ABCMdb

PMID: 20551307

Da Paula AC, Sousa M, Xu Z, Dawson ES, Boyd AC, Sheppard DN, Amaral MD
Folding and rescue of a cystic fibrosis transmembrane conductance regulator trafficking mutant identified using human-murine chimeric proteins.
J Biol Chem. 2010 Aug 27;285(35):27033-44. Epub 2010 Jun 15., 2010-08-27 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Lys584Glu We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. Login to comment 8 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu Introduction of the murine residue (Phe581 ) in cis with K584E in human CFTR rescued the processing and trafficking defects of K584E-CFTR. Login to comment 49 ABCC7 p.Lys584Glu Systematic mutagenesis of the divergent murine residues present in these chimeras followed by biochemical and functional studies revealed that K584E is responsible for the maturation defect of the hmNBD1 chimera. Login to comment 50 ABCC7 p.Lys584Glu Furthermore, replacement of Leu581 (interacting with Lys584 in the structure of hNBD1) by the corresponding murine amino acid (Phe581 ) rescued the maturation of K584E-CFTR. Login to comment 124 ABCC7 p.Ile539Thr ABCC7 p.Pro1290Thr ABCC7 p.Lys584Glu ABCC7 p.Glu528Gln ABCC7 p.Leu1367Ile ABCC7 p.Glu1409Asp ABCC7 p.Ser1311Lys ABCC7 p.Lys1302Gln ABCC7 p.Glu527Gln ABCC7 p.Cys1344Tyr ABCC7 p.Val1338Thr ABCC7 p.Gln1309Lys ABCC7 p.Arg1325Lys ABCC7 p.Asp1394Gly ABCC7 p.Tyr1307Asn ABCC7 p.Thr1263Ile ABCC7 p.Ser531Thr ABCC7 p.Lys536Gln Thus, we identified six residues in 12b-NBD1 (E527Q, E528Q, S531T, K536Q, I539T, and K584E) and 12 residues in 114c-NBD2 (T1263I, P1290T, K1302Q, Y1307N, Q1309K, S1311K, R1325K, V1338T, C1344Y, L1367I, D1394G, and E1409D) (see supplemental Fig. 1 and supplemental Table 1, A and B). Login to comment 126 ABCC7 p.Lys584Glu With one exception (K584E; Fig. 2A, lane 8), all point mutants derived from 12b-NBD1 were processed (lanes 3-7) similarly to WT-hCFTR (Fig. 2A, lane 1). Login to comment 127 ABCC7 p.Lys584Glu Like F508del (Fig. 2A, lane 2), K584E (lane 8) only generated immature CFTR protein (band B; 150-kDa). Login to comment 129 ABCC7 p.Lys584Glu These data suggest that all CFTR constructs except K584E are delivered to the cell surface. Login to comment 133 ABCC7 p.Lys584Glu Like BHK cells expressing F508del-CFTR (Fig. 2C and supplemental Fig. 2A), those expressing K584E-CFTR failed to elicit an efflux of I- when treated with forskolin and genistein (Fig. 2C and supplemental Fig. 2C). Login to comment 134 ABCC7 p.Lys584Glu The likely explanation for this result is the lack of K584E-CFTR expression at the cell membrane as suggested by the absence of mature CFTR protein FIGURE 1. Login to comment 147 ABCC7 p.Pro1290Thr ABCC7 p.Pro1290Thr ABCC7 p.Glu528Gln ABCC7 p.Glu528Gln For most NBD1 and NBD2 mutants, the magnitude of I-efflux was less than that of WT-CFTR; for two mutants in NBD2 (P1290T- and D1394D-CFTR), it was the same; and for one NBD1 mutant (E528Q-CFTR), the magnitude of I-efflux was greater than that of WT-CFTR (Fig. 2C; supplemental Fig. 2, B, D, and F; and Table 1). Login to comment 150 ABCC7 p.Ser531Thr ABCC7 p.Lys536Gln For example, compare the data for S531T and K536Q in Fig. 2, C and D. Taken together and consistent with the WB data (Fig. 2, A and B, and Table 1), our iodide efflux data suggest that most of the NBD1 and NBD2 mutants exhibited channel activity, arguing that they reach the cell surface. Login to comment 152 ABCC7 p.Lys584Glu By contrast, the agonists had barely any effect on BHK cells expressing K584E-CFTR (Fig. 2C), a result that is consistent with the trafficking defect of this mutant (Fig. 2A). Login to comment 153 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu