ABCMdb

PMID: 23104983

He L, Kota P, Aleksandrov AA, Cui L, Jensen T, Dokholyan NV, Riordan JR
Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.
FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26., [PubMed]

Sentences

No. Mutations Sentence Comment

11 ABCC7 p.Gly551Asp The effectiveness of this strategy is most convincingly demonstrated by the small-molecule VX-770 (Kalydeco) that overcomes the dysfunction of G551D CFTR present in a significant subset of patients and improves their epithelial salt and fluid homeostasis and lung function (5). Login to comment 13 ABCC7 p.Gly551Asp However, as yet, these so-called correctors have been much less effective in early clinical trials on patients with the èc;F508 mutation than VX-770 is on patients with the G551D mutation (5). Login to comment 43 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr Metabolic pulse chase BHK cells stably expressing èc;F508 CFTR with or without I539T were treated with VX-809 (3 òe;M; Vertex Pharmeceuti- cals, Cambridge, MA, USA) for 24 h at 27&#b0;C. Cells expressing wild-type (WT) CFTR were grown at 37&#b0;C. Long-term pulse-chase experiments were performed in the absence (WT CFTR) or the presence of VX-809 (èc;F508 and èc;F508/I539T CFTR) to follow the lifetime of mature CFTR (14). Login to comment 44 ABCC7 p.Ile539Thr Briefly, BHK cells stably expressing WT CFTR, èc;F508 CFTR, and èc;F508/I539T CFTR were grown in 60-mm-diameter dishes and labeled for 8 h in 1.5 ml methionine-free medium supplemented with 10% normal growth medium, 10% FBS, and 66 òe;Ci/ml 35 S-methionine (Perkin-Elmer, Waltham, MA), at 27 or 37&#b0;C. Cells were then washed 2 times and chased at 37&#b0;C with growth medium supplemented with 10 mM unlabeled methionine. Login to comment 62 ABCC7 p.Ile539Thr The transport capacity of the structural unit ᐹॹᐺ for èc;F508/I539T CFTR with unstable gating kinetics and variable conductive state at 35&#b0;C was estimated as total charge transported in 10 min (area under the trace), divided by the potential difference applied and normalized per second so as to be an exact analog of ॹPo used for the channels with stable and well-defined open state. Login to comment 77 ABCC7 p.Ile539Thr Furthermore, even when the modestly effective second-site suppressor mutation, I539T (22, 23) also was present in VX-809-treated èc;F508-CFTR-ex- Figure 1. Login to comment 82 ABCC7 p.Ile539Thr While these lifetimes as reflections of the stability of the protein in cells do not necessarily equate to the functional thermal stability of its ion channel activity, only very transient channel activity with low open probability was observed in èc;F508/I539T-CFTR-expressing cells treated with VX-809 (Fig. 2B). Login to comment 83 ABCC7 p.Ile539Thr Thus, the shortened functional lifetime of the èc;F508-CFTR channel described previously (14, 25, 26) is not extended by the I539T substitution. Login to comment 84 ABCC7 p.Ile539Thr We used the èc;F508/I539T- CFTR variant as it, in contrast to the èc;F508 CFTR (14), has reasonably stable functional activity at 25&#b0;C, although it inactivates at higher temperatures (24). Login to comment 85 ABCC7 p.Ser492Pro Therefore, it serves as a basis for the comparison of the influence of the VX-809 compound with that of a known stabilizing second-site mutation, S492P (24). Login to comment 86 ABCC7 p.Ile539Thr For VX-809 treatment, the èc;F508/I539T CFTR variant was continuously exposed to the compound during cell growth, membrane vesicle isolation, and channel assay. Login to comment 88 ABCC7 p.Ile539Thr ABCC7 p.Ser492Pro This behavior strongly contrasted that of the èc;F508/I539T variant with the stabilizing S492P mutation added, where transport capacity increased, and full conductance state persisted up to 35&#b0;C (compare tracings in Fig. 2Biii, iv). Login to comment 92 ABCC7 p.Ile539Thr We employed this assay to compare the folded state of èc;F508 CFTR that had matured under the influence of the strongly NBD1-stabilizing 4PT modification (prolines introduced at 4 mobile sites: S422, S434, S492, and A534 plus I539T); or exposure of cells to VX-809 at 27&#b0;C. Login to comment 96 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr A) BHK cells stably expressing èc;F508 CFTR with or without I539T were treated with VX-809 (3 òe;M) for 24 h at 27&#b0;C. Cells expressing WT CFTR were grown at 37&#b0;C. Long-term pulse-chase experiments were performed in the absence of VX-809 (WT CFTR) or presence of VX-809 (èc;F508 and èc;F508/I539T CFTR) to follow the lifetime of CFTR, as described in Materials and Methods. Login to comment 98 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Ser492Pro B) Single-channel recordings of èc;F508/I539T CFTR (èc;F/T) rescued by 3 òe;M VX-809 at 35&#b0;C (i) and 25&#b0;C (ii) and of èc;F508/I539T/S492P CFTR (èc;F/PT) as an example of an already known (24) alternative type of èc;F508/I539T, thermally stabilized by proline substitutions at 35&#b0;C (iii) and 25&#b0;C (iv). Login to comment 99 ABCC7 p.Ile539Thr Six independent experiments of 38 min total time were used to estimate transport capacity ᐹॹᐺ afd; 1.46 afe; 0.28 for èc;F508/I539T at 25&#b0;C; data are shown as means afe; se. Login to comment 100 ABCC7 p.Ile539Thr Five independent experiments of 34 min total time were used to estimate ᐹॹᐺ afd; 0.35 afe; 0.14 for èc;F508/I539T at 35&#b0;C. Login to comment 101 ABCC7 p.Ile539Thr ABCC7 p.Ser492Pro Two sets of 4 independent experiments of 35 and 38 min total time were used to estimate transport capacity ᐹॹᐺ afd; 0.85 afe; 0.26 at 25&#b0;C (iii) and ᐹॹᐺ afd; 3.14 afe; 0.32 at 35&#b0;C (iv) for èc;F508/I539T/S492P CFTR. Login to comment 122 ABCC7 p.Ile539Thr ABCC7 p.Ala534Pro ABCC7 p.Ser434Pro ABCC7 p.Ser422Pro ABCC7 p.Ser492Pro 4PT, S422P/S434P/S492P/ A534P/I539T. Login to comment 126 ABCC7 p.Val510Cys ABCC7 p.Gly1069Cys ABCC7 p.Gln1280Cys HEK293 cells were transiently transfected with Cys-less CFTR or Cys-less èc;F508 CFTR with Cys pairs 276C/Q1280C or V510C/G1069C, introduced at NBD2/CL2 and NBD1/CL4 interfaces, respectively (13). Login to comment 134 ABCC7 p.Ile539Thr VX-809 treatment of the combined NBD1 signature suppressor mutations together with the I539T substitution (èc;F/4S) caused substantial further enhancement of maturation at both temperatures. Login to comment 135 ABCC7 p.Ile539Thr With the strongly stabilizing regulatory insertion deletion (èc;RI), the compound caused large increments that were of equivalent magnitude at both temperatures, and a similar effect was observed with the proline insertions in the context of I539T variant (4PT). Login to comment 138 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp The R1070W substitution in CL4 may do so by contributing to interactions among a cluster of aromatic residues at the interface that is weakened by the absence of F508 from the NBD1 surface (11), whereas the V510D mutation was proposed to provide a salt bridge with R1070 (31). Login to comment 139 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp The V510D mutant on the NBD1 side of the interface is very sensitive to further enhancement of maturation by the compound, whereas that on the CL4 side (R1070W) responds only rather weakly (Fig. 5B). Login to comment 140 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp This difference may reflect the fact that the V510D substitution stabilizes isolated NBD1 (32) in the absence of the rest of CFTR, as well as influencing the interface, whereas R1070W has only the latter effect. Login to comment 141 ABCC7 p.Arg1070Trp One might speculate that the less substantial influence of VX-809 on R1070W/èc;F508 CFTR could possibly reflect similar effects of either the tryptophan residue or the aromatic small molecule to partially fill the void left by the absence of the F508 residue. Login to comment 142 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Val510Asp Interestingly, as has already been observed by others (9), the influence of the combined V510D and R1070W substitutions is similar to that of V510D alone, which would not be expected if V510D were forming a salt bridge with R1070 but might be if V510D acted primarily to stabilize the NBD1 domain, as has been observed (32). Login to comment 148 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Ala534Pro ABCC7 p.Ser434Pro ABCC7 p.Ser422Pro ABCC7 p.Ser492Pro A) èc;F508 with NBD1-stabilizing mutations: 4S, I539T/G550E/R553M/R555K; èc;RI, deletion of amino acid residues 404-435; 4PT, S422P/S434P/S492P/A534P/I539T. Login to comment 149 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp B) èc;F508 with NBD1/CL4 interface substitutions R1070W and/or V510D. Login to comment 157 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp Interestingly, the patterns of enhancement by the compounds were remarkably similar for each of the three classes of NBD1 stabilizing mutations (èc;F/èc;RI, èc;F4S, and èc;F/4PT) with or without one of the interface substitutions (R1070W or V510D). Login to comment 167 ABCC7 p.Arg560Thr The èc;I507 mutant that also causes severe disease (36) was among those, as was the R560T missense mutation, prominent in some ethnic populations (37). Login to comment 181 ABCC7 p.Arg560Thr ABCC7 p.Arg258Gly ABCC7 p.Ser945Leu ABCC7 p.His139Arg B, C) HEK293 cells were transiently transfected with CFTR with misprocessing mutations located in CL1 (H139R), CL2 (R258G), and CL3 (S945L) (B), or in NBD1 (èc;I507 and R560T; C). Login to comment 197 ABCC7 p.Arg560Thr Two other severe disease-causing mutations in NBD1 (èc;I507 and R560T) that also were not temperature sensitive were unresponsive to VX-809. Login to comment