ABCMdb

PMID: 23493553

Woodward OM, Tukaye DN, Cui J, Greenwell P, Constantoulakis LM, Parker BS, Rao A, Kottgen M, Maloney PC, Guggino WB
Gout-causing Q141K mutation in ABCG2 leads to instability of the nucleotide-binding domain and can be corrected with small molecules.
Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5223-8. doi: 10.1073/pnas.1214530110. Epub 2013 Mar 14., [PubMed]

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0 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Gout-causing Q141K mutation in ABCG2 leads to instability of the nucleotide-binding domain and can be corrected with small molecules Owen M. Woodwarda,1,2 , Deepali N. Tukayea,1 , Jinming Cuia , Patrick Greenwella , Leeza M. Constantoulakisa , Benjamin S. Parkera , Anjana Raoa , Michael K&#f6;ttgenb , Peter C. Maloneya , and William B. Gugginoa,2 a Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; and b Renal Division, University Medical Centre Freiburg, 79106 Freiburg, Germany Edited by Maurice B. Burg, National Heart, Lung, and Blood Institute, Bethesda, MD, and approved February 19, 2013 (received for review August 22, 2012) The multidrug ATP-binding cassette, subfamily G, 2 (ABCG2) transporter was recently identified as an important human urate transporter, and a common mutation, a Gln to Lys substitution at position 141 (Q141K), was shown to cause hyperuricemia and gout. Login to comment 1 ABCG2 p.Gln141Lys The nature of the Q141K defect, however, remains undefined. Login to comment 2 ABCG2 p.Gln141Lys Here we explore the Q141K ABCG2 mutation using a comparative approach, contrasting it with another disease-causing mutation in an ABC transporter, the deletion of Phe-508 (ƊF508) in the cystic fibrosis transmembrane conductance regulator (CFTR). Login to comment 3 ABCG2 p.Gln141Lys We found, much like in ƊF508 CFTR, that the Q141K mutation leads to instability in the nucleotide-binding domain (NBD), a defect that translates to significantly decreased protein expression. Login to comment 4 ABCG2 p.Gln141Lys However, unlike the CFTR mutant, the Q141K mutation does not interfere with the nucleotide-binding domain/intracellular loop interactions. Login to comment 6 ABCG2 p.Gln141Lys Finally, we have demonstrated the utility of using small molecules to correct the Q141K defect in expression and function as a possible therapeutic approach for hyperuricemia and gout. Login to comment 14 ABCG2 p.Gln141Lys Additionally, we characterized the single nucleotide polymorphism resulting in a Gln to Lys substitution at position 141 (Q141K), as causal for gout resulting from a loss of urate transport function in ABCG2 (10). Login to comment 15 ABCG2 p.Gln141Lys The majority of work on ABCG2 [also know as the breast cancer resistance protein (BCRP)] has focused on its role as a multidrug transporter and specifically on its role as a xenotoxin and chemotherapeutic transport molecule, work that has included a description of the Q141K mutation`s effect on ABCG2 trafficking and function. Login to comment 16 ABCG2 p.Gln141Lys Multiple groups reported that the Q141K mutation decreases protein expression and, coincidentally, xenotoxin transport function (11-16). Login to comment 17 ABCG2 p.Gln141Lys This decrease of expression and function appears to be partially caused by the enhancement of the Q141K mutant`s susceptibility to proteasomal degradation (11-13). Login to comment 18 ABCG2 p.Gln141Lys There is also recent evidence suggesting that a significant portion of the mutant Q141K protein accumulates in perinuclear aggresomes before being degraded (17). Login to comment 19 ABCG2 p.Gln141Lys Conversely, the Q141K mutation has also been reported to reduce ABCG2 ATPase activity, suggesting that the mutation disrupts xenotoxin transport independently of expression or trafficking abnormalities (14, 16). Login to comment 20 ABCG2 p.Gln141Lys However, how