ABCMdb

PMID: 17911111

Wang Y, Loo TW, Bartlett MC, Clarke DM
Correctors promote maturation of cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by binding to the protein.
J Biol Chem. 2007 Nov 16;282(46):33247-51. Epub 2007 Oct 2., 2007-11-16 [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Val510Ala ABCC7 p.Val510Cys Although replacement of the 18 endogenous cysteines of CFTR with Ser or Ala yields a Cys-less mutant that does not mature at 37 °C, we found that maturation could be restored if Val510 was changed to Ala, Cys, Ser, Thr, Gly, Ala, or Asp. Login to comment 4 ABCC7 p.Val510Asp The V510D mutation also promoted maturation of ⌬F508 CFTR. Login to comment 5 ABCC7 p.Val510Ala The Cys-less/V510A mutant was used for subsequent cross-linking analysis as it yielded relatively high levels of mature protein that was functional in iodide efflux assays. Login to comment 6 ABCC7 p.Val510Ala We tested for cross-linking between cysteines introduced into TM6 and TM7 of Cys-less CFTR/V510A because cross-linking between TM6 and TM7 of P-glycoprotein, the sister protein of CFTR, was inhibited with the corrector VRT-325. Login to comment 7 ABCC7 p.Val510Ala Cys-less CFTR/V510A mutant containing cysteines at I340C(TM6) and S877C(TM7) could be cross-linked with a homobifunctional cross-linker. Login to comment 23 ABCC7 p.Cys590Leu ABCC7 p.Cys592Leu Cys-less CFTR was constructed by replacing Cys590 and Cys592 with leucine (9) and the other 16 endogenous cysteines at positions 76, 126, 225, 276, 343, 491, 524, 657, 832, 866, 1344, 1355, 1395, 1400, 1410, and 1458 with alanine. Login to comment 25 ABCC7 p.Val510* The Cys-less CFTR cDNA was also modified to include the V510X mutations. Login to comment 26 ABCC7 p.Val510Ala Mutations I340C(TM6) and S877C(TM7) were inserted into Cys-less CFTR/V510A singly or together. Login to comment 27 ABCC7 p.Val510* The V510X mutations were then introduced into ⌬F508 CFTR as described above. Login to comment 28 ABCC7 p.Val510Ala ABCC7 p.Val510Ala The ⌬F508 mutation was also introduced into the Cys-less/V510A and I340C(TM6)/S877C(TM7)/Cys-less/ V510A mutants. Login to comment 46 ABCC7 p.Val510Ala Disulfide Cross-linking Analysis-CFTR mutant I340C(TM6)/ S877C(TM7) Cys-less/V510A CFTR was expressed in HEK 293 cells. Login to comment 65 ABCC7 p.Val510Cys Fig. 2A shows that only the V510C mutation promoted maturation of Cys-less CFTR at 37 °C. Login to comment 67 ABCC7 p.Val510Pro ABCC7 p.Val510Thr Cys-less mutants in which residue Val510 was changed to Pro, Thr, Tyr, Gly, Ala, Ser, Leu, Asp, Phe, or Trp were constructed. Login to comment 68 ABCC7 p.Val510Ala ABCC7 p.Val510Thr ABCC7 p.Val510Gly The mutants were expressed in HEK 293 cells at 37 °C, and whole cell extracts were subjected to immunoblot analysis. Fig. 2B shows that changing Val510 to Thr, Gly, Ala, Ser, or Asp promoted maturation of Cys-less CFTR. Login to comment 70 ABCC7 p.Val510Ala ABCC7 p.Val510Cys ABCC7 p.Val510Asp ABCC7 p.Val510Gly ABCC7 p.Val510Ser Stable BHK cell lines expressing mutants V510A, V510C, V510S, V510G, or V510D were generated for use in iodide efflux assays. Login to comment 72 ABCC7 p.Val510Ala ABCC7 p.Val510Asp The results of the most active (V510A) and least active (V510D) mutants are shown in Fig. 2C. Login to comment 73 ABCC7 p.Val510Cys ABCC7 p.Val510Gly ABCC7 p.Val510Ser The results of mutants V510C, V510S, or V510G are not shown for clarity. Login to comment 82 ABCC7 p.Val510Ala ABCC7 p.Val510Cys ABCC7 p.Val510Gly ACCELERATED PUBLICATION: CFTR TM Domain Cross-linking 33248 To test whether the Val510 changes could also promote maturation of ⌬F508 CFTR (in a wild-type background), we introduced the Val510 mutations that promoted maturation of Cys-less CFTR (Val510 to Cys, Gly, Ala, Ser, Asp, or Thr) into ⌬F508 CFTR. Login to comment 83 ABCC7 p.Val510* The ⌬F508 CFTR/V510X mutants were expressed in HEK 293 cells at 37 °C, and whole cell extracts were subjected to immunoblot analysis. Fig. 2D shows that introduction of an Asp mutation at position 510 was most effective in promoting maturation of ⌬F508 CFTR. Login to comment 84 ABCC7 p.Val510Ala The Cys-less/V510A CFTR mutant was then used for disulfide cross-linking analysis to test whether correctors directly interacted with the protein. Since corrector VRT-325 inhibited cross-linking of P-gp mutant L339C(TM6)/F728C(TM7), we aligned TM segments (6 and 7) of P-gp and CFTR to identify the equivalent residues in CFTR. Login to comment 87 ABCC7 p.Val510Ala The 16 double cysteine mutants were initially constructed in a Cys-less CFTR lacking V510A, so they required expression at 27 °C for maturation of CFTR (data not shown). Login to comment 90 ABCC7 p.Val510Ala The I340C(TM6)/S877C(TM7) mutations