ABCMdb

PMID: 20053923

Pedemonte N, Tomati V, Sondo E, Galietta LJ
Influence of cell background on pharmacological rescue of mutant CFTR.
Am J Physiol Cell Physiol. 2010 Apr;298(4):C866-74. Epub 2010 Jan 6., [PubMed]

Sentences

No. Mutations Sentence Comment

13 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Most potentiators were also effective on two mutations, G551D and G1349D, that cause a purely gating defect. Login to comment 17 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Most potentiators were also effective on two mutations, G551D and G1349D, that cause a purely gating defect. Login to comment 27 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Class 3 mutations, such as G1349D and G551D (2, 11), have normal trafficking but are affected by a gating defect more severe than that of F508del. Login to comment 28 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp G551D and G1349D alter two highly conserved glycines in NBD1 and NBD2, respectively. Login to comment 31 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Class 3 mutations, such as G1349D and G551D (2, 11), have normal trafficking but are affected by a gating defect more severe than that of F508del. Login to comment 32 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp G551D and G1349D alter two highly conserved glycines in NBD1 and NBD2, respectively. Login to comment 49 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In brief, FRT cells were first stably transfected with the plasmid pTRACER (Invitrogen) containing the cDNA encoding CFTR (wild type or mutant F508del, G1349D, or G551D) and selected in Zeocin (0.6 mg/ml). Login to comment 53 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In brief, FRT cells were first stably transfected with the plasmid pTRACER (Invitrogen) containing the cDNA encoding CFTR (wild type or mutant F508del, G1349D, or G551D) and selected in Zeocin (0.6 mg/ml). Login to comment 76 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp FRT cells expressing mutant G1349D or G551D were grown at 37°C (90% humidity; 5% CO2) for 18-24 h. Login to comment 80 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp FRT cells expressing mutant G1349D or G551D were grown at 37°C (90% humidity; 5% CO2) for 18-24 h. Login to comment 85 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Potentiator activity on G551D- and G1349D-CFTR. Login to comment 86 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp A: original traces recorded in FRT cells expressing G1349D-CFTR (top) and G551D-CFTR (bottom), showing quenching of cellular YFP fluorescence by I- addition after stimulation with forskolin (20 ␮M) alone or forskolin ϩ indicated compounds (20 ␮M and 45 ␮M for G1349D and G551D, respectively). Login to comment 87 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp B: dose-response analysis of indicated compounds in G1349D-CFTR (top) and G551D-CFTR (bottom) cells (means Ϯ SE, n ϭ 6). Login to comment 88 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp C: maximal effect obtained from dose response of potentiators in G1349D-CFTR (left) and G551D-CFTR (right) cells, normalized to genistein effect (means Ϯ SE, n ϭ 9). Login to comment 89 ABCC7 p.Gly1349Asp Potentiator activity on G551Dand G1349D-CFTR. Login to comment 90 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp A: original traces recorded in FRT cells expressing G1349D-CFTR (top) and G551D-CFTR (bottom), showing quenching of cellular YFP fluorescence by I- addition after stimulation with forskolin (20 ␮M) alone or forskolin ϩ indicated compounds (20 ␮M and 45 ␮M for G1349D and G551D, respectively). Login to comment 91 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp B: dose-response analysis of indicated compounds in G1349D-CFTR (top) and G551D-CFTR (bottom) cells (means Ϯ SE, n ϭ 6). Login to comment 92 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp C: maximal effect obtained from dose response of potentiators in G1349D-CFTR (left) and G551D-CFTR (right) cells, normalized to genistein effect (means Ϯ SE, n ϭ 9). Login to comment 93 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Note that the concentrations needed to stimulate the G551D mutant are always 5-10 times higher than those effective on F508del or G1349D. Login to comment 94 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp D: correlation between potentiator activity on G1349D-CFTR and G551D-CFTR. Login to comment 95 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Each symbol represents the activity of a single potentiator on G1349D (left)- and G551D (right)- vs. F508del-CFTR. Login to comment 97 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Note that the concentrations needed to stimulate the G551D mutant are always 5-10 times higher