Cystic fibrosis (CF) is most commonly caused by deletion of a residue (ΔF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) protein. The misfolded mutant protein is retained in the ER (endoplasmic reticulum) and is not trafficked to the cell surface (misprocessed mutant). Corrector molecules such as corr-2b or corr-4a are small molecules that increase the amount of functional CFTR at the cell surface. Correctors may function by stabilizing CFTR at the cell surface or by promoting folding in the ER. To test whether correctors promoted folding of CFTR in the ER, we constructed double-cysteine CFTR mutants that would be retained in the ER and only undergo cross-linking when the protein folds into a native structure. The mature form, but not the immature forms, of M348C(TM6)/T1142C(TM12) (where TM is transmembrane segment), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants were efficiently cross-linked. Mutations to the COPII (coatamer protein II) exit motif (Y563KDAD567) were then made in the cross-linkable cysteine mutants to prevent the mutant proteins from leaving the ER. Membranes were prepared from the mutants expressed in the absence or presence of correctors and subjected to disulfide cross-linking analysis. The presence of correctors promoted folding of the mutants as the efficiency of cross-linking increased from approx. 2–5% to 22–35%. The results suggest that correctors interact with CFTR in the ER to promote folding of the protein into a native structure.

INTRODUCTION

The CFTR (cystic fibrosis transmembrane conductance regulator) protein functions as a cAMP-activated Cl channel at the apical surface of epithelial cells. Processing mutations in the CFTR protein, especially deletion of Phe508 (ΔF508 CFTR), are the major cause of CF (cystic fibrosis) [1]. The mutant CFTR proteins are retained in the ER (endoplasmic reticulum) and are not trafficked to the cell surface. The processing mutations appear to inhibit folding of CFTR so that it accumulates in the ER as a partially folded intermediate [24]. The misfolded CFTR is then degraded by the ERAD (ER-associated degradation) system [57]. The lack of functional CFTR on the cell surface of airways and submucosal glands results in the production of sticky mucus that becomes chronically infected with bacteria that eventually results in lung failure [8].

A potential treatment for CF would be to promote folding of CFTR-processing mutants to increase the amount of functional CFTR delivered to the cell surface. This can be achieved by perturbing molecular chaperones [9], expression of the processing mutants at low temperature [10], expression in the presence of non-specific chemical chaperones such as glycerol [11] or 4-phenylbutyrate [12], or expression in the presence of correctors [1315]. Correctors are small molecules that may interact directly with CFTR to promote protein folding and or enhance the stability of CFTR at the cell surface. Some corrector molecules, such as corr-2b, corr-4a and VRT-532, appear to be more specific for CFTR and therefore act as pharmacological chaperones in rescuing CFTR-processing mutants [1517]. Pharmacological chaperones may be more useful as therapeutic agents as they would be less likely to perturb other metabolic pathways. Most corrector compounds, however, cause only a modest increase in the maturation of CFTR-processing mutants so that the yield of mature CFTR is 5–10% of that of wild-type CFTR.

In order to develop more effective pharmacological chaperones, it is important to understand their mechanism of action in repairing folding defects in CFTR. It was proposed that corrector molecules interact with CFTR in the ER to promote packing of the TMs (transmembrane segments) because the structure of the mature CFTR at the cell surface was different from that of the immature protein [18]. It has been shown, however, that CFTR undergoes rapid endocytosis and degradation in the lysosome [19]. Therefore correctors may function by stabilizing mature CFTR at the cell surface by preventing endocytosis and subsequent degradation. Indeed, a recent study showed that correctors such as corr-4a or VRT-325 stabilized the ΔF508 CFTR at the cell surface [20].

