ABCMdb

PMID: 12124395

Derand R, Bulteau-Pignoux L, Becq F
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.
J Biol Chem. 2002 Sep 27;277(39):35999-6004. Epub 2002 Jul 17., 2002-09-27 [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Gly551Asp 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* Received for publication, June 20, 2002 Published, JBC Papers in Press, July 17, 2002, DOI 10.1074/jbc.M206121200 Renaud De´rand, Laurence Bulteau-Pignoux, and Fre´de´ric Becq‡ From the From LBSC, CNRS UMR 6558, Universite´ de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers, France Loss of cystic fibrosis transmembrane conductance regulator (CFTR) channel activity explains most of the manifestations of the cystic fibrosis (CF) disease. Login to comment 1 ABCC7 p.Gly551Asp To understand the consequences of CF mutations on CFTR channel activity, we compared the pharmacological properties of wild-type (wt) and G551D-CFTR. Login to comment 5 ABCC7 p.Gly551Asp In G551D-CFTR cells, channel activity was recovered by co-application of forskolin and genistein in a dose-dependent manner. Login to comment 6 ABCC7 p.Gly551Asp A further stimulation of G551D-CFTR channel activity was measured at concentrations from 30 ␮M to 1 mM. Login to comment 13 ABCC7 p.Gly551Asp The glycine-to-aspartic acid missense mutation at codon 551 (G551D) is a class III mutation located within the nucleotide binding domain 1 (NBD1) (5). Login to comment 15 ABCC7 p.Gly551Asp G551D is one of the five most frequent CF mutations with a frequency of 2-5% depending of the population of origin but is associated with a severe CF phenotype (5). Login to comment 17 ABCC7 p.Gly551Asp The G551D mutated protein is fully glycosylated, correctly located at the apical membrane (i.e. normal biosynthesis, trafficking, and processing), and normally phosphorylated at the R domain by cAMP-dependent protein kinase (6). Login to comment 18 ABCC7 p.Gly551Asp However, G551D proteins have a decreased nucleotide binding (7) and a reduced ATPase activity at NBD1 (8, 9). Login to comment 22 ABCC7 p.Gly551Asp In this work, we compared the effect of genistein on both activation and inhibition of wt-CFTR and G551D-CFTR chloride channels. Login to comment 23 ABCC7 p.Gly551Asp Analyzing dose-response relationships pointed to a novel type of alteration due to the disease-causing mutation G551D. Login to comment 26 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp EXPERIMENTAL PROCEDURES Cell Culture-Chinese hamster ovary (CHO) cells stably transfected with pNUT vector alone (pNUT CHO) or containing wild-type CFTR (CFTR(ϩ) CHO) or G551D (G551D CHO) mutation were provided by J. R. Riordan and X.-B. Chang, Scottsdale, AZ (2, 6). Login to comment 109 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Perturbation of the non-Michaelis-Menten Behavior of CFTR by the G551D Mutation-The glycine-to-aspartic acid missense mutation at codon 551 (G551D) is a class III mutation located within the NBD1 domain (5). Login to comment 110 ABCC7 p.Gly551Asp We used CHO cells stably expressing G551D-CFTR to analyze the effect of genistein. Login to comment 112 ABCC7 p.Gly551Asp The addition of up to 100 ␮M genistein failed to activate chloride transport in G551D-CFTR-expressing cells. Login to comment 113 ABCC7 p.Gly551Asp We also noticed that up to 10 ␮M forskolin failed to stimulate G551D-CFTR channels (not shown). Login to comment 115 ABCC7 p.Gly551Asp To compare the effect of genistein on G551D-CFTR with our study with wt-CFTR cells, we generated dose-response relationships for genistein in the presence of 10 ␮M forskolin. Login to comment 117 ABCC7 p.Gly551Asp Complete dose-response relationships for activation of G551D-CFTR using concentrations of genistein from 0.1 ␮M to 1000 ␮M were generated from six separate experiments. Login to comment 125 ABCC7 p.Gly551Asp Second, at concentrations that normally induced an inhibition of wt-CFTR (i.e. Ͼ 30 ␮M), genistein did not inhibit G551D-CFTR chloride