Mutations were introduced into ΔF508-CFTR, ΔF508/ΔNBD2 (Δ1173−1480)-CFTR, or ΔF508/ΔNBD2 (Δ1197−1480)-CFTR cDNAs containing an A52 epitope tag at the C-terminal end (10) by site-directed mutagenesis as described by Kunkel (11). The mutant CFTRs were transiently expressed in HEK 293 cells as described previously (5). HEK 293 cells were transfected with the cDNAs, and the medium was changed 4 h later to fresh medium (Dulbecco’s modified Eagle’s medium containing 10% (v/v) calf serum). Cells were harvested 18−36 h after the change in medium. Whole cell extracts of cells expressing untagged or A52-tagged CFTRs were subjected to immunoblot analysis using 6.5% (w/v) acrylamide gels and a CFTR polyclonal antibody or monoclonal antibody A52. Generation of stable BHK cell lines expressing wild-type or mutant ΔF508/V510D-CFTRs was performed as described previously (9).

HEK 293 cells were transfected with W356C/W1145C-, ΔF508/W356C/W1145C-, or ΔF508/V510D/W356C/W1145C-CFTR cDNAs, and the cells were incubated for 4 h at 37 °C. The transfection medium was removed, and the cells were incubated in fresh medium overnight at 30 °C. The cells were then treated for 15 min at 20 °C with 3 μM BMH. The medium was removed, and fresh medium containing 0.1 mM cysteine was added. At various time points the cells were cooled on ice, harvested, and washed with PBS, and whole cell SDS extracts were prepared. The reaction mixtures were then subjected to SDS−PAGE (6.5% (w/v) polyacrylamide gels) and immunoblot analysis with a rabbit polyclonal antibody against CFTR (12). Intramolecular disulfide cross-linking between domains of CFTR can be detected because the cross-linked product migrates with a slower mobility on SDS−PAGE gels (12). The gel lanes were scanned and quantitated using the NIH Image Program and an Apple computer.

Pulse−chase experiments (13) or cell surface labeling with biotin-LC-hydrazide after oxidation of carbohydrate with sodium periodate (14) were performed using HEK 293 cells transfected with A52-tagged wild-type, ΔF508-, or ΔF508/V510D-CFTR cDNAs as described previously. Pulse−chase assays were performed using transfected HEK 293 cells maintained at 37 °C. For cell surface labeling, the medium was replaced 4 h after transfection, and the cells were grown at 30 °C for 18 h. The cells were biotinylated and then grown in fresh media at 37 °C for various intervals.

Measurement of cAMP-stimulated iodide efflux was performed on baby hamster kidney (BHK) cells or BHK cells expressing wild-type or mutant ΔF508/V510D-CFTRs as described previously (9,15).

Models of CFTR structure predict that the segment in NBD1 containing Phe508 to Val510 would lie close to the fourth intracellular loop (ICL4) in TMD2 that connects transmembrane segments 10 and 11 (Figure 1, panels A and B). Recent crystal structures of wild-type NBD1 and ΔF508 NBD1 showed that deletion of Phe508 caused only local structural changes in residues 509−511 such as increased exposure of Val510 to solvent (16) (Figure 1, panels C and D). In a modeling study, it was predicted that the negative charge of the V510D mutation promoted maturation of ΔF508-CFTR because it restores defective NBD1−TMD2 interactions by forming a salt bridge with Arg1070 (TMD2) (17). We tested this prediction by introducing the charged amino acids Glu or Arg into ΔF508-CFTR. HEK 293 cells were transfected with the cDNA of wild-type CFTR (Figure 1E) or mutants ΔF508 (Figure 1, panels E−H), V510D/ΔF508 (Figure 1F), V510E/ΔF508 (Figure 1G), and V510R/ΔF508 (Figure 1H), and whole cell SDS extracts were subjected to immunoblot analysis 18 h after transfection. Mature and immature forms of CFTR can be distinguished in SDS−PAGE gels because of differences in glycosylation. The V510E mutation also promoted maturation, but it was less efficient than the V510D mutation (12% and 35%, respectively) (Figure 1, panels F and G). Little mature CFTR was observed in mutant ΔF508 (Figure 1, panels E-H) or V510R/ΔF508 (Figure 1H).