The goal of the present study was to test whether correctors could also function by promoting folding of CFTR in the ER. Our approach was to introduce pairs of cysteine residues into the TMDs (transmembrane domains) of a cysteine-less CFTR (Figure 1A) to generate a mutant that shows cross-linking only when it folds into a native conformation. A problem with a previous cross-linking study [18] was that treatment with cross-linkers caused the immature form of CFTR to form high-molecular-mass aggregates [3]. Another desirable property of the mutants was that the cross-linkable cysteine residues should lie outside the corrector-binding sites so that the correctors would not inhibit cross-linking. To test whether the correctors promoted folding of CFTR into a ‘native-like’ conformation in the ER, mutations were then made to the COPII (coatamer protein II) exit motif (Y563KDAD567) in NBD (nucleotide-binding domain) 1 of CFTR. It was shown previously that mutation in this motif prevented exit of CFTR from the ER [21]. The mutants were then expressed in the presence or absence of correctors followed by cross-linking analysis. We show that correctors such as corr-4a can promote folding of CFTR in the ER.

Model of CFTR

Figure 1
Model of CFTR

(A) The numbered cylinders represent the TMs and the branched lines represent glycosylation sites. NBD1 and NBD2 represent the N- and C-terminal NBDs respectively. TMD1 and TMD2 contain TM1–TM6 and TM7–TM12 respectively. ® represents the regulatory domain. The location of the V510A and cross-linkable cysteine residues [(M348C(TM6)/T1142C(TM12) (dashed line), T351C(TM6)/T1142C(TM12) (solid line) and W356C(TM6)/W1145C(TM12) (dotted line) mutants] are indicated. (B) Chemical structures of corr-4a, glibenclamide and benzbromarone.

Figure 1
Model of CFTR

(A) The numbered cylinders represent the TMs and the branched lines represent glycosylation sites. NBD1 and NBD2 represent the N- and C-terminal NBDs respectively. TMD1 and TMD2 contain TM1–TM6 and TM7–TM12 respectively. ® represents the regulatory domain. The location of the V510A and cross-linkable cysteine residues [(M348C(TM6)/T1142C(TM12) (dashed line), T351C(TM6)/T1142C(TM12) (solid line) and W356C(TM6)/W1145C(TM12) (dotted line) mutants] are indicated. (B) Chemical structures of corr-4a, glibenclamide and benzbromarone.

MATERIALS AND METHODS

Construction of mutants

Mutations were introduced into CFTR cDNAs using the method of Kunkel [22]. A cysteine-less CFTR was constructed by replacing Cys590 and Cys592 with leucine and by changing all other endogenous cysteine residues to alanine [23]. The cysteine-less CFTR also contained a V510A mutation (cysteine-less/V510A) that promoted maturation at 37 °C [17]. The double-cysteine mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) that were shown to be cross-linkable with M8M (3,6-dioxaoctane-1,8-bismethanethiosulfonate) cross-linker [3] were introduced into the cysteine-less/V510A CFTR. The COPII exit motif (Y563KDAD567) was disrupted by introducing the Y563N or D565A/D567A changes into wild-type CFTR [21] or into the double-cysteine mutants. The ΔF508 mutation was also introduced into the Y563N double-cysteine mutants.

Expression of mutants and disulfide cross-linking analysis

The CFTR mutants were transiently expressed in HEK-293 (human embryonic kidney) cells as described previously [18]. After 24 h at 37 °C, the cells were incubated with fresh medium in the absence or presence of 15 μM corrector corr-4a (Figure 1B) for another 24 h. Corr-4a was added from a 30 mM stock that was prepared in DMSO. DMSO was also added to mock-transfected cells (controls). The final concentration of DMSO was 0.05%, and cell growth was not impaired at this concentration.

In whole-cell cross-linking experiments, the HEK-293 cells expressing the double-cysteine mutants were harvested, washed twice and suspended in PBS (10 mM sodium phosphate, pH 7.4, and 150 mM NaCl). The cells were then pre-incubated for 20 min at 20 °C in the presence or absence of corrector molecules (VRT-325, VRT-640, corr-2b, corr-3a or corr-4a), channel blockers (benzbromarone, glibenclamide or CFinh-172) or potentiators (SF-03 or P5), then chilled on ice and treated with various concentrations of M8M cross-linker. The reactions were stopped by addition of 2× SDS sample buffer [125 mM Tris/HCl (pH 6.8), 20% (v/v) glycerol and 4% (w/v) SDS] containing 50 mM EDTA and no reducing agent. Samples were then subjected to immunoblot analysis by SDS/7.5% PAGE using a rabbit polyclonal anti-CFTR antibody [24] followed by enhanced chemiluminescence detection.

