mutation delF508 because this mutation belongs to a different class of mutation but causes, like G551D and G1349D, a severe CF phenotype. Login to comment 140 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Finally, similar experiments were conducted with CFTR chloride channels having the class II CF mutation delF508 because this mutation belongs to a different class of mutation but causes, like G551D and G1349D, a severe CF phenotype. Login to comment 145 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Inconclusion,wehavestudiedthepharmacologyofCFTR chloride channel and the consequence of the three severe CF mutations, delF508, G551D and G1349D. Login to comment 146 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp We found a dramatic modification of the pharmacological behaviour of CFTR only with the class III mutations G551D and G1349D that appears to be restricted to genistein. Login to comment 147 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp We found a dramatic modification of the pharmacological behaviour of CFTR only with the class III mutations G551D and G1349D that appears to be restricted to genistein. Login to comment 153 ABCC7 p.Gly551Asp Our previous study on G551D, the symmetrical glycine mutation located in the LSGGQ signature sequence of NBD1, showed that the mutation abolished only the inhibitory genistein effect [19]. Login to comment 154 ABCC7 p.Gly551Asp Our previous study on G551D, the symmetrical glycine mutation located in the LSGGQ signature sequence of NBD1, showed that the mutation abolished only the inhibitory genistein effect [19]. Login to comment 160 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Thus, deleting the phenylalanine at position 508, which alters the gating mechanism of the channel and the intracellular trafficking process of the protein, has no apparent effect -6 -5 -4 -3 0.00 0.05 0.10 0.15 0.20 wt-CFTR G551D G1349D 2GD Log [MPB-91] (M) kpeak-kbasal(min -1 ) Fig. 3. Login to comment 161 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Effect of the benzoquinolizinium activator MPB-91 on GFP-tagged wt-, G551D-, G1349D- and 2GD-CFTR chloride channel activity. Login to comment 162 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Effect of the benzoquinolizinium activator MPB-91 on GFP-tagged wt-, G551D-, G1349D- and 2GD-CFTR chloride channel activity. Login to comment 165 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Calculated EC50 are 29 Æ 1:4 mM (wt-CFTR), 22 Æ 1:4 mM (G551D-), 22 Æ 1:3 mM (G1349D-), and 36 Æ 1:1 mM (2GD-CFTR). Login to comment 166 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Calculated EC50 are 29 1:4 mM (wt-CFTR), 22 1:4 mM (G551D-), 22 1:3 mM (G1349D-), and 36 1:1 mM (2GD-CFTR). Login to comment 167 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Some error bars are smaller than the symbol. 0 2 4 6 8 0.0 0.1 0.2 0.3 +1 µM Gst +10 µM Gst +3 µM Gst +30 µM Gst Fsk 10 µM time (min) k(min -1 ) G551D-CFTR 0 2 4 6 8 0.0 0.1 0.2 0.3 Fsk 500 nM +3 µM Gst +10 µM Gst +30 µM Gst time (min) k(min-1 ) wt-CFTR 0 2 4 6 8 0.0 0.1 0.2 0.3 Fsk 500 nM +30 µM Gst +100 µM Gst +300 µM Gst time (min) k(min-1 ) 0 2 4 6 8 0.0 0.1 0.2 0.3 +100 µM Gst +30 µM Gst +300 µM Gst Fsk 10 µM time (min) k(min -1 ) 0 2 4 6 8 0.0 0.1 0.2 0.3 Fsk 10µM + 30 µM Gst time (min) k(min -1 ) 2GD-CFTR 0 2 4 6 8 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 +1 µM Gst +3 µM Gst +10 µM Gst +30 µM Gst +100 µM Gst +300 µM Gst Fsk 10µM time (min) k(min -1 ) G1349D-CFTR -6 -5 -4 -3 0.0 0.1 0.2 0.3 G551D CFTRwt G1349D Log[Gst] (M) kpeak-kbasal(min -1 ) (A) (B) (C) (D) (E) Fig. 4. Login to comment 168 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Effect of genistein on GFP-tagged wt-, G551D-, G1349D- and 2GD-CFTR chloride channel activity. Login to comment 169 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Iodide efflux curves calculated in response to forskolin and various concentrations of genistein with COS-7 cells transiently expressing GFP-CFTR channels: wt- (A), G551D- (B), G1349D- (C) and 2GD- (D). Login to comment 170 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Effect of genistein on GFP-tagged wt-, G551D-, G1349D- and 2GD-CFTR chloride channel activity. Login to comment 171 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Iodide efflux curves calculated in response to forskolin and various concentrations of genistein with COS-7 cells transiently expressing GFP-CFTR channels: wt- (A), G551D- (B), G1349D- (C) and 2GD- (D). Login to comment 188 ABCC7 p.Gly551Asp With the G551D mutant, the glycine G1349 being still free to accept a molecule of genistein, the channel can be activated without inhibition at high concentration (because the inhibitory glycine at position 551 has been mutated into aspartate). Login to comment 190 ABCC7 p.Gly1349Asp On the contrary, with the mutant G1349D the activation of CFTR was abolished. Login to comment 191 ABCC7 p.Gly551Asp With the G551D mutant, the glycine G1349 being still free to accept a molecule of genistein, the channel can be activated without inhibition at high concentration (because the inhibitory glycine at position 551 has been mutated into aspartate). Login to comment 193 ABCC7 p.Gly1349Asp On the contrary, with the mutant G1349D the activation of CFTR was abolished. Login to comment