It was possible that the high level of expression of V510D/ΔF508 in the transiently transfected HEK 293 cells was also a factor in enhancing maturation of the mutant. To examine the extent of maturation at lower levels of expression, HEK 293 cells were transfected with lower concentrations of plasmid containing V510D/ΔF508 cDNA and whole cell extracts subjected to immunoblot analysis 42 h after transfection. The time of incubation was increased so CFTR could be detected after transfection with low cDNA concentrations. It was observed that expression of CFTR was reduced when cells were transfected with lower concentrations of plasmid (Figure 1I) but the relative steady-state level of mature V510D/ΔF508-CFTR remained at about 50% of total CFTR (Figure 1J).

To test if the mutant was active, BHK cell lines were generated that stably expressed wild-type or V510D/ΔF508-CFTRs for use in iodide efflux assays. The relative expression level of mature to total CFTR for mutant V510D/ΔF508 in BHK cells was also found to be about 50% (data not shown). Iodide efflux assays were performed rather than chloride channel measurements because very few channels other than CFTR can conduct iodide ions (18). Cells expressing wild-type or V510D/ΔF508-CFTRs exhibited iodide efflux upon addition of forskolin (Figure 2).

Wild-type CFTR lacking NBD2 shows efficient maturation (19). It was recently reported that rescue of ΔF508-CFTR by other suppressor mutations in NBD1 (I539T, G550E, R553M, R555K) was drastically reduced in wild-type CFTR lacking NBD2 (ΔNBD2) (20). It was concluded that these suppressor mutations likely promoted maturation through interactions with NBD2 as well as other domains. We introduced the V510D mutation into ΔF508-CFTR lacking NBD2 (truncated after residue 1196 and containing the epitope for monoclonal antibody A52 (Δ1197−1480)-A52) to test if it still promoted maturation. Immunoblot analysis of whole cell SDS extracts (Figure 3, left panel) showed that ΔF508/V510D/ΔNBD2-A52 (Δ1197−1480) had a maturation efficiency (about 50%) that was similar to that of the full-length ΔF508/V510D mutant (about 50%; Figure 1, panel F).

These results suggest that the V510D suppressor mutation differs from other NBD1 suppressor mutations because the absence of NBD2 did not reduce its effect. A potential problem with this interpretation, however, is that the two studies used different ΔF508/ΔNBD2 constructs. This study used a construct truncated after residue 1196 (Δ1197−1480) whereas the other study used a construct truncated after residue 1172 (Δ1173−1480) (20). To test if the position of the truncation affected maturation, mutant ΔF508/V510D/ΔNBD2-A52 (Δ1173−1480) was constructed and expressed in HEK293 cells. Immunoblot analysis of whole cell extracts showed that the V510D mutation promoted maturation of the truncation mutant but the relative level of mature was only about 20% of total CFTR protein.

It was predicted in a modeling study (17) that the V510D mutation may promote NBD1−TMD2 interactions in ΔF508-CFTR by forming a salt bridge with positively charged residue Arg1070 (TMD2). We tested the contribution of Arg1070 by introducing the R1070A change into ΔF508/V510D. As shown in Figure 4A, mature protein was not detectable in mutant ΔF508/V510D/R1070A. To test if the R1070A mutation alone affected maturation of ΔF508, mutants ΔF508/R1070A and ΔF508 were expressed at 30 °C. Maturation of both mutants was similar (about 20%) (Figure 4B). Therefore, it was unlikely that the R1070A mutant caused further misfolding of ΔF508-CFTR. This is consistent with the observation that introduction of the R1070A mutation into wild-type CFTR also did not affect its maturation (data not shown).