Characterization of Processing Defect of K584E-CFTR-The lack of maturation and of detectable activity for the K584E-CFTR mutant led us to investigate the surrounding environment of Lys584 in the published structure of hNBD1 (12). Login to comment 154 ABCC7 p.Lys584Glu The rationale behind this approach was to uncover which residues near or interacting with Lys584 (hNBD1) might contribute to the putative conformational change caused by the K584E mutation. Login to comment 155 ABCC7 p.Lys584Glu We found that in the hNBD1 structure Lys584 interacts with Leu581 (Fig. 3, A and B) and that predictively in K584E the glutamic acid residue at position 584 becomes solvent-exposed (Fig. 3B). Login to comment 157 ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe Thus, we postulated that replacing Leu581 by a phenylalanine residue (as in mNBD1) might improve CFTR folding and rescue the processing defect of K584E-CFTR. Login to comment 158 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe To test this hypothesis, we introduced L581F in cis with K584E-CFTR by site-directed mutagenesis (L581F/K584E-CFTR) and generated the corresponding stable BHK cell line as well as one expressing L581F-CFTR. Login to comment 159 ABCC7 p.Lys584Glu We then analyzed these cell lines in parallel with that expressing K584E-CFTR by biochemical (Fig. 4, A and C-E), cell biology (Fig. 5), and functional approaches (Figs. Login to comment 174 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe Error bars correspond to S.E. Folding of CFTR Chimeras AUGUST 27, 2010•VOLUME 285•NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 27037 Fig. 4A demonstrates that both L581F- (lane 2) and L581F/ K584E- (lane 3) were detected as their fully glycosylated forms (band C) in contrast to K584E-CFTR (lane 1) for which only immature protein could be detected. Login to comment 175 ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe We interpret these data to suggest that K584E is rescued by L581F, possibly mediated by the side chain of phenylalanine 581 that most probably fills the empty space previously occupied by the side chain of Lys584 (Fig. 3B) as occurs in the structure of mNBD1 (Fig. 3C) (11). Login to comment 176 ABCC7 p.Lys584Glu Data from WB also show that the trafficking defect of K584E-CFTR was rescued by incubation of cells at 26 °C (Fig. 4A, lane 7), similarly to F508del-CFTR (lane 6). Login to comment 177 ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe The magnitude of I-efflux elicited by L581F-CFTR (Fig. 4B and supplemental Fig. 3A) was smaller than that of WT-CFTR. Login to comment 178 ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe However, that of L581F/K584E-CFTR was intermediate between L581F- and WT-CFTR (Fig. 4B and supplemental Fig. 3A). Login to comment 179 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu Furthermore, when cells expressing K584E-CFTR were incubated at 26 °C for 24 h (Fig. 4B and supplemental Fig. 3B), they generated an efflux of I- similar to that of F508del-CFTR-expressing cells incubated at 26 °C for 24 h; albeit this response of K584E-CFTR was 5-fold smaller than that of WT-CFTR at 37 °C. Login to comment 180 ABCC7 p.Lys584Glu These data together with those of Fig. 4A (lane 6) indicate that, like F508del-CFTR (Fig. 4A, lane 6) (33), the processing of K584E-CFTR is temperature-sensitive. Login to comment 181 ABCC7 p.Lys584Glu At 26 °C, some K584E-CFTR protein is delivered to the cell membrane because the trafficking defect of this mutant was (at least partially) reverted at this permissive temperature. Login to comment 182 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe To learn more about the stability