channel activity. Login to comment 139 ABCC7 p.Gly551Asp Classical Michaelis-Menten kinetics of G551D-CFTR channel activated by genistein. Login to comment 140 ABCC7 p.Gly551Asp A, example traces of iodide efflux showing the stimulation of G551D-CFTR genistein in the presence of 10 ␮M forskolin. Login to comment 151 ABCC7 p.Gly551Asp Several inhibitors were also used to further characterize G551D-CFTR chloride channel activity. Login to comment 152 ABCC7 p.Gly551Asp Fig. 6, C-E, shows that in the presence of 100 ␮M glibenclamide, the activation of G551D-CFTR currents is abolished. Login to comment 154 ABCC7 p.Gly551Asp A summary of the effect of four different inhibitors on the rate of iodide efflux stimulated by FSK ϩ GST is also presented in Fig. 6F, showing the inhibition of G551D-CFTR channel activity by 100 ␮M glibenclamide and 250 ␮M DPC but not by 200 ␮M DIDS nor by 100 nM calixarene. Login to comment 155 ABCC7 p.Gly551Asp DISCUSSION We described here the mechanism of action and kinetic parameters of wt-CFTR chloride channel activated by genistein and the profound alteration resulting from the G551D mutation. Login to comment 156 ABCC7 p.Gly551Asp This study reveals two important results: (i) a non- Michaelis-Menten behavior describes the pharmacological effect of genistein on wt-CFTR, suggesting the presence of one activatory site and one inhibitory site; (ii) the non-Michaelis-Menten behavior of wt-CFTR is absent and transformed into a classical Michaelis-Menten behavior by the CF mutation G551D. Login to comment 163 ABCC7 p.Gly551Asp Our results support a model with two binding sites for genistein on wt-CFTR but only one binding site on G551D-CFTR. Login to comment 172 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Activation of G551D-CFTR chloride currents using whole cell patch clamp recording from G551D CHO cells. Login to comment 173 ABCC7 p.Gly551Asp G551D-CFTR currents were stimulated with 10 ␮M FSK and 30 ␮M GST. Login to comment 180 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp F, histograms summarizing the inhibition of genistein-mediated 125 I efflux in G551D CHO cells. Login to comment 188 ABCC7 p.Gly551Asp Indeed, one major difference concerning the phosphorylation process is that with G551D-CFTR, much higher concentrations of forskolin are required to achieve the stimulation by genistein of the CFTR channel activity. Login to comment 189 ABCC7 p.Gly551Asp The G551D-CFTR protein is a class III mutation that produced a fully glycosylated protein correctly located at the apical membrane (i.e. normal biosynthesis, trafficking, and processing) and normally phosphorylated at the R domain by cAMP-dependent protein kinase (6). Login to comment 190 ABCC7 p.Gly551Asp However, G551D proteins have decreased nucleotide binding (7) and a reduced ATPase activity at NBD1 (8, 9). Login to comment 191 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The mutation G551D also disrupts activation and regulation of CFTR at the plasma membrane since phosphorylation alone is not able to achieve efficient stimulation of chloride transport in G551D cells (12, 29, 30). Login to comment 192 ABCC7 p.Gly551Asp Our study of the activity of G551D-CFTR mutant demonstrates that the inhibitory component of the dual mechanism of the action of genistein on wt-CFTR is lacking. Login to comment 195 ABCC7 p.Gly551Asp In addition, our study reveals that the G551D mutation profoundly alters the pharmacological behavior of CFTR. Login to comment 200 ABCC7 p.Gly551Asp We have described here a similar effect with the class III CF mutation G551D and showed that it abolishes the inhibitory response to genistein. Login to comment 203 ABCC7 p.Gly551Asp In conclusion, our results show that the high affinity stimulatory site for genistein, located in NBD2, is dependent on the phosphorylation status of CFTR and preserved by G551D mutation. Login to comment 204 ABCC7 p.Gly551Asp The second site is lacking in G551D-CFTR cells. Login to comment