Mutant ΔF508/V510R/R1070D was constructed to reverse the positions of the charged residues. Figure 4C shows that the V510R/R1070D mutations promoted maturation of ΔF508-CFTR. Both mutations were required because the presence of V510R (Figure 1H) or R1070D (Figure 4D) alone did not promote maturation of ΔF508-CFTR. These results suggest that V510D may form a salt bridge with Arg1070 in ΔF508-CFTR.

The ΔF508 mutation reduces the stability of CFTR at the cell surface (7). To test if the V510D mutation increased the stability of ΔF508-CFTR, a cross-linking assay was used to measure the half-life of mature CFTR at the cell surface. We previously showed that cysteines introduced into TM segments 6 (W356C) and 12 (W1145C) could be cross-linked in mature CFTR when whole cells were treated with bifunctional thiol cross-linkers (14,15). Cross-linked mature CFTR was readily detected because it migrates with a slower mobility on SDS−PAGE gels. Cross-linking of immature CFTR was not detected. Therefore, the cross-linking can be used as a reporter to monitor turnover of mature CFTR at the cell surface. Accordingly, the W356C and W1145C mutations were introduced into wild-type, ΔF508-, and ΔF508/V510D-CFTRs. Transfected cells were incubated for 24 h at 30 °C and then treated with 3 μM 1,6-bismaleimidohexane (BMH) for 15 min at 21 °C. BMH was used instead of the bifunctional methanethiosulfonate cross-linkers (14) because maleimide-type cross-links are not reduced by thiol-reducing compounds such as dithiothreitol. BMH was then removed, and fresh medium containing 0.1 mM cysteine was added. Cells were harvested at various intervals and whole cell extracts subjected to immunoblot analysis. Panels A and B of Figure 5 show that the half-lives of cross-linked wild-type/W356C/W1145C-, ΔF508/W356C/W1145C-, and ΔF508/V510D/W356C/W1145C-CFTRs were about 12, 3, and 14 h, respectively. These results indicate that the stability of mutant V510D/ΔF508-CFTR resembled that of wild-type protein.

A potential problem with the cross-linking assay is that it required introduction of a pair of cysteines into the TM segments of CFTR. It was possible that cysteines W356C and W1145C may influence the stability of the protein. To examine the effect of the V510D mutation on turnover of ΔF508-CFTR without the introduced cysteines, a pulse−chase assay was performed. It was observed that the rate of conversion of immature wild-type CFTR to the mature form was nearly complete by 2−4 h (Figure 6A, top panel). By contrast, the conversion of immature to mature form in mutant ΔF508/V510D was slower since there was considerable amount of immature CFTR still present at 2−4 h (Figure 6A, bottom panel). No mature protein was detected in mutant ΔF508-CFTR because there is little maturation when expression is performed at 37 °C (Figure 6A, middle panel).

After an 8 h chase, the majority of labeled CFTR in wild-type and mutant ΔF508/V510D was present as the mature protein (Figure 6A). Therefore, we used this time point to compare the lifetimes of the mature protein in wild-type and mutant ΔF508/V510D. Figure 6 (panels B and C) shows that the half-lives of both proteins were similar (12−14 h). The results indicate that V510D stabilizes ΔF508-CFTR.

To test if V510D stabilizes ΔF508-CFTR at the cell surface, cells expressing wild-type, ΔF508-, or ΔF508/V510D-CFTRs were first incubated at 30 °C for 18 h so that ΔF508-CFTR would be present at the cell surface. CFTR at the cell surface was then biotinylated. The cells were washed to remove biotin-LC-hydrazide, and cells were harvested at various time points after labeling. Labeled CFTR was immunoprecipitated, samples were subjected to SDS−PAGE, and CFTR was detected with streptavidin conjugated to horseradish peroxidase. It was found that mature forms of wild-type or ΔF508/V510D-CFTRs had similar half-lives of about 12 h while ΔF508-CFTR had a short half-life of about 2 h (Figure 7).