ABCMdb

PMID: 9849891

Wei L, Vankeerberghen A, Cuppens H, Droogmans G, Cassiman JJ, Nilius B
Phosphorylation site independent single R-domain mutations affect CFTR channel activity.
FEBS Lett. 1998 Nov 13;439(1-2):121-6., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln All mutations were found in cystic fibrosis (CF) patients (H620Q, E822K and E826K). Login to comment 2 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln The macroscopic CFTR chloride conductance induced by phosphorylation was significantly enhanced in Xenopus oocytes injected with mRNA of H620Q but reduced in the E822K and E826K mutants compared to wild type CFTR. Login to comment 4 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln Cell attached single channel studies in COS cells revealed that both open channel probability and/or the number of functional channels were either higher (H620Q) or lower (E822K and E826K) than in wild type CFTR. Login to comment 25 ABCC7 p.Glu826Lys ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln ABCC7 p.His620Gln Three di¡erent mutations, t1992g (= H620Q), g2596a (= E822K) and g2608a (= E826K), were introduced using the Transformer Site-Directed Mutagenesis kit (Clontech). Login to comment 74 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln COS cells were transfected with wt CFTR, E822K CFTR (top), H620Q CFTR (bottom) and E826K CFTR (bottom) and selected for 2 weeks with G418. Login to comment 76 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln COS cells were transfected with wt CFTR, E822K CFTR (top), H620Q CFTR (bottom) and E826K CFTR (bottom) and selected for 2 weeks with G418. Login to comment 86 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln In this study, the maturation pattern of three mutant R-domain proteins (H620Q-CFTR, E822K-CFTR and E826K-CFTR) has been characterized. Login to comment 88 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln In this study, the maturation pattern of three mutant R-domain proteins (H620Q-CFTR, E822K-CFTR and E826K-CFTR) has been characterized. Login to comment 90 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln Whole cells currents of mutants in Xenopus oocytes Whole cell membrane currents were recorded from Xenopus oocytes injected with RNA transcribed from either wild type or mutant (H620Q, E822K, E826K) constructs. Login to comment 92 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln Whole cells currents of mutants in Xenopus oocytes Whole cell membrane currents were recorded from Xenopus oocytes injected with RNA transcribed from either wild type or mutant (H620Q, E822K, E826K) constructs. Login to comment 98 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys The two R-domain proteins E822K and E826K, in which a negatively charged glutamic acid was exchanged for a positively charged lysine, showed a signi'cantly smaller phos-cock activated conductance. Login to comment 99 ABCC7 p.His620Gln Oocytes expressing the mutant R-domain protein (H620Q), in which a predominantly positively charged histidine was substituted by a less charged glutamine (at pH 7.2), showed a much larger conductance activated by application of phos-cock. Login to comment 100 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys The two R-domain proteins E822K and E826K, in which a negatively charged glutamic acid was exchanged for a positively charged lysine, showed a signi'cantly smaller phos-cock activated conductance. Login to comment 101 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln Oocytes expressing the mutant R-domain protein (H620Q), in which a predominantly positively charged histidine was substituted by a less charged glutamine (at pH 7.2), showed a much larger conductance activated by application of phos-cock. Login to comment 102 ABCC7 p.His620Gln The conductance activated by phos-cock in H620Q expressed cells was 27.84 &#fe; 5.7 WS (n = 6). Login to comment 103 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys The conductance for E822K and E826K was 3.55 þ 0.44 WS (n = 6) and 4.24 þ 0.37 WS (n = 6), as compared to 7.57 þ 0.65 WS (n = 14) in the wild type. Login to comment 104 ABCC7 p.His620Gln The conductance activated by phos-cock in H620Q expressed cells was 27.84 þ 5.7 WS (n = 6). Login to comment 132 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln We found that the open probability of the E822K and E826K mutants was signi'cantly lower than that of wild type CFTR, whereas that of the H620Q mutant was strongly enhanced compared to wild type (not shown). Login to comment 134 ABCC7 p.Glu826Lys ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln ABCC7 p.His620Gln We found that the open probability of the E822K and E826K mutants was signi'cantly lower than that of wild type CFTR, whereas that of the H620Q mutant was strongly enhanced compared to wild type (not shown). Login to comment 135 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys The di&#a1;erences between wild type CFTR and E822K and E826K are signi'- cant (P 6 0.05). Login to comment 136 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln The average number of activated channels is 2.6 þ 0.306 (n = 10) for wild-type CFTR, 3.51 þ 0.428 (n = 6) for H620Q, 1.0 þ 0.000 (n = 4) for E822K and 1.66 þ 0.211 (n = 6) for E826K. Login to comment 137 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys The di¡erences between wild type CFTR and E822K and E826K are signi'- cant (P 6 0.05). Login to comment 139 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys All the mutations studied here are located outside the phosphorylation sites, which may suggest that other regions in the R-domain, especially the highly conserved regions where E822K and E826K were l,ocated are important for the regulation of the CFTR Cl3 channel. Login to comment 141 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys All the mutations studied here are located outside the phosphorylation sites, which may suggest that other regions in the R-domain, especially the highly conserved regions where E822K and E826K were l,ocated are important for the regulation of the CFTR Cl3 channel. Login to comment 143 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln A comparison of the three R-domain mutants leads to a remarkable conclusion: both mutations, E822K and E826K, in which negatively charged glutamic acids were replaced by positively charged lysine had a signi'cantly reduced CFTR channel activity, whereas the H620Q mutation, in which positively charged histidine was replaced by the more neutral amino acid glutamine, had a much higher channel activity. Login to comment 145 ABCC7 p.Glu826Lys ABCC7 p.Glu822Lys ABCC7 p.His620Gln A comparison of the three R-domain mutants leads to a remarkable conclusion: both mutations, E822K and E826K, in which negatively charged glutamic acids were replaced by positively charged lysine had a signi'cantly reduced CFTR channel activity, whereas the H620Q mutation, in which positively charged histidine was replaced by the more neutral amino acid glutamine, had a much higher channel activity. Login to comment 153 ABCC7 p.Glu822Lys However, it may be not enough for interpreting a mutant like E822K, in which only one positively charge mutation in the R-domain almost completely eliminates single channel activity. Login to comment 155 ABCC7 p.Glu822Lys However, it may be not enough for interpreting a mutant like E822K, in which only one positively charge mutation in the R-domain almost completely eliminates single channel activity. Login to comment