ABCMdb

PMID: 12727866

Kogan I, Ramjeesingh M, Li C, Kidd JF, Wang Y, Leslie EM, Cole SP, Bear CE
CFTR directly mediates nucleotide-regulated glutathione flux.
EMBO J. 2003 May 1;22(9):1981-9., 2003-05-01 [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Arg347Asp ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala We show that CFTR-expressing membrane vesicles mediate nucleotide-activated GSH ¯ux, which is disrupted in the R347D pore mutant, and in the Walker A K464A and K1250A mutants. Login to comment 7 ABCC7 p.Arg347Asp Keywords: CFTR/glutathione/puri®ed protein/R347D pore mutant/Walker A mutants Introduction Cystic ®brosis (CF) is a lethal autosomal recessive disease caused by mutations within the cystic ®brosis transmembrane conductance regulator (CFTR) gene (Boat et al., 1989). Login to comment 63 ABCC7 p.Arg347Asp As seen in Figure 3A (insert), the R347D variant was expressed well in Sf9 membranes. Login to comment 64 ABCC7 p.Arg347Asp We compared [35S]GSH uptake by vesicles containing either phosphorylated wild-type or R347D CFTR proteins in the presence of 1 mM GSH, as this concentration is close to the Km(GSH) and is known not to have any non-speci®c effects on non-pore regions of CFTR. Login to comment 88 ABCC7 p.Arg347Asp In the current study, we found that [35S]GSH uptake was signi®cantly decreased in membrane vesicles expressing the R347D mutant protein, relative to those expressing wild-type CFTR, with rates of GSH ¯ux of 207 and 498 pmol/mg CFTR/h, respectively (Figure 3A; P < 0.0001). Login to comment 91 ABCC7 p.Arg347Asp Kinetic analyses revealed that vesicles with the R347D mutation exhibited an ~75% decrease in the Vmax of GSH uptake compared with vesicles with wild-type CFTR, corresponding to 176 and 612 pmol GSH/mg CFTR/h, respectively (Table I). Login to comment 92 ABCC7 p.Arg347Asp These analyses also suggest that GSH interaction with the pore of the R347D mutant is ~4 times stronger than that with the wild-type pore, with Km(GSH) values of 0.11 and 0.47 mM, respectively. Login to comment 93 ABCC7 p.Arg347Asp Modi®ed af®nity of GSH for the mutant channel is consistent with previous reports, suggesting altered interaction of GSH with the R347D pore (Kogan et al., 2001). Login to comment 94 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala To assess the nucleotide dependence of GSH permeation through CFTR, we determined the consequences of lysine mutations in the conserved Walker A consensus motifs for ATP binding in NBD1 and NBD2: K464A and K1250A, respectively. Login to comment 96 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala In Figure 4, we show that both the K464A and K1250A mutants exhibit similar signi®cant reductions in GSH ¯ux. Login to comment 97 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala We observed that GSH uptake in both the K464A and K1250A membrane vesicles was 3to 4-fold lower than in vesicles expressing wild-type CFTR protein, yielding permeability values of 132 and 120 pmol/mg CFTR/h, respectively (P < 0.001). Login to comment 100 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp Kinetic parameters of GSH uptake by Sf9 membrane vesicles containing PKA-phosphorylated wild-type or R347D CFTR proteins, in the presence of MgAMP-PNP Variables Wild type R347D Vmax (pmol/mg CFTR/h) 612 176 Km (mM) 0.47 0.11 Fig. 4. Login to comment 102 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala Membrane vesicles expressing phosphorylated wild-type, K464A or K1250A CFTR were incubated with 20 nM [35S]GSH and 1 mM cold GSH in CFTR transport buffer, in the presence of MgAMPPNP. Login to comment 104 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala Values shown represent the mean activity (T SEM; for K464A and K1250A, n = 4; for wild-type CFTR, n = 5). Login to comment 105 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala Inset: expression of CFTR in membranes from Sf9 cells transfected with wild-type, K464A or K1250A CFTR constructs. Login to comment 108 ABCC7 p.Arg347Asp Comparison of GSH ¯ux by wild-type CFTR versus CFTR R347D protein. Login to comment 109 ABCC7 p.Arg347Asp (A) Membrane vesicles expressing phosphorylated wild-type or R347D CFTR proteins were incubated with 20 nM [35S]GSH and 1 mM cold GSH, in the presence of MgAMP-PNP. Login to comment 111 ABCC7 p.Arg347Asp Values are expressed as the mean T SEM (n = 5 for wild-type CFTR, n = 2 for R347D). Login to comment 112 ABCC7 p.Arg347Asp Inset: expression of CFTR in membranes from Sf9 cells transfected with wild-type or R347D CFTR constructs. Login to comment 115 ABCC7 p.Arg347Asp (B) Effect of increasing substrate concentration on [35S]GSH uptake by vesicles expressing phosphorylated wild-type CFTR or the R347D variant, in the presence of MgAMP-PNP. Login to comment 117 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp [35S]GSH uptake by vesicles expressing the R347D protein was also obtained by subtracting GSH uptake values of vesicles with no CFTR from those of vesicles expressing the R347D variant. Login to comment 118 ABCC7 p.Arg347Asp Curve ®tting was performed by non-linear regression analysis, using the Michaelis±Menten equation, to yield the following kinetic parameters for the R347D mutant: Km = 0.11 mM, Vmax = 176 pmol GSH/mg CFTR/h, r2 = 0.97. Login to comment 194 ABCC7 p.Arg347Asp ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala An Sf9 cell pellet (0.5 l) expressing either recombinant CFTR-His proteins (wild type or mutant: R347D, K464A, K1250A) or no CFTR was solubilized in 30 ml of homogenization buffer containing 250 mM sucrose, 50 mM Tris±HCl, 0.25 mM CaCl2 pH 7.5 and protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). Login to comment