does the Q141K mutation affect urate transport? Login to comment 21 ABCG2 p.Gln141Lys In our work, we found urate transport was significantly reduced by the Q141K mutation in Xenopus oocytes when transport was normalized to expression levels (10). Login to comment 22 ABCG2 p.Gln141Lys In contrast, subsequent work by Matsuo et al. (18) confirmed ABCG2 as a urate transporter and that Q141K results in a loss of function. Login to comment 23 ABCG2 p.Gln141Lys However, they postulated that the loss of urate transport function for the Q141K was specifically due to the reduction in protein expression (18) . Login to comment 24 ABCG2 p.Gln141Lys Interestingly, the possibility that the Q141K mutation disrupts both function and trafficking/expression suggests intriguing parallels to other human disease causing ABC transporter mutations, namely the deletion of Phe-508 (ƊF508) in the cystic fibrosis transmembrane conductance regulator (CFTR). Login to comment 26 ABCG2 p.Gln141Lys However, it remains unclear why the Q141K mutation causes increased ubiquitination and proteasomal degradation or accumulation in aggresomes. Login to comment 27 ABCG2 p.Gln141Lys In this paper, we explore the nature of the Q141K defect and, specifically, its effect on expression in mammalian cells. Login to comment 30 ABCG2 p.Gln141Lys Finally, we demonstrate that correcting the gout causing Q141K ABCG2 defect can be accomplished with small molecules in a manner similar to the successful correction of the CFTR molecule as a treatment for cystic fibrosis. Login to comment 41 ABCG2 p.Gln141Lys 13 | 5223-5228 Results Temperature Correction of Q141K. Login to comment 42 ABCG2 p.Gln141Lys Previously, we showed that the Q141K mutation in ABCG2 was causal for gout and resulted in a 53% loss of urate transport function (10, 22). Login to comment 44 ABCG2 p.Gln141Lys To investigate the opposing observations and to explore the possibility that both a loss of function and a expression defect could be caused by the Q141K mutation, we expressed our ABCG2 constructs in mammalian cell expression systems. Login to comment 45 ABCG2 p.Gln141Lys We found, much like previous reports (12, 13, 15, 18), that, in multiple mammalian cell lines, transient expression of the Q141K mutation resulted in significantly less total protein (Fig. S1 A and B and Fig. 2D: 63.4 &#b1; 0.1% decrease), surface protein (Fig. 1D and Fig. S1 C and D), and function (Fig. S1E: 63.08 &#b1; 3.0% decrease) than wild-type ABCG2 [although with only slightly lower levels of unglycosylated protein (Fig. S2A): 27.3 &#b1; 0.1% reduction; see Fig. S2B for ABCG2 glycosylation states]. Login to comment 46 ABCG2 p.Gln141Lys In both the oocytes and mammalian cell lines, we used the same human ABCG2 constructs; thus the differences in relative expression may give a clue as to the nature of the Q141K defect. Login to comment 48 ABCG2 p.Gln141Lys To test the possibility that the Q141K mutant is being stabilized by the low incubation temperatures of the oocytes, we injected oocytes with mutant and wild-type ABCG2 mRNA and incubated the cells at either 16 &#b0;C or 32 &#b0;C (Fig. 1A). Login to comment 51 ABCG2 p.Gln141Lys Fig. 1 B and C shows that the lowering of the incubation temperature of mammalian cells expressing wild-type ABCG2 and the Q141K mutant increases the expression of both; however, the percentage change in expression level is significantly greater in the mutant protein (Fig. 1C), a result consistent with low-temperature enhancement of folding and stabilizing mutant protein. Login to comment 53 ABCG2 p.Gln141Lys Low temperature was also able to rescue the surface expression of the Q141K mutant (Fig. 1D), but again the majority of the rescued surface protein is the unglycosylated version of the protein (Fig. 1D, black arrows). Login to comment 56 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys We found that