ABCMdb

PMID: 17098864

Roxo-Rosa M, Xu Z, Schmidt A, Neto M, Cai Z, Soares CM, Sheppard DN, Amaral MD
Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms.
Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17891-6. Epub 2006 Nov 10., 2006-11-21 [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Arg516Lys ABCC7 p.Arg29Lys ABCC7 p.Arg766Lys Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms Mo´ nica Roxo-Rosa*† , Zhe Xu‡ , Andre´ Schmidt*† , Ma´rio Neto*, Zhiwei Cai‡ , Cla´udio M. Soares§ , David N. Sheppard‡ , and Margarida D. Amaral*†¶ *Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, Campo Grande, 1749-016 Lisbon, Portugal; †Centre of Human Genetics, National Institute of Health Dr. Ricardo Jorge, Avenida Padre Cruz, 1649-016 Lisbon, Portugal; ‡Department of Physiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom; and §Institute of Chemistry and Biological Technology, New University of Lisbon, 2781-901 Oeiras, Portugal Communicated by Michael J. Welsh, University of Iowa College of Medicine, Iowa City, IA, September 22, 2006 (received for review June 9, 2006) The revertant mutations G550E and 4RK [the simultaneous mutation of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K, and R766K] rescue the cell surface expression and function of F508del-cystic fibrosis (CF) transmembrane conductance regulator (-CFTR), the most common CF mutation. Login to comment 1 ABCC7 p.Arg560Thr ABCC7 p.Arg560Thr ABCC7 p.Val562Ile ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Here, we investigate their mechanism of action by using biochemical and functional assays to examine their effects on F508del and three CF mutations (R560T, A561E, and V562I) located within a conserved region of the first nucleotide-binding domain (NBD1) of CFTR. Like F508del, R560T and A561E disrupt CFTR trafficking. Login to comment 2 ABCC7 p.Arg560Thr ABCC7 p.Gly550Glu ABCC7 p.Ala561Glu G550E rescued the trafficking defect of A561E but not that of R560T. Login to comment 3 ABCC7 p.Val562Ile ABCC7 p.Val562Ile Of note, the processing and function of V562I were equivalent to that of wild-type (wt)-CFTR, suggesting that V562I is not a disease-causing mutation. Login to comment 4 ABCC7 p.Val562Ile ABCC7 p.Gly550Glu Biochemical studies revealed that 4RK generates higher steady-state levels of mature CFTR (band C) for wtand V562I-CFTR than does G550E. Login to comment 6 ABCC7 p.Gly550Glu Thus, our data suggest that the revertants G550E and 4RK might rescue F508del-CFTR by distinct mechanisms. Login to comment 7 ABCC7 p.Gly550Glu G550E likely alters the conformation of NBD1, whereas 4RK allows F508del-CFTR to escape endoplasmic reticulum retention͞retrieval mediated by AFTs. Login to comment 21 ABCC7 p.Gly550Glu DeCarvalho et al. (26) demonstrated that substitution of G550 by a negatively charged amino acid (G550E) promoted the maturation of F508del-CFTR and enhanced CFTR-mediated Cl- permeability (26). Login to comment 22 ABCC7 p.Arg555Lys ABCC7 p.Arg516Lys ABCC7 p.Arg29Lys ABCC7 p.Arg766Lys Moreover, Chang et al. (25) rescued the trafficking and function of F508del-CFTR with the simultaneous mutation of the four arginine-framed tripeptides (AFTs) (R29QR31, R516YR518, R553AR555, and R764RR766) present in CFTR termed 4RK (R29K, R516K, R555K, and R766K). Login to comment 25 ABCC7 p.Gly550Glu To better understand how revertant mutants rescue F508del-CFTR, we selected for study the revertants G550E and 4RK for several reasons. Login to comment 26 ABCC7 p.Gly550Glu First, they represent two very distinct types of amino acid residues (G550E, acidic; 4RK, basic). Login to comment 27 ABCC7 p.Gly550Glu Second, they reside in distinct functional motifs (G550E, the LSGGQ motif of NBD1; 4RK, the AFTs). Login to comment 28 ABCC7 p.Gly550Glu Third, they are likely to have different effects on the structure of CFTR because only two AFTs lie in NBD1 (like G550E), whereas the other two are on the N terminus and regulatory domain, respectively. Login to comment 30 ABCC7 p.Gly550Glu Together, these reasons led us to hypothesize that G550E Author contributions: M.R.