To test for cross-linking in membranes, the cells expressing M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant with the COPII mutations were treated for 24 h in the absence or presence of 15 μM corr-4a. Membranes were then prepared as described previously [25] and treated with 20 μM [M348C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12)] or 200 μM [T351C(TM6)/T1142C(TM12)] M8M cross-linker for 10 min at 20 °C. The membrane preparations were enriched in ER as the method for preparing membranes was as described for assaying the activity of SERCA1 (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1) [26]. The reactions were stopped by addition of 2× SDS sample buffer containing 50 mM EDTA and no reducing agent. Samples were then subjected to immunoblot analysis with rabbit anti-CFTR antibody. The gel lanes were scanned, and the amount of cross-linked product was measured using the NIH Image program (available at http://rsb.info.nih.gov/nih-image) and an Apple computer.

Cell-surface labelling of CFTR

Wild-type CFTR or COPII exit motif mutant Y563N or D565A/D567A was transiently expressed in HEK-293 cells. The cells were harvested and washed three times with ice-cold PBS (50 mM sodium phosphate, pH 8.0, and 150 mM NaCl). All subsequent labelling steps were performed on ice. The cells were then suspended in PBS and treated with 1 mM sulfo-NHS-SS-biotin [sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate] (Pierce) for 30 min. The reaction was stopped by addition of 1% (w/v) BSA in PBS. After 10 min, the cells were washed three times with PBS containing 50 mM Tris/HCl (pH 8.0). The cells were then suspended in PBS and lysed by addition of 1 vol. of PBS containing 2% (w/v) dodecyl-β-D-maltoside. Insoluble material was removed by centrifugation at 25000 g for 10 min. Streptavidin–agarose beads (Pierce) were added to the cleared lysates and incubated for 2 h. The beads were washed four times with TBS (Tris-buffered saline: 10 mM Tris/HCl, pH 7.4, and 150 mM NaCl) containing 0.1% dodecyl-β-D-maltoside. Labelled CFTR was then released from the beads by addition of SDS sample buffer containing 2% (v/v) 2-mercaptoethanol. Samples were then subjected to immunoblot analysis with rabbit anti-CFTR antibody followed by enhanced chemiluminescence detection.

RESULTS

Identification of cross-links that distinguish native and non-native forms of CFTR

CFTR-processing mutations, such as ΔF508, cause defective folding of the protein. The mutant proteins accumulate in the ER as incompletely folded biosynthetic intermediates [10]. It has been shown that the majority of wild-type CFTR also fails to fold into a native state as approx. 60–80% of the newly synthesized protein is degraded [27]. The incompletely folded forms of wild-type and processing mutants, however, are structurally similar [2,24]. Therefore synthesis of CFTR appears to yield a mixture of partially and completely folded molecules in the ER. To test whether correctors enhance folding of misfolded CFTR into a native-like state, we needed to be able to monitor these structures in the ER. A structural difference between the native and non-native forms of CFTR is observed in the folding of the TMs [3]. It was shown that pairs of cysteine residues introduced into TM6 and TM12 [M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12)] of CFTR (Figure 1) could be cross-linked with MTS (methanethiosulfonate) cross-linkers when the protein matured and was delivered to the cell surface. All of the mutants showed channel activity at the cell surface [3]. Cross-linking between cysteine residues located in TMD1 and TMD2 can be detected readily because the cross-linked product migrates with lower mobility in SDS/PAGE gels [3,28]. Cross-linked products were not detected when single-cysteine mutants M348C(TM6), T351C(TM6), W356C(TM6), T1142(TM12) and W1145C(TM12) were treated with cross-linkers [3]. Therefore the mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) are potential reporters for monitoring folding of the TMDs in CFTR. A problem in the previous cross-linking studies, however, was that almost all of the immature form of CFTR becomes aggregated after treatment with cross-linker and was detected in the stacking gel after immunoblot analysis [3,18]. The aggregates probably formed because of cross-linking between the 18 endogenous cysteine residues as the M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutations were introduced into a wild-type CFTR background. A cysteine-less CFTR (all cysteine residues changed to serine) could not be used in the initial study because the mutant protein failed to mature at 27 °C or 37 °C [3].