the low-temperature incubation again preferentially increased the dimer mutant Q141K protein expression compared with wild type (Fig. 1E: dimer Q141K expression increased 242.98 &#b1; 89.8% relative to wild type; n = 3). Login to comment 57 ABCG2 p.Gln141Lys These results support the hypothesis that the Q141K mutant is unstable and that expression can be rescued with low-temperature incubation. Login to comment 58 ABCG2 p.Gln141Lys Nature of the Q141K Defect. Login to comment 59 ABCG2 p.Gln141Lys Human disease, loss of function, reduced expression of mature protein, and temperature correction are characteristics that the Q141K mutation shares with exactly one other mutant ABC protein: ƊF508 CFTR, the most common cystic fibrosis-causing mutation (25). Login to comment 61 ABCG2 p.Gln141Lys However, the deletion of F508 in CFTR produces no mature glycosylated protein (25), whereas the Q141K defect in ABCG2 only reduces the amount of mature protein; thus the defects may be similar but not precisely homologous. Login to comment 65 ABCG2 p.Gln141Lys These results support our model and the similar natures of the defects between Q141K ABCG2 and ƊF508 CFTR. Login to comment 70 ABCG2 p.Gln141Lys To test whether or not Q141K decreases stability of the ABCG2 NBD, we introduced stabilizing mutations into the signature sequence to try to rescue protein expression. Login to comment 71 ABCG2 p.Arg193Lys ABCG2 p.Arg193Lys ABCG2 p.Gly188Glu ABCG2 p.Gly188Glu We separately mutated two residues in the Q141K background-a Gly to Glu substitution at position 188 (G188E) and an Arg to Lys substitution at 193 (R193K) (homologous to the Gly-550 and Arg-555 residues of CFTR, shown in Fig. 3A)-and found that the G188E mutation acted as a suppressor mutation, significantly increased the amount of the Q141K total protein (Fig. 3 B-E and Fig. S4 E and G), and increased the dimer protein (Fig. 3 C and E and Fig. S4 F and H) and the surface protein (Fig. 3F); the R193K mutation did not act as a suppressor (P < 0.41; n = 10). Login to comment 73 ABCG2 p.Gln141Lys Temperature correction of Q141K ABCG2 expression defect. Login to comment 75 ABCG2 p.Gln141Lys (B) Western blot of transient expression of wild-type (WT) and Q141K ABCG2 in CHO cells incubated for 48 h at 27 &#b0;C or 37 &#b0;C. Login to comment 76 ABCG2 p.Gln141Lys (C) Summary data of temperature rescue of protein expression (WT: n = 6; Q141K: n = 8). Login to comment 78 ABCG2 p.Gln141Lys (E) Dimer expression of WT and Q141K ABCG2 after 48 h of incubation at 27 &#b0;C or 37 &#b0;C (n = 4). Login to comment 80 ABCG2 p.Gly188Glu G188E mutation introduced into the wild-type protein produced no change in expression (Fig. 3G) and did not recue the ƊF142 mutation (Fig. S2 E and F), supporting the hypothesis that the F142 deletion disrupts both stability and interdomain interactions. Login to comment 81 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys In contrast, stabilizing the NBD domain of Q141K ABCG2 rescues protein expression and suggests that primarily NBD stability may underlie the Q141K defect. Login to comment 82 ABCG2 p.Gln141Lys Even with considerable evidence suggesting that Q141K causes NBD instability, there remains the possibility that interdomain interactions may also be disrupted, as in the ƊF142 mutant and in the CFTR F508 deletion. Login to comment 87 ABCG2 p.Lys473Glu The reversal of charge with a K473E substitution also resulted in reduced expression and dimerization (Fig. S5 A-D). Login to comment 89 ABCG2 p.Gln141Lys ABCG2 p.Lys473Trp Significantly, introduction of the K473W substitution on the Q141K background did not rescue total, mature, or dimer levels (all were actually significantly less) (Fig. 4 A-C and Fig. S5E). Login to comment 90 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Lys473Trp The failure of the K473W mutation to rescue Q141K expression supports the