-R. and Z.X. contributed equally to this work; D.N.S. and M.D.A. designed research; M.R.-R., Z.X., A.S., M.N., and C.M.S. performed research; Z.C. analyzed data; and M.R.-R., D.N.S., and M.D.A. wrote the paper. Login to comment 37 ABCC7 p.Arg560Thr ABCC7 p.Arg560Thr ABCC7 p.Val562Ile ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu 47 ͉ 17891-17896 and 4RK act by different mechanisms. To explore this possibility, we tested the effects of G550E and 4RK on three additional CF mutations within NBD1: R560T, A561E, and V562I.ʈ We selected for study these CF mutants because (i) these residues constitute a hot spot for disease-causing mutations (seven mutations are associated with these three residues࿣ ); (ii) A561E and R560T are the second and fourth most frequent mutations among Portuguese and Irish CF patients, respectively (28); (iii) like G550E and R555K (one of the 4RK mutants), these mutations affect residues located between the LSGGQ and Walker B motifs of NBD1, which are highly conserved across species; and (iv) they all lie within the same ␣-helix (H5; G550-Y563) within the ATP-binding cassette ␣-subdomain of NBD1 (29, 30). Login to comment 38 ABCC7 p.Arg560Thr ABCC7 p.Val562Ile ABCC7 p.Gly550Glu ABCC7 p.Ala561Glu To test the hypothesis that G550E and 4RK act by different mechanisms, we used biochemical and functional assays to investigate how these revertants rescue F508del-, R560T-, A561E-, and V562I-CFTR. Login to comment 39 ABCC7 p.Arg560Thr ABCC7 p.Val562Ile Results R560T, but Not V562I, Disrupts the Processing of CFTR. Like F508del-CFTR, many CF mutations disrupt the processing of CFTR and its delivery to the cell surface. Login to comment 41 ABCC7 p.Arg560Thr ABCC7 p.Val562Ile ABCC7 p.Ala561Glu For the reasons outlined in the Introduction, we chose to analyze the CF mutations R560T, A561E, and V562I. Login to comment 42 ABCC7 p.Ala561Glu Although we previously demonstrated that A561E is processed defectively (31), no trafficking data are available for mutations at R560 and V562. Login to comment 43 ABCC7 p.Arg560Thr ABCC7 p.Val562Ile Therefore, we first investigated the processing of R560T and V562I by using Western blot (WB). Login to comment 44 ABCC7 p.Arg560Thr ABCC7 p.Ala561Glu Fig. 1A demonstrates that like F508del- and A561E-CFTR, R560T-CFTR generated only a discrete Ϸ145 kDa band (band B). Login to comment 45 ABCC7 p.Val562Ile In contrast, band B and a diffuse Ϸ180 kDa band (band C) were observed for both V562I-CFTR and wt-CFTR (Fig. 1A). Login to comment 46 ABCC7 p.Arg560Thr ABCC7 p.Val562Ile These data suggest that R560T, disrupts the trafficking of CFTR, whereas V562I-CFTR is delivered to the cell surface. Login to comment 47 ABCC7 p.Arg560Thr ABCC7 p.Gly550Glu ABCC7 p.Ala561Glu Next, we investigated whether the revertants G550E and 4RK rescue the defective biosynthesis of R560T- and A561E-CFTR. Login to comment 49 ABCC7 p.Gly550Glu ABCC7 p.Ala561Glu Fig. 1A shows that G550E and 4RK were much less effective at rescuing A561E-CFTR compared with their effects on F508del-CFTR. Login to comment 50 ABCC7 p.Gly550Glu ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu In fact, band C of both A561E-4RK- and A561E- G550E-CFTR was detected only after a 24 h incubation with 2 mM 4-phenylbutyrate (4-PB), an enhancer of CFTR expression through transcriptional activation (Fig. 1B) (32). Login to comment 51 ABCC7 p.Ala561Glu Quantification of the data reveals that, after this treatment with 4-PB, Ϸ30% of A561E-CFTR was rescued by either revertant, whereas for F508del-CFTR the value was Ϸ65%. Login to comment 55 ABCC7 p.Arg560Thr ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Ala561Glu The mature form of CFTR (band C) was clearly observed for F508del- and A561E-CFTR (Fig. 1D) in the presence of G550E and 4RK, but G550E failed to correct the defective biosynthesis of R560T-CFTR (Fig. 1 A and D). Login to comment 56 ABCC7 p.Gly550Glu Thus, the revertants G550E and 4RK rescue some, but not all, CF mutations in NBD1. Login to comment 57 ABCC7 p.Gly550Glu Effect of the G550E and 4RK Revertants on the Turnover and Processing of CFTR Variants. Login to comment 58 ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Gly550Glu Fig. 1A demonstrates that steady-state levels of band C for both wtand V562I-CFTR were increased by 4RK but not by G550E, most strikingly for V562I-CFTR. Login to comment 59 ABCC7 p.Gly550Glu In contrast, the revertants had the opposite effect on F508del-CFTR, with G550E generating higher steady-state levels of band C than 4RK (Fig. 1A). Login to comment 60 ABCC7 p.Gly550Glu These data suggest that G550E and 4RK might alter the processing of CFTR in different ways. Login to comment 61 ABCC7 p.Val562Ile To investigate this, we examined the turnover rate of band B and the efficiency of its processing into band C for wt-, F508del- and V562I-CFTR alone or in cis with the revertants by pulse-chase analysis, followed by IP (Fig. 2 A-C). Login to comment 62 ABCC7 p.Gly550Glu For wt-CFTR, the presence of the revertants, 4RK or G550E (dotted and dashed lines in Fig. 2 D and G, respectively), did not alter either the turnover rate of band B (solid line in Fig. 2D) or the efficiency of its conversion into band C (compare dotted and dashed lines with solid line in Fig. 2G). Login to comment 63 ABCC7 p.Gly550Glu Similarly, the turnover rate of band B of either F508del-4RK- or F508del-G550E-CFTR (dotted and dashed lines in Fig. 2E) was the same as that of F508del-CFTR (solid line, Fig. 2E). Login to comment 64 ABCC7 p.Gly550Glu Moreover, Fig. 2H demonstrates identical processing efficiencies for F508del-4RK- and F508del-G550E-CFTR (compare the dotted and dashed lines). Login to comment 65 ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Gly550Glu For V562I-CFTR, the turnover rate of V562I-G550E was slightly, but not significantly (P Ͼ 0.05), reduced compared with those of V562I- and V562I-4RK-CFTR (Fig. 2F). Login to comment 66 ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Gly550Glu Furthermore, the efficiency of processing of V562I-4RK (dotted line, Fig. 2I) was slightly increased relative to those of V562I and V562I-G550E (solid and dashed lines, Fig. 2I, respectively). Login to comment 67 ABCC7 p.Val562Ile ABCC7 p.Val562Ile Finally, Fig. 2 shows that the turnover rates of V562I- and wt-CFTR were equivalent (slope of solid lines in D and F; P Ͼ 0.05), whereas the processing of V562I- was slightly faster than that of wt-CFTR (solid lines in G and I).