were then introduced into the Cys-less/V510A CFTR background. Login to comment 91 ABCC7 p.Val510Ala The I340C(TM6)/S877C(TM7) Cys-less/ V510A CFTR mutant was expressed in HEK 293 cells, and samples were treated with methanethiosulfonate cross-linkers of various sizes and subjected to immunoblot analysis. Login to comment 93 ABCC7 p.Val510Ala Fig. 3A shows that mutant I340C(TM6)/ S877C(TM7) Cys-less/V510A CFTR could be cross-linked with various methanethiosulfonate cross-linkers. Login to comment 98 ABCC7 p.Val510Ala ABCC7 p.Val510Asp C, iodide efflux assays were performed on stable BHK cell lines expressing Cys-less/V510A CFTR (open circles) or Cys-less/V510D CFTR (closed squares). Login to comment 103 ABCC7 p.Val510Ala Disulfide cross-linking of mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR. Login to comment 104 ABCC7 p.Val510Ala A, HEK 293 cells expressing mutant I340C(TM6)/ S877C(TM7) Cys-less/V510A CFTR were treated with (ϩ) or without (-) 0.1 mM of the indicated cross-linkers for 16 min at 22 °C. Login to comment 108 ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.Val510Ala B, immunoblot analysis of HEK 293 cells expressing Cys-less/V510A CFTR, mutants I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR, I340C(TM6) Cys-less/V510A CFTR, or S877C(TM7) Cys-less/V510A CFTR were treated with (ϩ) or without (-) cross-linker M11M. Login to comment 113 ABCC7 p.Val510Ala The I340C(TM6)/S877C(TM7) Cys-less/ V510A CFTR mutant was used as a reporter molecule to study CFTR interactions with CFTR correctors such as corr-4a (4), VRT-325 (5, 17), and VRT-532 (5, 6). Login to comment 114 ABCC7 p.Val510Ala We first tested the system by using a compound that binds within the CFTR channel pore (at the interface between the two TMDs) and to see whether it could block cross-linking of mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR. Login to comment 116 ABCC7 p.Val510Ala Accordingly, HEK 293 cells expressing mutant I340C(TM6)/ S877C(TM7) Cys-less/V510A CFTR were preincubated with various concentrations of benzbromarone and then treated with M11M cross-linker. Login to comment 120 ABCC7 p.Val510Ala Verapamil (Fig. 4E) and demecolcine (Fig. 4F) did not inhibit cross-linking of mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR. Login to comment 122 ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.Val510Ala We could not test whether correctors blocked cross-linking in the ⌬F508/ I340C(TM6)/S877C(TM7)/Cys-less/V510A mutant because mature CFTR was not detected in mutants ⌬F508/Cys-less/ V510A or ⌬F508/I340C(TM6)/S877C(TM7)/Cys-less/V510A even when expressed at 27 °C (Fig. 4, G and H). Login to comment 124 ABCC7 p.Val510Asp Position 510 appears to be particularly important for CFTR maturation as introduction of the suppressor mutation V510D into ⌬F508 CFTR also promoted maturation of the protein (Fig. 2D). Login to comment 128 ABCC7 p.Val510Asp The V510D mutation may counter the effects of ⌬F508 by acting as a suppressor mutation, perhaps by forming a salt bridge with a positive amino acid located in one of the intracellular loops. Login to comment 131 ABCC7 p.Val510Ala ABCC7 p.Val510Asp Although V510D was the most efficient suppressor mutation because it promoted maturation of both Cys-less and ⌬F508 CFTRs, it was less useful than the V510A change in Cys-less CFTR because it showed reduced iodide efflux activity. Login to comment 133 ABCC7 p.Val510Ala The ability of VRT-325, VRT-532, and corr-4a (Fig. 4) to block cross-linking of I340C(TM6)/S877C(TM7) Cys-less/ V510A CFTR suggests that they interact directly with CFTR. Login to comment 136 ABCC7 p.Val510Asp The proposed effects of the V510D mutation and correctors on maturation of ⌬F508 CFTR are shown in the models in supplemental Fig. 1. Login to comment 138 ABCC7 p.Val510Ala Effect of CFTR modulators and correctors on disulfide cross-linking of CFTR mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR. Login to comment 139 ABCC7 p.Val510Ala HEK 293 cells expressing mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR were incubated at 22 °C for 30 min in the absence (None) or presence of theindicatedcompounds.Thesampleswerethentreatedwith(ϩ)orwithout (-) 0.1 mM M11M at 22 °C for 16 min. Login to comment 141 ABCC7 p.Val510Ala ABCC7 p.Val510Ala Immunoblot analysis was also performed on mutants Cys-less/V510A (G) or I340C(TM6)/S877C(TM7)/Cys-less/V510A (H) with (⌬F508) or without (-) the ⌬F508 mutation that were transiently expressed in HEK 293 cells for 24 h at 27 °C. Login to comment 143 ABCC7 p.Val510Asp The V510D suppressor mutation is predicted to overcome the effects of the ⌬F508 CFTR mutation by promoting interactions between TMD1 and NBD1 (supplemental Fig. 1B). Login to comment