ABCMdb

PMID: 17535157

Wang Y, Loo TW, Bartlett MC, Clarke DM
Additive effect of multiple pharmacological chaperones on maturation of CFTR processing mutants.
Biochem J. 2007 Sep 1;406(2):257-63., 2007-09-01 [PubMed]

Sentences

No. Mutations Sentence Comment

33 ABCC7 p.His1085Arg MATERIALS AND METHODS Construction and expression of mutants The construction of F508 and H1085R CFTR cDNAs and insertion into the pcDNA3 vector (Invitrogen Canada, Burlington, ON, Canada) was described previously [23]. Login to comment 34 ABCC7 p.His1085Arg The CFTR cDNAs coding for NBD2 (where NBD2 is nucleotide-binding domain 2), H1085R CFTR (residues 1-1196) as well as P-gp cDNAs for mutant P709G P-gp and P-gp truncation mutants NBD2 P-gp (residues 1-1023), TMD1+2 {TMD1 [N-terminal TMD (TM domain) containing TM segments 1-6] and TMD2 (C-terminal TMD containing TM segments 7-12)}, P-gp (residues 1-379 plus 681-1025) and TMD1 P-gp (residues 1-379) were modified to contain the epitope for monoclonal antibody A52 at the C-terminal ends and subcloned into the mammalian expression vector pMT21 as described previously [21]. Login to comment 103 ABCC7 p.His1085Arg ABCC7 p.His1085Arg Figure 4 Effect of correctors on maturation of CFTR processing mutant H1085R CFTR processing mutant H1085R was expressed for 24 h in the absence (-) or presence (+) of 3 µM VRT-325, 10 µM corr-2b, 10 µM corr-4a, combination of 3 µM VRT-325 and 10 µM corr-2b, combination of 3 µM VRT-325 and 10 µM corr-4a, or combination of 10 µM corr-2b and 10 µM corr-4a respectively. Login to comment 112 ABCC7 p.His1085Arg First, we tested the correctors on a CFTR processing mutant (H1085R) with a mutation in the fourth cytoplasmic loop (CL4) connecting TM segments 10 and 11 in the CO2H half of the molecule, where a relatively large number of clinically relevant processing mutations are found [35]. Login to comment 113 ABCC7 p.His1085Arg The H1085R CFTR mutant was selected for use in our initial studies because expression of its cDNA in HEK-293 cells yields a small amount of mature protein (approx. Login to comment 115 ABCC7 p.His1085Arg Therefore the use of mutant H1085R in the presence of combinations of correctors would enable us to determine if the compounds increase or decrease the efficiency of maturation. Login to comment 116 ABCC7 p.His1085Arg CFTR processing mutant H1085R was transiently expressed in HEK-293 cells and incubated in the presence or absence of 3 µM VRT-325, 10 µM corr-2b and 10 µM corr-4a, combination of VRT-325 and corr-2b, combination of VRT-325 and corr-4a, or combination of corr-2b and corr-4a for 24 h. Whole cell extracts were subjected to immunoblot analysis. Login to comment 119 ABCC7 p.His1085Arg Expression of mutant H1085R CFTR in the presence of a combination of VRT-325 and corr-2b caused the largest increase in maturation efficiency as 48% of total CFTR was present as mature protein. Login to comment 122 ABCC7 p.His1085Arg ABCC7 p.His1085Arg It has been postulated that correctors promote maturation of CFTR processing mutants by promoting interactions between the Figure 5 Effect of correctors on maturation of CFTR truncation mutant NBD2 H1085R HEK-293 cells were transfected with NBD2 H1085R followed by a 24 h incubation in the absence (-) or presence (+) of 3 µM VRT-325, 10 µM corr-2b, 10 µM corr-4a, combination of 3 µM VRT-325 and 10 µM corr-2b, or combination of 3 µM VRT-325 and 10 µM corr-4a respectively. Login to comment 126 ABCC7 p.His1085Arg ABCC7 p.His1085Arg ABCC7 p.His1085Arg To determine if interactions between the NBDs were required for the enhanced maturation of mutant H1085R in the presence of VRT-325 together with corr-2b or corr-4a, we utilized an H1085R mutant that lacked NBD2 ( NDB2 H1085R CFTR) (Figure 1B). Login to comment 127 ABCC7 p.His1085Arg The NDB2 H1085R CFTR mutant was transfected into HEK-293 cells and expressed in the presence of VRT-325, corr-2b, corr-4a, VRT-325 plus corr-2b, VRT-325 plus corr-4a or no correctors for 24 h. Whole cell extracts were then subjected to immunoblot analysis. Login to comment