࿣The CFTR Mutation Database: http://www.sickkids.on.ca/cftr. Login to comment 70 ABCC7 p.Arg560Thr ABCC7 p.Val562Ile ABCC7 p.Gly550Glu ABCC7 p.Ala561Glu (A) WB of total protein (30 ␮g) from BHK cells stably expressing wt-, F508del-, R560T-, A561E-, or V562I-CFTR, alone or in cis with 4RK and G550E. Login to comment 75 ABCC7 p.Arg560Thr ABCC7 p.Val562Ile ABCC7 p.Gly550Glu ABCC7 p.Ala561Glu (D) BHK cells expressing F508del-, R560T-, or A561E-CFTR alone or in cis with the revertants 4RK and G550E were analyzed by CFTR IP after pulse-labeling for 3 h. Labeled arrows indicate the positions of bands A, B, and C. Thus, the higher steady-state levels of band C for 4RK variants of both wtand V562I-CFTR (Fig. 1A) are explained only in part by a slight (but not significant) increase in the efficiency of processing band B to band C. Surprised that the revertants did not exert stronger effects on the processing of CFTR, we wondered how they might influence CFTR Cl-channel function. Login to comment 76 ABCC7 p.Val562Ile WTand V562I-CFTR Have Equivalent Functions. Login to comment 79 ABCC7 p.Arg560Thr ABCC7 p.Val562Ile ABCC7 p.Ala561Glu Consistent with the biochemical data (Fig. 1), these agonists had no effect on F508del-, R560T-, or A561E-CFTR (Fig. 3 B-D) but evoked a striking efflux of I- from V562I-CFTR (Fig. 3E), which has a time course equivalent to that of wt-CFTR and 1.3-fold greater (Fig. 3F). Login to comment 80 ABCC7 p.Val562Ile A likely explanation for this enhanced efflux of I- from V562I-CFTR-expressing cells is their greater membrane abundance of CFTR (Fig. 1A). Login to comment 82 ABCC7 p.Gly550Glu In contrast, G550E, which produced lower steady-state levels of band C than wt-CFTR (Fig. 1A), generated Ϸ1.5-fold higher efflux of I- (Fig. 3 A and F). Login to comment 83 ABCC7 p.Gly550Glu Consistent with previous work (24) and our biochemical data (Figs. 1 and 2), both 4RK and G550E restored CFTR function to F508del, although the time course of the F508del-4RK response was delayed by 1 min compared with that of wt-CFTR (Fig. 3 B and F). Login to comment 84 ABCC7 p.Arg560Thr ABCC7 p.Gly550Glu ABCC7 p.Ala561Glu In contrast to F508del-CFTR, G550E did not restore CFTR function to R560T and both revertants rescued only modestly the CFTR function of A561E (Fig. 3 C, D, and F). Login to comment 85 ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Gly550Glu Interestingly, V562I-G550E generated an efflux of I- equivalent in magnitude and time-course to that of V562I-CFTR (Fig. 3 E and F) despite its lower steady-state levels of band C. However, 4RK, which generated higher steady-state levels of band Fig. 2. Login to comment 86 ABCC7 p.Val562Ile ABCC7 p.Gly550Glu Turnover and processing of wt-, F508del-, and V562I-CFTR alone or in cis with 4RK and G550E. Login to comment 87 ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu (A-C) BHK cells expressing wt-, G550E- and 4RK-CFTR (A); F508del-, F508del-4RK-, and F508del-G550E-CFTR (B); and V562I-, V562I-4RK-, and V562I-G550E-CFTR (C) were pulse-labeled for 20 min with 100 ␮Ci͞ml [35S]methionine and then chased for 0, 0.5, 1, 3, and 5 h. Login to comment 88 ABCC7 p.Val562Ile (D-F) Turnover of band B for wt- (D), F508del- (E), and V562I-CFTR (F) in the absence and presence of revertant mutations. Login to comment 91 ABCC7 p.Val562Ile (G and I) Efficiency of processing into band C for wt-CFTR (G) and V562I-CFTR (I) in the absence and presence of revertant mutations and for revertants of F508del-CFTR (H). Login to comment 96 ABCC7 p.Arg560Thr ABCC7 p.Val562Ile ABCC7 p.Gly550Glu ABCC7 p.Ala561Glu (A-E) Time courses of I-efflux from BHK cells stably expressing wt- (A), F508del- (B), R560T- (C), A561E- (D), and V562I- (E) CFTR in the absence and presence of the 4RK and G550E mutations. Login to comment 102 ABCC7 p.Val562Ile C (Fig. 