hypothesis that the Q141K mutation does not interfere with interdomain interactions or dimerization of the ABCG2 protein. Login to comment 91 ABCG2 p.Gln141Lys Furthermore, we found that the Q141K dimer protein expression, as a fraction of wild-type expression, is greater than the total expression (Fig. 4D), not less, suggesting that the mutant dimerizes normally (or marginally more efficiently than wild type; P ࣘ 0.03; n = 7). Login to comment 92 ABCG2 p.Gln141Lys Finally, modeling of ABCG2 with the Q141K substitution did not alter the F142 orientation or position in respect to the ICL residues, and K141 adopted the same orientation as Q141 (Fig. 4E). Login to comment 95 ABCG2 p.Gln141Lys Taken together, our findings support the hypothesis that the Q141K mutation does not interfere with interdomain interactions or with dimerization of the ABCG2 protein. Login to comment 96 ABCG2 p.Gln141Lys Small-Molecule Correction of Q141K. Login to comment 100 ABCG2 p.Gln141Lys Q141K mutation occurs in mutational hotspot of ABC protein NBD. Login to comment 103 ABCG2 p.Gln141Lys Western blots of transient total (B) and dimer expression (C) of WT, Q141K, and ƊF142 ABCG2 in HEK293 cells. Login to comment 107 ABCG2 p.Arg193Lys ABCG2 p.Arg193Lys ABCG2 p.Gly188Glu ABCG2 p.Gly188Glu ABCG2 WT 141K 188E -- -- 188E 76k ABCG2 GAPDH surface ABCG2 Na/K atpase WT 141K 188E 193K -- WT 141K 188E 193K -- 76k ABCG2 GAPDH ABCG2 monomer ABCG2 dimer 140k WT 141K 188E 193K -- A B C 0.0 0.5 1.0 Normalized ABCG2 protein WT 141K G188E R193K -- ** 0.0 0.5 1.0 1.5 2.0 2.5 Normalized ABCG2 dimer protein WT 141K G188E R193K -- * E F G D CFTR Fig. 3. Login to comment 108 ABCG2 p.Gln141Lys Suppressor mutation correction of Q141K expression defect. Login to comment 112 ABCG2 p.Gln141Lys Western blot of transient total (B) and dimer (C) expression in CHO cells of suppressor mutations in the Q141K background. Login to comment 113 ABCG2 p.Gln141Lys Summary data of suppressor rescue of Q141K ABCG2 total (D; n = 10) and dimer protein expression (E; n = 4). Login to comment 114 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gly188Glu (F) Surface expression of biotinylated ABCG2 and Q141K variants and Na+ /K+ ATPase, demonstrating strong rescue with the Q141K G188E mutation (n = 6). Login to comment 115 ABCG2 p.Gly188Glu (G) The G188E mutation does not affect WT ABCG2 protein expression (n = 3). Login to comment 116 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys All means are &#b1; SEM; *P < 0.05; **P < 0.01. this principle and to learn more about the mechanism of dysfunction in the Q141K ABCG2 mutation, we attempted to correct the Q141K defect with small molecules. Login to comment 118 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys We found that application of 4-PBA increased significantly the expression of the Q141K protein (Fig. 5 A and B) but had no effect on the wild type, consistent with the hypothesis that the ERAD pathway targets the Q141K protein. Login to comment 119 ABCG2 p.Gln141Lys The Q141K monomer expression levels, even after correction, were not at levels comparable to the wild type (Fig. 5A). Login to comment 120 ABCG2 p.Gln141Lys Conversely, the dimer form of the Q141K protein when treated with 4-PBA was corrected to expression levels not different from wild type (P < 0.147, n = 4), suggesting that the effect of 4-PBA is critical for the creation of the dimer form of the protein (Fig. 5C). Login to comment 121 ABCG2 p.Gln141Lys ABCG2 p.Gly188Glu ABCG2 p.Gly188Glu Interestingly, we found that 4-PBA was also able to increase the amount of Q141K/G188E protein (Fig. 5 A-C), demonstrating that the G188E suppressor mutation is not a full rescue and that some protein is still targeted for degradation. Login to comment 125 ABCG2 p.Gln141Lys We found that incubation with the VRT-325 molecule significantly increased the amount of total (Fig. 5 D