than those effective on F508del or G1349D. Login to comment 98 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Gray lines indicate correlation lines (R ϭ 0.96 for G1349D and R ϭ 0.95 for G551D). Login to comment 153 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Potentiators were also tested on FRT cells expressing the G1349D and the G551D mutants with the YFP assay (Fig. 3). Login to comment 154 ABCC7 p.Gly551Asp As expected, CFTR activity in G551D cells was undetectable despite the use of a maximal forskolin concentration (Fig. 3A). Login to comment 155 ABCC7 p.Gly1349Asp In contrast, the gating defect of the G1349D mutant was less severe, allowing a significant cAMP-activated anion transport. Login to comment 156 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp CFTR activity in G551D and G1349D cells was strongly stimulated in the presence of potentiators (Fig. 3A). Login to comment 157 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Potentiators were also tested on FRT cells expressing the G1349D and the G551D mutants with the YFP assay (Fig. 3). Login to comment 158 ABCC7 p.Gly551Asp As expected, CFTR activity in G551D cells was undetectable despite the use of a maximal forskolin concentration (Fig. 3A). Login to comment 159 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In contrast, the gating defect of the G1349D mutant was less severe, allowing a significant cAMP-activated anion transport. Login to comment 160 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp CFTR activity in G551D and G1349D cells was strongly stimulated in the presence of potentiators (Fig. 3A). Login to comment 161 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Interestingly, the order of potency for active potentiators in G551D and G1349D correlated well with the order of potency in F508del (Fig. 3D). Login to comment 162 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp However, the Ka values for G551D were consistently 5-10 times higher than those for G1349D and F508del. Login to comment 163 ABCC7 p.Gly551Asp Other compounds showed partial activity, particularly on G551D cells. Login to comment 165 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Interestingly, the order of potency for active potentiators in G551D and G1349D correlated well with the order of potency in F508del (Fig. 3D). Login to comment 166 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp However, the Ka values for G551D were consistently 5-10 times higher than those for G1349D and F508del. Login to comment 236 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Potentiators were also tested on two classical CF mutations belonging to class 3 (gating defect), namely, G551D and G1349D. Login to comment 237 ABCC7 p.Gly551Asp Such mutations are particularly interesting because they cause a drastic reduction in CFTR activity (with G551D being the most severe mutation) and because they involve two highly conserved glycine residues in the first and second NBDs, respectively. Login to comment 239 ABCC7 p.Gly551Asp In particular, the G551D mutant was almost totally insensitive to these three compounds. Login to comment 240 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Potentiators were also tested on two classical CF mutations belonging to class 3 (gating defect), namely, G551D and G1349D. Login to comment 241 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Such mutations are particularly interesting because they cause a drastic reduction in CFTR activity (with G551D being the most severe mutation) and because they involve two highly conserved glycine residues in the first and second NBDs, respectively. Login to comment 242 ABCC7 p.Gly551Asp Although the order of potency was maintained, the Ka values for G551D were consistently 5-10 times higher than those of the other two mutants. Login to comment 243 ABCC7 p.Gly551Asp In particular, the G551D mutant was almost totally insensitive to these three compounds. Login to comment 244 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Other potentiators were also less effective in activating G551D, such as the sulfonamide P3. Login to comment 245 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In general, the order of potency of the active potentiators was similar for G551D, G1349D, and F508del. Login to comment 246 ABCC7 p.Gly551Asp Although the order of potency was maintained, the Ka values for G551D were consistently 5-10 times higher than those of the other two mutants. Login to comment 248 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Our findings suggest that all potentiators having activity on G551D, G1349D, and F508del (P1-P7 and DHPs) act with the same mechanism of action, possibly through binding to the same site on CFTR protein. Login to comment