1A), decreased the magnitude of the V562I-evoked I-efflux to a level equivalent to that of wt-CFTR (Fig. 3 E and F). Login to comment 103 ABCC7 p.Val562Ile Taken together, our iodide efflux data strongly suggest that the increased steady-state levels of band C (and the slight increase in CFTR processing efficiency) observed for the 4RK variants of wtand V562I-CFTR (Figs. 1 and 2) do not result in increased CFTR channel activity. Login to comment 104 ABCC7 p.Val562Ile ABCC7 p.Gly550Glu In contrast, the data suggest that G550E compensates for a decreased steady-state level of band C by enhancing the channel activity of wtand V562I-CFTR. Login to comment 105 ABCC7 p.Gly550Glu A similar mechanism might explain how G550E rescues the activity of CF mutants. Login to comment 106 ABCC7 p.Gly550Glu 4RK and G550E Rescue F508del-CFTR Channel Gating with Different Efficacy. Login to comment 107 ABCC7 p.Val562Ile ABCC7 p.Gly550Glu To test the idea that the revertant G550E enhances the Cl-channel function of wt and CF mutants, we used the patch-clamp technique to study the single-channel behavior of wt-, V562I-, and F508del-CFTR in the absence and presence of the revertants. Login to comment 108 ABCC7 p.Val562Ile Fig. 4 demonstrates that the single-channel properties of V562I-CFTR were identical to those of wt-CFTR. Login to comment 109 ABCC7 p.Val562Ile Like wt-CFTR (2), the gating behavior of V562I-CFTR was characterized by bursts of channel openings interrupted by brief flickery closures and separated by longer closures between bursts (Fig. 4A). Login to comment 110 ABCC7 p.Val562Ile ABCC7 p.Val562Ile Strikingly, V562I-CFTR had the same open probability (Po), mean burst duration (MBD), interburst interval (IBI), and ATP-dependence [V562I, ATP concentration for half maximal activation (Km) ϭ 252 ␮M; wt, Km ϭ 210 ␮M] as wt-CFTR (Fig. 4 B-D and data not shown). Login to comment 111 ABCC7 p.Val562Ile ABCC7 p.Val562Ile Together with our observation that V562I-CFTR does not affect the efficiency of CFTR processing (Figs. 1 and 2), these data raise the possibility that V562I is not a disease-causing mutation. Login to comment 112 ABCC7 p.Gly550Glu Before examining the rescue of F508del-CFTR channel function by the revertants, we quantified the deficit in CFTR function caused by F508del and the effects of 4RK and G550E on wt-CFTR. Login to comment 116 ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu The revertants 4RK and G550E enhanced wt-CFTR channel gating but with strikingly different efficacies. 4RK caused a small but not significant increase in Po by lengthening MBD 2-fold, whereas G550E increased the Po of wt-CFTR 1.5-fold by prolonging MBD 4.5-fold without altering IBI (Fig. 4 B-D). Login to comment 117 ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu Moreover, G550E dramatically enhanced the ATP-sensitivity of wt-CFTR (wt-CFTR, Km ϭ 210 ␮M; G550E, Km ϭ 19 ␮M; data not shown). Login to comment 118 ABCC7 p.Val562Ile ABCC7 p.Gly550Glu Like their effects on wt-CFTR, 4RK and G550E enhanced V562I-CFTR channel gating and rescued that of F508del-CFTR with different efficacies (Fig. 4). Login to comment 119 ABCC7 p.Gly550Glu For example, Fig. 4A demonstrates that F508del-4RK- and F508del-G550E-CFTR both have prolonged channel openings compared with those of wtand F508del-CFTR. Login to comment 120 ABCC7 p.Gly550Glu But more strikingly, the IBI for F508del-G550E-CFTR is dramatically shorter than that of F508del-CFTR and approaches that of wt-CFTR (Fig. 4A). Login to comment 121 ABCC7 p.Gly550Glu Fig. 4C demonstrates that both revertants prolonged strikingly