and E) and surface Q141K protein expression (Fig. S6A); VRT-325 had no effect on wild-type ABCG2 expression (P < 0.193, n = 3). Login to comment 126 ABCG2 p.Gln141Lys Both low temperature and VRT-325 correction of Q141K produced increased levels of mature glycosylated monomer protein (Fig. 5D); however, unlike the low-temperature correction, VRT-325 did not increase the amount of immature unglycosylated protein (Fig. 5D and Fig. S6B). Login to comment 127 ABCG2 p.Gln141Lys This suggests that VRT-325 may allow more of the Q141K protein to traffic normally, become glycosylated, and reach the cell membrane. Login to comment 128 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys In HEK293 cells expressing the Q141K mutant and treated with VRT-325, we also found that the accumulation of C-14-labeled uric acid was significantly less than in the untreated Q141K-expressing cells (Fig. 5F; P ࣘ 0.004; n = 11, 10), demonstrating partial rescue of function along with expression of the Q141K mutant. Login to comment 130 ABCG2 p.Gln141Lys Surprisingly, corr-4a (Fig. 5 G and H) increased the total protein levels of the Q141K ABCG2 mutant, but not wild-type protein, although the corr-4a (20 bc;M) correction appears to be less than the VRT-325 (10 bc;M) correction (Fig. S6C). Login to comment 131 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Taken together, our results show that the Q141K protein level can be rescued using small corrector molecules, which is consistent with the hypothesis that the Q141K mutation is a misfolding mutation that increases degradation and consistent with the principle that the gout-causing Q141K mutation can be corrected with small molecules. Login to comment 132 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys GAPDH monomer dimer 141K -- P E W 473: 141K -- P E W 473: * A B Normalized ABCG2 dimer protein 0.0 0.5 1.0 Q141K -- P E 473: W ** ** ** C ABCG2 Q141K Q141K protein expression as fraction of wild type 0 0.25 0.5 0.75 Dimer Total * D E Fig. 4. Login to comment 133 ABCG2 p.Gln141Lys Q141K does not disrupt interdomain interactions or dimerization. Login to comment 134 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Lys473Trp Western blots of total (A) and dimer (B) expression of Q141K ABCG2 with secondary mutations at K473 demonstrating that K473W cannot rescue Q141K expression. Login to comment 135 ABCG2 p.Gln141Lys Summary data of K473 mutation on total (C; n = 4-6) Q141K expression. Login to comment 136 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys (D) Expression of total or dimer Q141K as a fraction of wild type (total or dimer); a greater proportion of total Q141K exists as a dimer than does wild type (n = 7). Login to comment 137 ABCG2 p.Gln141Lys (E) Superimposition of ABCG2 NBD model (green, with Q141 as orange sticks and F142 as red sticks) and the model with Q141K substitution (blue, with K141 and F142 as cyan sticks). Login to comment 140 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys 76k ABCG2 GAPDH WT 141K 188E 193K -- -- +4-PBA WT 141K 188E 193K -- -- control A ** ** 0 100 200 300 WT 141K 188E -- % Change in expression after 4-PBA B 76k ABCG2 GAPDH 141K WT -- -- +VRT 325 +VRT 325 27&#b0;C 27&#b0;C D -50 0 100 200 % Change with VRT-325 141K WT * E WT 141K 188E 193K -- -- control +4-PBA WT 141K 188E 193K -- -- Ɗ142 Ɗ142 C 150k 76k ABCG2 GAPDH F Q141K Q141K + VRT-325 ** % Change in C-14 uric acid accumulation as compared to WT 0 20 40 WT + VRT-325 % Change with Corr-4a -50 0 50 100 150 141K WT ** H G WT -- +Corr 4a 76k ABCG2 GAPDH 141K -- +Corr 4a Fig. 5. Login to comment 141 ABCG2 p.Gln141Lys Small-molecule correction of gout-causing Q141K ABCG2 expression defect. Login to comment 143 ABCG2 p.Gln141Lys (B) Summary data of 4-PBA rescue of Q141K expression (n = 3-6); 4-PBA has no effect on WT ABCG2 expression (P < 0.28; n = 4) compared with zero. Login to comment 