ABCMdb

PMID: 15657297

Wang W, Oliva C, Li G, Holmgren A, Lillig CH, Kirk KL
Reversible silencing of CFTR chloride channels by glutathionylation.
J Gen Physiol. 2005 Feb;125(2):127-41. Epub 2005 Jan 18., [PubMed]

Sentences

No. Mutations Sentence Comment

43 ABCC7 p.Ser660Ala The S660A/⌬R-CFTR mutant and all but one of the alanine-substituted cysteine mutants were provided by M. Welsh (University of Iowa, Iowa City, IA) (Rich et al., 1991; Cotton and Welsh, 1997) and were subcloned into the pCDNA3 expression vector (Invitrogen). Login to comment 44 ABCC7 p.Cys1458Ala C1458A-CFTR was gener- ated by PCR mutagenesis and subcloned into pCDNA3. Login to comment 45 ABCC7 p.Cys590Leu A CFTR construct lacking all 18 cysteines (16CS C590L/592L) was provided by D. Gadsby (Rockefeller University, New York, NY) (Mense et al., 2004). Login to comment 51 ABCC7 p.Ser660Ala HEK-293T cells that were transfected with S660A/⌬R-CFTR or with the cys-free constructs were grown overnight at 27ЊC because these mutants are temperature-sensitive ER processing mutants as determined by immunoblot analysis. Login to comment 169 ABCC7 p.Cys1344Ala All CFTR constructs, with the exception of C1344A-CFTR, were markedly inhibited by diamide/GSH (Fig. 7, B and C). Login to comment 170 ABCC7 p.Cys1344Ala C1344A-CFTR was largely, although not completely, resistant to each of the three glutathione species at the indicated concentrations (Fig. 7, B and D). Login to comment 203 ABCC7 p.Ser660Ala Fig. 9 (A and B) shows that S660A/⌬R-CFTR, which was previously shown to have moderate channel activity in the absence of PKA (Rich et al., 1991), was substantially and reversibly inhibited by diamide/GSH in the absence of added kinase. Login to comment 205 ABCC7 p.Ser660Ala Interestingly, Grx could rescue completely the currents mediated by S660A/⌬R-CFTR, which indicates that the Grx-insensitive oxidation previously observed for the WT channel (Fig. 4) requires an intact R domain. Login to comment 212 ABCC7 p.Cys1344Ala CFTR channels that lack cys-1344 (C1344A-CFTR) are largely resistant to inhibition by reactive glutathione species. Login to comment 219 ABCC7 p.Cys1344Ala (C) Representative current trace showing resistance of C1344A-CFTR to inhibition by diamide/GSH. Login to comment 220 ABCC7 p.Cys1344Ala (D) Mean data comparing the sensitivities of WT CFTR and C1344A-CFTR to inhibition by diamide/ GSH (20 ␮M), GSNO (200 ␮M), and GSSG (20 mM). Login to comment 227 ABCC7 p.Cys1344Ala However, the fact that C1344A-CFTR was the only cysteine mutant that was highly resistant to inhibition by all three reactive glutathione species (GSNO, GSSG, and diamide/GSH) indicates that this cysteine likely is the functionally important site for glutathionylation. Login to comment 253 ABCC7 p.Gly551Asp For example, disease-associated mutations within the NBD1 signature sequence (e.g., G551D) dramatically inhibit the rate of CFTR channel opening (Li et al., 1996). Login to comment 259 ABCC7 p.Ser660Ala (A) Inhibitory effect of 20 ␮M diamide/ GSH on macroscopic current mediated by ⌬R-S660A-CFTR in patch excised from transfected HEK-293T cell. Login to comment 260 ABCC7 p.Ser660Ala (B) Mean data for ⌬R-S660A-CFTR showing inhibition by diamide/GSH (20 ␮M) and recovery by E. coli Grx1 (4 ␮M) plus 1 mM GSH. Login to comment