of the K584E-, L581F-, and L581F/K584E-CFTR variants, we examined the turnover rate of FIGURE 3. Login to comment 187 ABCC7 p.Ile539Thr ABCC7 p.Pro1290Thr ABCC7 p.Lys584Glu ABCC7 p.Glu528Gln ABCC7 p.Leu1367Ile ABCC7 p.Glu1409Asp ABCC7 p.Ser1311Lys ABCC7 p.Lys1302Gln ABCC7 p.Glu527Gln ABCC7 p.Cys1344Tyr ABCC7 p.Val1338Thr ABCC7 p.Gln1309Lys ABCC7 p.Arg1325Lys ABCC7 p.Asp1394Gly ABCC7 p.Tyr1307Asn ABCC7 p.Thr1263Ile ABCC7 p.Ser531Thr ABCC7 p.Lys536Gln ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe TABLE 1 Summary information of CFTR point mutants analyzed in present study CFTR variants Clinical dataa Band C/band Bb (؎S.E., n ‫؍‬ 5) Processingc Normalized processingd Normalized iodide efflux functione (؎S.E., n ‫؍‬ 6) Iodide efflux to processed proteinf % % % % peak intensity % WT-CFTR -g 83 Ϯ 3 77 100 100 Ϯ 8 - Murine - 86 Ϯ 5 66 86 74 Ϯ 4 86 Ϯ 4 E527Q Mild CF 64 Ϯ 5 49 63 46 Ϯ 4 73 Ϯ 4 E528Q - 86 Ϯ 5 79 102 135 Ϯ 16 132 Ϯ 10 S531T - 87 Ϯ 6 81 105 71 Ϯ 5 67 Ϯ 5 K536Q - 69 Ϯ 3 42 54 51 Ϯ 4 94 Ϯ 3 I539T Revertant 112 Ϯ 5 81 105 49 Ϯ 6 46 Ϯ 5 L581F - 118 Ϯ 3 83 107 72 Ϯ 5 67 Ϯ 3 L581F/K584E - 125 Ϯ 2 77 100 100 Ϯ 12 100 Ϯ 8 T1263I Mild CF 75 Ϯ 3 76 98 31 Ϯ 8 31 Ϯ 5 P1290T Asymptomatic 87 Ϯ 3 82 106 92 Ϯ 10 86 Ϯ 6 K1302Q - 72 Ϯ 3 77 100 37 Ϯ 2 37 Ϯ 2 Y1307N - 82 Ϯ 2 76 98 70 Ϯ 5 71 Ϯ 3 Q1309K - 79 Ϯ 4 77 100 26 Ϯ 2 26 Ϯ 3 S1311K - 73 Ϯ 4 72 93 33 Ϯ 7 35 Ϯ 5 R1325K - 64 Ϯ 6 78 101 47 Ϯ 2 46 Ϯ 4 V1338T - 88 Ϯ 2 77 100 37 Ϯ 11 37 Ϯ 6 C1344Y - 71 Ϯ 4 76 98 86 Ϯ 4 87 Ϯ 4 L1367I - 72 Ϯ 5 80 103 36 Ϯ 5 34 Ϯ 5 D1394G - 78 Ϯ 4 86 111 93 Ϯ 12 83 Ϯ 8 E1409D - 70 Ϯ 3 70 90 43 Ϯ 5 47 Ϯ 4 a Data from the CFTR mutation database. Login to comment 194 ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe Pulse-chase analyses followed by CFTR immunoprecipitation (Fig. 4C) demonstrate that the turnover rates of band B of both L581F- (Fig. 4D, open squares) and L581F/K584E-CFTR (Fig. 4D, filled triangles) were the same as those of WT-CFTR (Fig. 4D, filled circles). Login to comment 195 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe However, the turnover rate of K584E-CFTR (Fig. 4D, filled diamonds) was significantly (p Ͻ 0.05) reduced compared with those of L581F-, L581F/K584E-, WT-, and F508del-CFTR (Fig. 4D, open squares, filled triangles, filled circles, and open circles, respectively). Login to comment 196 ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe Consistent with the data in Fig. 4D, the processing efficiencies of L581F- (Fig. 4E, open squares) and L581F/ K584E-CFTR (Fig. 4E, filled triangles) were not significantly different from those of WT-CFTR (Fig. 4D, filled circles). Login to comment 197 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe Taken together, these data suggest that the immature form of K584E-CFTR is significantly stabilized compared with those of L581F-, L581F/K584E-, WT-, and even F508del-CFTR. Login to comment 198 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe Localization of K584E-, L581F-, and L581F/K584E-CFTR by Immunofluorescence-Our biochemical studies demonstrated that K584E-CFTR is only detected in its immature form, suggesting that it is misfolded and retained in the ER. Login to comment 199 ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe To test this possibility and learn whether the biochemically detected rescue of K584E-CFTR by L581F-CFTR (mature form) indeed corresponds to protein present at the cell surface, we used immunofluorescence and confocal microscopy. Login to comment 200 ABCC7 p.Lys584Glu Immunodetection of CFTR showed that the K584E-CFTR mutant (Fig. 5, C1) was predominately located intracellularly like F508del-CFTR (B1). Login to comment 201 ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe By contrast, L581F- (D1), L581F/ K584E- (E1), and WT-CFTR (A1) were mostly found at the plasma membrane co-localizing with wheat germ agglutinin FIGURE 4. Login to comment 202 ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe Rescue of trafficking defect of K584E-CFTR by L581F-CFTR. Login to comment 203 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe A, biochemical analysis by Western blot of total protein extract from BHK cells stably expressing K584E (lane 1), L581F (lane 2), L581F/K584E (lane 3), WT (lane 4), F508del (lane 5), F508del at 26 °C (lane 6), and K584E at 26 °C (lane 7). Login to comment 209 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe C, turnover and processing of WT-, F508del-, K584E-, L581F-, and L581F/K584E-CFTR determined in BHK cells stably expressing these CFTR variants by pulse-chase experiments followed by immunoprecipitation. Login to comment 217 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe We interpret these data to suggest that L581F rescues the cell surface expression of K584E, confirming our biochemical and functional data, which argue that L581F-CFTR is a revertant mutant for K584E-CFTR. Login to comment 218 ABCC7 p.Gly550Glu ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Lys584Glu Single Channel Behavior of Processing Mutant K584E-CFTR-In previous research, we demonstrated that revertant (e.g. G550E-CFTR (24)) and solubilizing mutations (e.g. F429S/F494N/Q637R (13)) rescue defects in CFTR channel gating in addition to promoting the cell surface expression of F508del-CFTR. Login to comment 219 ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe We therefore speculated that L581F-CFTR might augment the single channel activity of K584E-CFTR. Login to comment 221 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe Fig. 6A demonstrates that addition of ATP (1 mM) and protein kinase A (75 nM) to the intracellular solution bathing excised inside-out membrane patches from cells expressing K584E-, L581F-, and L581F/K584E-CFTR cultured at 37 °C activated single channels with properties and regulation characteristic of wild-type CFTR. Login to comment 224 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe Consistent with the behavior of other CFTR variants containing point mutations in the NBDs (2), K584E-, L581F-, and L581F/K584E-CFTR had values of i similar to that of WT-CFTR (Fig. 6B). Login to comment 227 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe Visual inspection of single channel records suggested that the gating behavior of the CFTR variants K584E-, L581F-, and L581F/K584E-CFTR all resembled that of WT-CFTR (Fig. 6A). Login to comment 228 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe Consistent with this idea, the Po of L581F/ K584E-CFTR was the same as that of WT-CFTR, whereas those of K584E- and L581F-CFTR were slightly, albeit significantly (p Ͻ 0.05), reduced (Fig. 6C). Login to comment 229 ABCC7 p.Lys584Glu Thus, K584E-CFTR profoundly FIGURE 5. Login to comment 234 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe Single channel analysis of CFTR constructs K584E-, L581F-, and L581F/K584E-CFTR. Login to comment 237 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe For WT-, F508del-, K584E-, and L581F/ K584E-CFTR, membrane patches contained a single active channel, but for L581F-CFTR, the membrane patch contained two active channels. Login to comment 244 ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe Of note, these effects of K584E-CFTR were rescued by the revertant mutation L581F-CFTR. Login to comment 255 ABCC7 p.Lys584Glu Indeed, the point mutant that we found to cause a failure in CFTR