ABCMdb

ABCC7 p.Lys584Glu

Predicted by SNAP2:	A: D (63%), C: D (66%), D: D (80%), E: D (53%), F: D (75%), G: D (71%), H: D (59%), I: D (66%), L: D (71%), M: D (63%), N: D (66%), P: D (80%), Q: D (59%), R: N (93%), S: D (59%), T: D (63%), V: D (66%), W: D (80%), Y: D (75%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: N, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: N, R: N, S: D, T: D, V: D, W: D, Y: D,

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[hide] Folding and rescue of a cystic fibrosis transmembr... J Biol Chem. 2010 Aug 27;285(35):27033-44. Epub 2010 Jun 15. 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 [PMID:20551307]

Abstract [show]
Impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel causes cystic fibrosis, a fatal genetic disease. Here, to gain insight into CFTR structure and function, we exploited interspecies differences between CFTR homologues using human (h)-murine (m) CFTR chimeras containing murine nucleotide-binding domains (NBDs) or regulatory domain on an hCFTR backbone. Among 15 hmCFTR chimeras analyzed, all but two were correctly processed, one containing part of mNBD1 and another containing part of mNBD2. Based on physicochemical distance analysis of divergent residues between human and murine CFTR in the two misprocessed hmCFTR chimeras, we generated point mutations for analysis of respective CFTR processing and functional properties. We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. No single mutation was identified in NBD2 that disrupts protein processing. However, a number of NBD2 mutants altered channel function. Analysis of structural models of CFTR identified that although Lys(584) interacts with residue Leu(581) in human CFTR Glu(584) interacts with Phe(581) in mouse CFTR. Introduction of the murine residue (Phe(581)) in cis with K584E in human CFTR rescued the processing and trafficking defects of K584E-CFTR. Our data demonstrate that human-murine CFTR chimeras may be used to validate structural models of full-length CFTR. We also conclude that hmCFTR chimeras are a valuable tool to elucidate interactions between different domains of CFTR.

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No. Sentence Comment
4 We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. Login to comment
8 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 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 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 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 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 Like F508del (Fig. 2A, lane 2), K584E (lane 8) only generated immature CFTR protein (band B; 150-kDa). Login to comment
129 These data suggest that all CFTR constructs except K584E are delivered to the cell surface. Login to comment
133 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 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
152 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 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 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 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 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 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 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 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 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 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
178 However, that of L581F/K584E-CFTR was intermediate between L581F- and WT-CFTR (Fig. 4B and supplemental Fig. 3A). Login to comment
179 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 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 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 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 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 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 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 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 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 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 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 Immunodetection of CFTR showed that the K584E-CFTR mutant (Fig. 5, C1) was predominately located intracellularly like F508del-CFTR (B1). Login to comment
201 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 Rescue of trafficking defect of K584E-CFTR by L581F-CFTR. Login to comment
203 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 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 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 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 We therefore speculated that L581F-CFTR might augment the single channel activity of K584E-CFTR. Login to comment
221 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 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 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 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 Thus, K584E-CFTR profoundly FIGURE 5. Login to comment
234 Single channel analysis of CFTR constructs K584E-, L581F-, and L581F/K584E-CFTR. Login to comment
237 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 Of note, these effects of K584E-CFTR were rescued by the revertant mutation L581F-CFTR. Login to comment
255 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 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
285 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
296 Characterization of K584E and Rescuing by Leu581 -Like F508del-CFTR (33), the trafficking defect of K584E-CFTR is temperature-sensitive. Login to comment
297 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 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 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 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
308 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 But this explanation has to be discarded because, by immunofluorescence, K584E-CFTR could not be detected at the plasma membrane. Login to comment
310 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 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 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 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 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 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 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 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 We identified an NBD1 trafficking mutant (K584E) that, despite being inefficiently processed, exhibits some activity as a regulated Cl-channel. Login to comment
327 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