We recently modified the cysteine-less CFTR so that it matured at 37 °C [17]. Cysteine-less CFTR, in which Cys590 and Cys592 were replaced with leucine and the remaining cysteine residues were changed to alanine [23], did not mature at 37 °C unless Val510 (in NBD1) was changed to alanine [17]. The cysteine-less/V510A CFTR mutant exhibited channel activity at the cell surface [17]. It has been shown that the single-channel conductance of cysteine-less CFTR is similar to that of wild-type protein [23]. To test whether the mature and immature forms of cysteine-less CFTR/V510A still exhibited structural differences, the M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutations were introduced into cysteine-less/V510A CFTR. The mutants were expressed in HEK-293 cells and were subjected to cross-linking with M8M cross-linker, followed by immunoblot analysis. When the mutations were introduced into the wild-type background, the mature form of CFTR was cross-linked with M8M cross-linker, whereas the immature form of CFTR almost completely disappeared owing to formation of cross-linked aggregates [18]. An example is the M348C(TM6)/T1142C(TM12) mutant shown in Figure 2(A). There was a little aggregation of immature CFTR, however, when cysteine-less/V510A containing the M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutations were treated with M8M cross-linker (Figure 2B). Cross-linking of the mature form of the mutants could still be detected. In contrast, no cross-linked product was detected when single-cysteine mutants M348C(TM6), T351C(TM6), W356C(TM6), T1142(TM12) and W1145C(TM12) in the cysteine-less/V510A background were each treated with M8M cross-linker (results not shown). These results suggest that the M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutations in the cysteine-less/V510A CFTR background could act as reporters for monitoring folding of the TMDs because only mature CFTR shows cross-linking.

Disulfide cross-linking of cysteine mutants in wild-type or cysteine-less/V510A CFTR background

Figure 2
Disulfide cross-linking of cysteine mutants in wild-type or cysteine-less/V510A CFTR background

Wild-type CFTR containing the M348C(TM6)/T1142C(TM12) mutations (A) or cysteineless/V510A CFTR containing M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutation (B) were expressed in HEK-293 cells. Whole-cell samples were treated with 0.2 mM M8M cross-linker for 10 min at 20 °C, and the reactions were stopped by addition of SDS sample buffer containing 50 mM EDTA and no thiol-reducing agent. Samples were subjected to immunoblot analysis with a rabbit polyclonal antibody against CFTR. The positions of cross-linked (X-link), mature and immature CFTRs are indicated.

Figure 2
Disulfide cross-linking of cysteine mutants in wild-type or cysteine-less/V510A CFTR background

Wild-type CFTR containing the M348C(TM6)/T1142C(TM12) mutations (A) or cysteineless/V510A CFTR containing M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutation (B) were expressed in HEK-293 cells. Whole-cell samples were treated with 0.2 mM M8M cross-linker for 10 min at 20 °C, and the reactions were stopped by addition of SDS sample buffer containing 50 mM EDTA and no thiol-reducing agent. Samples were subjected to immunoblot analysis with a rabbit polyclonal antibody against CFTR. The positions of cross-linked (X-link), mature and immature CFTRs are indicated.