maturation (K584E-CFTR) lies exactly in this region. Login to comment 274 ABCC7 p.Lys584Glu Processing and Activity of Point Mutants in NBD1 and NBD2-Biochemical studies of CFTR variants identified by physicochemical distance analysis of residues in 12b-NBD1 revealed that the mutation K584E disrupts the maturation of CFTR protein. Login to comment 278 ABCC7 p.Asn1419Ser Nevertheless, as discussed above, N1419S alone likely rescues the processing defect of 114c-NBD2. Login to comment 279 ABCC7 p.Asn1419Ser This argues that the interacting residues of N1419S cause the trafficking defect of the hmCFTR chimera 114c-NBD2. Login to comment 285 ABCC7 p.Ile539Thr ABCC7 p.Pro1290Thr ABCC7 p.Lys584Glu ABCC7 p.Glu527Gln ABCC7 p.Thr1263Ile ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe Among the 20 point mutants described in this study (the above 18 point mutants plus L581F and L581F/K584E), we found four that are listed in the CFTR mutation database (CFTR mutation database), namely E527Q, T1263I, and P1290T, which are described in association with mild CF, and I539T, which is described as an F508del-revertant mutation (Table 1). Login to comment 286 ABCC7 p.Pro1290Thr Our own data suggest that P1290T (associated with asymptomatic CF) is worthy of further study because it does not affect CFTR processing or function. Login to comment 287 ABCC7 p.Val562Ile ABCC7 p.Pro1290Thr This raises the interesting possibility that P1290T might be a sequence variation (polymorphism) like V562I (24). Login to comment 289 ABCC7 p.Ile539Thr ABCC7 p.Leu1367Ile ABCC7 p.Glu1409Asp ABCC7 p.Ser1311Lys ABCC7 p.Lys1302Gln ABCC7 p.Glu527Gln ABCC7 p.Val1338Thr ABCC7 p.Gln1309Lys ABCC7 p.Thr1263Ile ABCC7 p.Leu581Phe The most striking differences for the NBD1 mutants (Table 1 and Fig. 2, compare C with D) were (Iodide efflux to processed protein (%) far right column) E527Q (64/46), I539T (112/49), and L581F (118/72), whereas for the NBD2 mutants, they were T1263I (75/31), K1302Q (72/37), Q1309K (79/26), S1311K (73/33), V1338T (88/37), L1367I (72/36), and E1409D (70/43). Login to comment 290 ABCC7 p.Ile539Thr Curiously, the point mutant with the highest discrepancy was I539T, which rescues the trafficking defect of F508del-CFTR (43). Login to comment 292 ABCC7 p.Gly550Glu By contrast, the revertant mutation G550E, which enhances both CFTR trafficking and gating, was proposed to correct the defective folding of F508del-CFTR (24). Login to comment 296 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu Characterization of K584E and Rescuing by Leu581 -Like F508del-CFTR (33), the trafficking defect of K584E-CFTR is temperature-sensitive. Login to comment 297 ABCC7 p.Lys584Glu However, in contrast to F508del-CFTR, active K584E-CFTR Cl-channels could be detected in cells cultured at 37 °C using the single channel patch clamp, although this mutant could not be detected at the cell surface by immunocytochemistry or in its processed form by WB. Login to comment 298 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe To investigate this discrepancy, we used our processing (Table 1) and single channel data (Fig. 6) to calculate predicted macroscopic currents for K584E-, L581F-, and L581F/K584E-CFTR and compared the values obtained with the magnitude of iodide efflux generated by these different CFTR constructs (Fig. 2C). Login to comment 304 ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe Thus, these data provide a molecular explanation for the quantitative decrease in CFTR-mediated iodide efflux caused by K584E and for the rescuing action of L581F. Login to comment 305 ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Lys584Glu The different effects of K584E on CFTR