the MBD of F508del-CFTR compared with that of either wtor F508del-CFTR (4RK, 2.5-fold; G550E, 4-fold; both vs. F508del-CFTR). Login to comment 122 ABCC7 p.Gly550Glu However, only G550E rescued the IBI defect of F508del-CFTR, reducing its IBI from 8-fold longer than wt-CFTR to only 2-fold longer (Fig. 4D). Login to comment 123 ABCC7 p.Gly550Glu As a result, the Po of F508del-G550E-CFTR exceeded that of wt-CFTR, whereas that of F508del-4RK is only half that of wt-CFTR (Fig. 4B). Login to comment 124 ABCC7 p.Gly550Glu To summarize, our data demonstrate that the revertant G550E efficaciously rescues the gating defect of F508del-CFTR, whereas rescue by 4RK is modest. Login to comment 125 ABCC7 p.Gly550Glu We interpret these data to suggest that 4RK and G550E exert their effects on F508del by different mechanisms. Login to comment 127 ABCC7 p.Arg560Thr ABCC7 p.Val562Ile ABCC7 p.Ala561Glu Like F508del, R560T and A561E disrupt CFTR processing, whereas V562I traffics normally to the cell surface, forming a Cl-channel with properties indistinguishable from those of wt-CFTR. Login to comment 128 ABCC7 p.Arg560Thr ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Ala561Glu The revertants 4RK and G550E rescue the cell surface expression of A561E, albeit not as effectively as F508del, whereas G550E is without effect on R560T. Login to comment 129 ABCC7 p.Arg560Thr ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Ala561Glu Of note, G550E, but not 4RK, rescues the defective channel gating of F508del, suggesting that G550E and 4RK rescue the expression and function of F508del by distinct mechanisms. To understand the structural basis by which R560T and A561E disrupt the processing of CFTR (refs. Login to comment 132 ABCC7 p.Arg560Thr R560T likely disrupts the interactions of R560 with neighboring residues and, hence, the folding of NBD1. Login to comment 133 ABCC7 p.Arg560Gly The fact that other CF-causing mutations at R560 (e.g., R560G) also cause severe disease highlights the importance of this residue for the global structure of CFTR. Login to comment 136 ABCC7 p.Val562Ile ABCC7 p.Gly550Glu Single-channel activity of wt-, V562I-, and F508del-CFTR alone and in cis with 4RK and G550E. Login to comment 140 ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu Columns and error bars are means Ϯ SEM (wt: n ϭ 6 for all data; 4RK: Po, n ϭ 2; MBD and IBI, n ϭ 1; G550E: Po, n ϭ 3; MBD and IBI, n ϭ 1; V562I: Po, n ϭ 5; MBD and IBI, n ϭ 2; V562I-4RK: Po, n ϭ 4; MBD and IBI, n ϭ 3; V562I-G550E: Po, n ϭ 8; MBD and IBI, n ϭ 3; F508del: n ϭ 10 for all data; F508del-4RK: Po, n ϭ 13; MBD and IBI, n ϭ 10; F508del-G550E: Po, n ϭ 5; MBD and IBI, n ϭ 4). Login to comment 144 ABCC7 p.Val562Ile Surprisingly, our data demonstrate that V562I-CFTR is processed as efficiently as wt-CFTR (Fig. 2) and forms a Cl-channel functionally equivalent to wt-CFTR (Fig. 4). Login to comment 145 ABCC7 p.Val562Ile Indeed, our structural model predicts that V562I is without effect on the tertiary structure of NBD1. Login to comment 146 ABCC7 p.Val562Ile Based on these data, we do not consider V562I-CFTR to be a CF-causing variant. Login to comment 163 ABCC7 p.Gly550Glu DeCarvalho et al. (26) identified the revertant G550E by using STE6͞CFTR chimeras to search for F508del suppressor mutations. Login to comment 164 ABCC7 p.Gly550Glu The authors demonstrated that G550E (i) partially rescues the processing defect of F508del-CFTR, (ii) enhances F508del-CFTR Cl- currents in Fischer rat thyroid (FRT) epithelia, and (iii) increases the sensitivity of F508del-CFTR to stimulation by cAMP agonists (26). Login to comment 166 ABCC7 p.Gly550Glu Any difference in the level of F508del rescue between previous studies of 4RK (25) and G550E (26) and our own are most likely due to the use of different cell lines. Login to comment 167 ABCC7 p.Gly550Glu Of note, our single-channel data provide an explanation for the effects of G550E on F508del-CFTR activity. Login to comment 169 ABCC7 p.Gly550Glu This suggests that G550E might promote the binding of ATP to site 1 of the NBD dimer while slowing the hydrolysis of ATP at site 2, plausibly by stabilizing the NBD dimer (4). Login to comment 170 ABCC7 p.Gly550Glu Thus, we speculate that the gating behavior of F508del-G550E-CFTR might be a consequence of a (partial) correction the F508del-induced folding defect of NBD1. Login to comment 177 ABCC7 p.Ala561Glu This explains the observed rescue of the cell surface expression of F508del- and A561E-CFTR by the 4RK revertant. Login to comment 178 ABCC7 p.Ala561Glu However, such an explanation does not imply correction of the folding defects of F508del- and A561E-CFTR. Login to comment 179 ABCC7 p.Gly550Glu Consistent with this idea, our single-channel data demonstrate that 4RK incompletely rescues the gating defect of F508del-CFTR, unlike the G550E revertant. Login to comment 180 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu Jurkuvenaite et al. (42) recently studied R31C and R31L, two CF mutations in the N terminus of CFTR that affect the first AFT (R29QR31). Login to comment 182 ABCC7 p.Val562Ile However, the opposite effect was observed for both wtand V562I-CFTR when in cis with 4RK; therefore, the effects observed with the R31 mutants must result from the loss of a stabilizing role played by the N terminus rather than from interference with an AFT. Login to comment 185 ABCC7 p.Arg555Lys Interestingly, different patterns of channel gating have been reported for R555K-CFTR. Login to comment 186 ABCC7 p.Arg555Lys Teem et al. (24) demonstrated that R555K increases the activity of wt-CFTR by extending the duration of bursts without altering the closed time interval between bursts. Login to comment 187 ABCC7 p.Arg555Lys In contrast, Vergani et al. (4) showed that R555K slows the rate of channel opening by markedly prolonging the closed time interval between bursts. Login to comment 190 ABCC7 p.Arg555Lys ABCC7 p.Arg29Lys Of note, the work of Hegedus et al. (40), who studied F508del-CFTR in cis with R29K and R555K (called 2RK), suggests that the stability of F508del-2RK-CFTR is temperature-dependent. Login to comment 196 ABCC7 p.Gly550Glu In summary, our studies indicate that the location of CF mutations within the H5 ␣-helix of NBD1 determines the efficacy with which the revertants G550E and 4RK restore their function: Mutations located proximally are rescued better than those located distally. Login to comment 198 ABCC7 p.Gly550Glu Moreover, our data demonstrate that the revertants 4RK and G550E produce distinct effects on F508del-CFTR: 4RK affects mainly the efficiency of processing, enabling the mutant to escape AFT-mediated ER retention͞ retrieval, with only limited rescuing of folding. Login to comment 199 ABCC7 p.Gly550Glu In contrast, G550E appears to exert its effect directly on the conformation of NBD1, as it rescues efficaciously the gating defect of F508del-CFTR. Login to comment 205 ABCC7 p.Arg560Thr ABCC7 p.Val562Ile When compared with both wtand F508del-CFTR, clones expressing V562I- and R560T-CFTR expressed higher and lower levels of protein, respectively, precluding studies on cell lines with equivalent amounts of CFTR protein. Login to comment