ABCMdb

PMID: 12181609

Bulteau-Pignoux L, Derand R, Metaye T, Joffre M, Becq F
Genistein modifies the activation kinetics and magnitude of phosphorylated wild-type and G551D-CFTR chloride currents.
J Membr Biol. 2002 Aug 1;188(3):175-82., 2002-08-01 [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Gly551Asp We have studied the mechanism by which genistein activates cystic ®brosis transmembrane conductance regulator (CFTR) in CHO cells expressing wild type or G551D-CFTR. Login to comment 8 ABCC7 p.Gly551Asp Among the 900 mutations associated with a CF disease phenotype, the G551D mutation is the third most common one with a frequency of 2±5%, depending on the population of origin (Hamosh et al., 1992; Cashman et al., 1995). Login to comment 10 ABCC7 p.Gly551Asp G551D, localized in NBD1 of CFTR, interferes with ATP binding (Logan et al., 1994) and hydrolysis (Li et al., 1996). Login to comment 28 ABCC7 p.Gly551Asp In the present study, we further explored the mechanism by which genistein activates wild type and G551D-CFTR expressed in CHO cells. Login to comment 30 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Materials and Methods CELL CULTURE Chinese Hamster Ovary cells, stably transfected with pNUT vector containing wild-type CFTR (CFTR(+) CHO) or G551D mutation (G551D CHO) were provided by J.R. Riordan and X.-B. Chang, Scottsdale, AZ, USA (Tabcharani et al., 1991). Login to comment 31 ABCC7 p.Gly551Asp Cells cultured at 37°C in 0.5% CO2 were maintained in aMEM containing 7% fetal bovine serum, 0.5% L-glutamine, 0.5% antibiotics (50 IU/ml penicillin and 50 lg/ml streptomycin) and 100 or 20 lM methotrexate for, respectively, CFTR(+) or G551D CHO. Login to comment 60 ABCC7 p.Gly551Asp Results We compared activation of wild type and G551D-CFTR by low concentrations of forskolin and genistein, with two protocols of activation, using the whole-cell and perforated patch-clamp techniques. Login to comment 61 ABCC7 p.Gly551Asp In the ®rst protocol, cells were preincubated during 3 minutes with 2.5 lM (for CFTR(+) CHO) or 10 lM (for G551D CHO) forskolin, then 25 lM genistein was added in the presence of forskolin. Login to comment 83 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Since one of the main dierences between wild-type and G551D-CFTR is the lack of responsiveness of the mutant to cAMP agonists, we then performed the same study in G551D CHO cells (Fig. 4). Login to comment 84 ABCC7 p.Gly551Asp No current was activated in the presence of 10 lM fors- Fig. 4. Eect of genistein on forskolin-stimulated G551D-CFTR ClÀ channels. Representative current traces for CFTR. Login to comment 87 ABCC7 p.Gly551Asp Fig. 5. Eect of forskolin on genistein-stimulated G551D-CFTR ClÀ channels. Representative current traces for CFTR for cell ®rst incubated with 25 lM genistein (A), then in the presence of 10 lM forskolin (B). Login to comment 92 ABCC7 p.Gly551Asp In G551D CHO cells not treated with forskolin (Fig. 5), genistein failed to stimulate ClÀ current (Fig. 5A). Login to comment 97 ABCC7 p.Gly551Asp When genistein was ®rst added to the bath, subsequent addition of forskolin led to a current density $3-fold (for wild type) and $4-fold (for G551D) lower than when CFTR was ®rst stimulated by forskolin. Login to comment 98 ABCC7 p.Gly551Asp A time course of the activation of G551D-CFTR current is shown in Fig. 7. Login to comment 102 ABCC7 p.Gly551Asp Activation for G551D-CFTR current (for both conditions) is signi®cantly slower than for wild-type CFTR (compare time constants in Tables 1 and 2). Login to comment 108 ABCC7 p.Gly551Asp Here we show that low concentration of genistein (25 lM) dierentially activates wild type or G551D-CFTR channels depending on whether the protein is phosphorylated before or after genistein interaction with CFTR. Login to comment 110 ABCC7 p.Gly551Asp When forskolin was added after genistein preincubation, (1) whole-cell current density was reduced by $ 3-fold (for wild type) and $4-fold (for G551D), (2) time to activation was greatly delayed, and (3) time constant of activation was reduced when compared with genistein activation after phosphorylation of CFTR. Login to comment 111 ABCC7 p.Gly551Asp Direct interaction of genistein with CFTR has been shown by Randak et al. (1999) who demonstrated Table 2. Comparison of time constants determined at dierent conditions of activation in G551D CHO cells First activator Second activator Activation delay Time constant 1/K sec n P sec n P Fsk Gst 8 1 5 138 30 5 Gst Fsk 120 40 3 * 522 39 3 ** For methods of analysis, see Table 1. Login to comment 114 ABCC7 p.Gly551Asp Fig. 6. Comparison of wild-type and G551D-CFTR current densities at +40 mV holding potential. Login to comment 126 ABCC7 p.Gly551Asp We also found that the kinetic parameters of activation by genistein are similar in wild type and G551D CFTR cells. Login to comment