ABCMdb

PMID: 22138447

Loo TW, Bartlett MC, Shi L, Clarke DM
Corrector-mediated rescue of misprocessed CFTR mutants can be reduced by the P-glycoprotein drug pump.
Biochem Pharmacol. 2012 Feb 1;83(3):345-54. Epub 2011 Nov 28., [PubMed]

Sentences

No. Mutations Sentence Comment

14 ABCC7 p.His1085Arg ABCC7 p.Val232Asp Here, we tested our predictions that (1) other CFTR mutants such V232D and H1085R were more stable at the cell surface than DF508 CFTR after low temperature rescue and (2) the advantages of rescue with specific correctors (pharmacological chaperones) are that they may stabilize DF508 CFTR and increase the effectiveness of the correctors by bypassing drug pumps such as P-glycoprotein (P-gp) (increased bioavailability). Login to comment 15 ABCC7 p.His1085Arg ABCC7 p.Val232Asp It was found that the stability of mutants V232D and H1085R after low-temperature (30 8C) rescue was about 10-fold higher than DF508 CFTR. Login to comment 37 ABCC7 p.His1085Arg ABCC7 p.Val232Asp We show that rescue of DF508 CFTR with specific correctors resulted in a more stable protein and mutations such as V232D and H1085R are different from DF508 because they do not destabilize the rescued form of mature CFTR. Login to comment 67 ABCC7 p.His1085Arg To test the effect of P-gp on rescue of CFTR, cells were cotransfected with the H1085R CFTR processing mutant plus wild-type P-gp or an inactive P-gp mutant containing mutations to the catalytic carboxylate residues (E556Q/E1201Q) [26]. Login to comment 106 ABCC7 p.His1085Arg ABCC7 p.Val232Asp (B) Samples of cells expressing DF508, V232D or H1085R CFTR mutants or P-gp mutant G268V and grown in the absence (À) or presence (+) of 20 mM VX-809 for 18 h were subjected to immunoblot analysis. Login to comment 120 ABCC7 p.His1085Arg ABCC7 p.Val232Asp We also tested whether correctors would promote maturation of CFTR mutants DF508, V232D and H1085R in HEK 293 cells in parallel because it has been reported that CFTR rescue depends on the cell system used [38-41]. Login to comment 121 ABCC7 p.His1085Arg ABCC7 p.Val232Asp Mutants V232D (TMD1) and H1085R (TMD2) were included because they are processing mutations located in different domains of CFTR (Fig. 1A) and both yield active proteins after rescue [16,42,43]. Login to comment 181 ABCC7 p.His1085Arg ABCC7 p.Val232Asp Degradation of wild-type, V232D, and H1085R CFTRs was monitored over time at 37 8C. Login to comment 185 ABCC7 p.His1085Arg Fig. 8. Effect of wild-type P-gp expression (+P-gp) on rescue of CFTR mutant H1085R with corrector KM11060. Login to comment 203 ABCC7 p.His1085Arg ABCC7 p.Val232Asp To test if other CF mutations affect stability of CFTR, mutants V232D (TMD1) and H1085R (TMD2) were selected for study as both are processing mutations that yield active proteins after rescue [16,43]. Login to comment 204 ABCC7 p.His1085Arg ABCC7 p.Val232Asp Accordingly, we examined the stability of mutants V232D and H1085R after low-temperature rescue. Login to comment 205 ABCC7 p.His1085Arg ABCC7 p.Val232Asp Cells expressing wild-type, V232D, or H1085R CFTRs were expressed at low temperature to promote maturation of the protein. Protein synthesis was stopped by addition of cycloheximide, and turnover of the protein was monitored after incubation for 0-32 h at 37 8C (Fig. 7). Login to comment 206 ABCC7 p.His1085Arg ABCC7 p.Val232Asp It was observed that the half-lives of the mature forms of V232D and H1085R were at least 10-fold longer (about 14 and 12 h, respectively) than DF508 CFTR (about 1 h, Fig. 4). Login to comment 208 ABCC7 p.His1085Arg ABCC7 p.Val232Asp The V232D TMD1 and H1085R mutations may have less effect on the stability of mature CFTR because they have more localized effects on protein folding in the TMD1 and TMD2 domains, respectively. Login to comment 210 ABCC7 p.His1085Arg 3.4. Effect of P-gp expression on rescue of CFTR mutant H1085R We predicted that the efficiency of CFTR rescue with correctors would be reduced if the correctors were also substrates of drug pumps such as P-gp. Login to comment 215 ABCC7 p.His1085Arg To test if P-gp expression would alter CFTR rescue with KM11060, mutant H1085R was expressed in the presence of various concentrations of KM11060 in the presence of an inactive P-gp containing mutations to the catalytic carboxylate residues (E556Q/E1201Q) in the nucleotide binding domains [26] or in the presence of wild-type P-gp. Login to comment 216 ABCC7 p.His1085Arg CFTR mutant H1085R was tested because ithas a higher efficiency of rescue with correctors compared to DF508 CFTR [16] and the mature protein is more stable (Fig. 7). Login to comment 219 ABCC7 p.His1085Arg The R50 for KM11060 when H1085R CFTR was expressed with noexogenous P-gp was similar to that obtained with inactive P-gp (data not shown) suggesting that expression of P-gp did not adversely affect the cellular folding machinery. Login to comment 245 ABCC7 p.Val232Asp Corr-4a also may promote interactions between TMD1 and TMD2 as it restored folding of a processing mutant containing a charged residue (V232D) within TM4 [43] that is embedded in the lipid bilayer [56]. Login to comment 246 ABCC7 p.Val232Asp The V232D mutation is predicted to alter packing of the TM segments [56]. Login to comment 249 ABCC7 p.Gly551Asp There is evidence that 2b related arylaminothiazoles can directly bind to CFTR because they correct defective gating in mutants such as DF508 CFTR and G551D [57]. Login to comment 251 ABCC7 p.Gly551Asp The potentiator activity of cyanoquinolines differed from arylaminothiazoles because they activated chloride conductance of DF508 or G551D CFTRs within minutes. Login to comment