processing and Cl-channel function are reminiscent of A455E and P574H, two CF mutations associated with a milder clinical phenotype (44). Login to comment 306 ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu Although production of the mature forms of A455E and P574H was reduced and very much delayed, A455E had channel activity similar to and P574H had channel activity greater than that of WT-CFTR (44). Login to comment 307 ABCC7 p.Gly551Asp Conversely, the NBD1 CF mutant G551D is processed correctly but forms a Cl-channel with a profound gating defect that is not regulated by intracellular ATP (28, 45). Login to comment 308 ABCC7 p.Lys584Glu A further explanation for the discrepancy between the processing (WB) and single channel patch clamp data of K584E-CFTR is that this trafficking mutant might escape the ER via a non-conventional route (46). Login to comment 309 ABCC7 p.Lys584Glu But this explanation has to be discarded because, by immunofluorescence, K584E-CFTR could not be detected at the plasma membrane. Login to comment 310 ABCC7 p.Lys584Glu It thus seems likely that only very little (below biochemical/immunofluorescence detection levels) of the K584E-CFTR reaches the cell surface, but once correctly inserted, this CFTR variant has a significant capacity to transport Cl- . Login to comment 311 ABCC7 p.Lys584Glu To understand the structural basis by which K584E disrupts the processing and function of CFTR, we used a model of full-length CFTR.5 This trafficking mutant lies on the highly conserved region of NBD1 where Glu584 is solvent-exposed and 5 R. Ford, personal communication. Login to comment 312 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe TABLE 2 Comparison of predicted and measured CFTR-mediated iodide efflux for K584E-, L581F-, and L581F/K584E-CFTR N, the number of Cl-channels in the cell membrane; i, single-channel current; N ϫ i ϫ Po, the predicted CFTR-mediated iodide efflux based on CFTR processing and single channel data; ICFTR , measured CFTR-mediated iodide efflux. Login to comment 315 ABCC7 p.Lys584Glu ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe CFTR N i Po N ؋ i ؋ Po ICFTR % % % % % Wild type 100 100 100 100 100 F508del 5 104 12 1 0 K584E 1 90 73 1 0 L581F 107 88 81 75 72 L581F/K584E 100 91 103 93 100 Folding of CFTR Chimeras 27042 interacts with Leu581 . Login to comment 316 ABCC7 p.Lys584Glu Accordingly, we changed the interacting residue in human CFTR (Leu581 ) to the corresponding residue in murine CFTR (Phe581 ) and found that it rescued the trafficking defect of K584E. Login to comment 317 ABCC7 p.Lys584Glu In the crystal structure of NBD1 (11, 12), it is likely that K584E disrupts the interaction of Lys584 with neighboring residues and, hence, the folding of NBD1. Login to comment 318 ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe The introduction of the L581F mutation likely restores the processing defect of K584E possibly mediated by the side chain of Phe581 that fills the empty space left by the removal of Lys584 . Login to comment 319 ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe ABCC7 p.Leu581Phe Moreover, confirmation that both L581F and L581F/K584E are present at the cell surface was provided by functional studies (both iodide efflux and patch clamp). Login to comment 326 ABCC7 p.Lys584Glu We identified an NBD1 trafficking mutant (K584E) that, despite being inefficiently processed, exhibits some activity as a regulated Cl-channel. Login to comment 327 ABCC7 p.Lys584Glu ABCC7 p.Leu581Phe Moreover, by using the available structure of NBD1, we predicted that substitution of another NBD1 residue (L581F) should restore the processing defect of K584E. Login to comment