149 ABCG2 p.Gln141Lys (G) Western blot and summary data (H) of total Q141K ABCG2 expression rescue by 24-h treatment with 20 bc;M corr-4a (n = 4). Login to comment 154 ABCG2 p.Gln141Lys Seeing ABCG2 as an important urate transporter and the Q141K mutation as a loss-of-function mutation led naturally to a comparison with other disease-causing mutations in human ABC transporters, a comparison that lead inevitably to CFTR. Login to comment 155 ABCG2 p.Gln141Lys The parallels between the Q141K mutation in ABCG2 and the deletion of the Phe-508 in CFTR, a mutation that occurs in ~90% of all individuals with cystic fibrosis (25), are striking. Login to comment 158 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCC7 p.Arg555Lys ABCG2 p.Gly188Glu Here we tested homologous NBD mutations in Q141K ABCG2 and found the G188E mutation rescued Q141K expression, suggesting the Q141K mutation results in increased ABCG2 NBD instability. Login to comment 159 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCG2 p.Arg193Lys Interestingly, the R193K mutation, homologous to the suppressor mutation R555K in CFTR, didn`t increase Q141K ABCG2 expression; but did appear to decrease the insoluble fraction of Q141K protein (Fig. S6D). Login to comment 161 ABCG2 p.Arg193Lys It may be that R193K could not rescue mature protein but actually enhanced the degradation of misfolded protein by preventing the formation of the large insoluble aggresomes as reportedly seen by other groups (17). Login to comment 162 ABCG2 p.Gln141Lys Our CFTR ƊF508 and Q141K ABCG2 comparison also revealed the importance of F142 in ABCG2. Login to comment 163 ABCG2 p.Gln141Lys The F142 deletion causes a severe decrease in expression but, unlike the Q141K mutation, the ƊF142 cannot be rescued with temperature or with suppressor mutations. Login to comment 177 ABCG2 p.Gln141Lys Interestingly, Basseville et al. (17) recently found that a different suite of HDAC inhibitors rescued Q141K ABCG2 function and expression levels but did not observe any changes in Hsp 70 or Hsp 90 levels. Login to comment 184 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Thus, the VRT-325 molecule corrects in ƊF508 CFTR the exact defect that we describe here for Q141K ABCG2, and indeed, we found that VRT-325 significantly increased total, dimer, and surface expression of the Q141K protein. Login to comment 186 ABCG2 p.Gln141Lys The success of the VRT-325 and corr-4a molecules in correcting mutant Q141K protein expression clearly demonstrates that a better understanding of the way in which ABCG2 folds and dimerizes can lead to drug discovery of new therapies for gout and hyperuricemia. Login to comment 187 ABCG2 p.Gln141Lys Going forward, it will also be important to discern why the Q141K mutation leads to a functional defect, even after expression level is normalized (10, 16), and whether NBD instability reduces binding or hydrolysis of ATP. Login to comment 188 ABCG2 p.Gln141Lys Finally, the similarities between ƊF508 CFTR and Q141K ABCG2 extend beyond the work presented here. Login to comment 191 ABCG2 p.Gln141Lys For CFTR, the dysfunction of the ƊF508 mutation may provide some protection from cholera infection (54), whereas in ABCG2 the Q141K mutation may persist to increase blood urate levels of particular human populations beyond what even the loss of uricase function achieved. Login to comment 239 ABCG2 p.Gln141Lys Furukawa T, et al. (2009) Major SNP (Q141K) variant of human ABC transporter ABCG2 undergoes lysosomal and proteasomal degradations. Login to comment 248 ABCG2 p.Gln141Lys Imai Y, et al. (2002) C421A polymorphism in the human breast cancer resistance protein gene is associated with low expression of Q141K protein and low-level drug resistance. Login to comment 325 ABCC7 p.Gly550Glu Roxo-Rosa M, et al. (2006) Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms. Login to comment