Effects of correctors, channel blockers or potentiators on cross-linking of TM6/TM12 mutants

We have shown previously that cross-linking between TM6 and TM7 [I340C(TM6)/S877C(TM7)] could be blocked by correctors such as VRT-325 or corr-4a [17]. Therefore it was important to determine whether the presence of correctors would inhibit cross-linking between cysteine residues in the M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant. The first step was to determine the concentration-dependence of cross-linking with M8M cross-linker. Accordingly, cells expressing each mutant were treated for 4 min on ice with various concentrations of M8M cross-linker. The reactions were carried out on ice to reduce thermal motion in the proteins. The reactions were stopped with SDS sample buffer containing 50 mM EDTA and no reducing agent. Samples were then subjected to immunoblot analysis. Figure 3(A) shows that cross-linking of T351C(TM6)/T1142C(TM12) mutant required much higher concentrations of M8M. Cross-linked product in M348C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants was detected readily after treatment with 12.5 μM M8M, whereas detection of cross-linked product in T351C(TM6)/T1142C(TM12) mutant required approx. 100 μM M8M. The product migrating with lower mobility than the mature CFTR appears to contain a disulfide bond. When the cross-linked samples were exposed to 2-mercaptoethanol, there was a concomitant decrease in the cross-linked product and an increase in the amount of the mature CFTR (Figure 3B). Slow-migrating product was not detected when single-cysteine mutants M348C(TM6), T351C(TM6), W356C(TM6), T1142C(TM12) and W1145(TM12) in cysteine-less CFTR/V510A were each treated with M8M (results not shown)

Concentration-dependence of M8M cross-linking of cysteine mutants

Figure 3
Concentration-dependence of M8M cross-linking of cysteine mutants

(A) HEK-293 cells expressing M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant in the cysteine-less/V510A background were suspended in PBS. Samples were treated with various concentrations of M8M for 4 min on ice, and the reactions were stopped by addition of SDS sample buffer containing 50 mM EDTA and no thiol-reducing agent. (B) M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant was treated with 50, 200 or 50 μM M8M respectively. The reactions were stopped as described above. A sample of each was then treated with 2% (v/v) 2-mercaptoethanol (+/MSH) in SDS sample buffer. Samples were subjected to immunoblot analysis with a rabbit polyclonal antibody against CFTR. The positions of cross-linked (X-link), mature and immature CFTRs are indicated.

Figure 3
Concentration-dependence of M8M cross-linking of cysteine mutants

(A) HEK-293 cells expressing M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant in the cysteine-less/V510A background were suspended in PBS. Samples were treated with various concentrations of M8M for 4 min on ice, and the reactions were stopped by addition of SDS sample buffer containing 50 mM EDTA and no thiol-reducing agent. (B) M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant was treated with 50, 200 or 50 μM M8M respectively. The reactions were stopped as described above. A sample of each was then treated with 2% (v/v) 2-mercaptoethanol (+/MSH) in SDS sample buffer. Samples were subjected to immunoblot analysis with a rabbit polyclonal antibody against CFTR. The positions of cross-linked (X-link), mature and immature CFTRs are indicated.

We then examined whether correctors, channel blockers or potentiators inhibited cross-linking of M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants. The correctors (VRT-325, VRT-640, corr-2b, corr-3a and corr-4a) [1315], channel blockers predicted to interact with the pore of CFTR (benzbromarone, glibenclamide and CFinh-172) [2931] and potentiators predicted to interact with CFTR (SF-03 or P5) [32,33] were tested. Cells expressing M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant were pre-treated with 0.2 mM of the various compounds except for CFinh-172 (0.02 mM) and glibenclamide (1 mM). A lower concentration of CFinh-172 was used because its maximum solubility in water is approx. 0.02 mM, whereas a higher concentration of glibenclamide was used because its half-maximal inhibitory concentration is approx. 0.1 mM [29]. After 20 min at 20 °C, the samples were cooled on ice and treated with M8M cross-linker. The reactions were stopped by addition of SDS sample buffer containing no reducing agent, and samples were subjected to immunoblot analysis. Figure 4 shows that the channel blockers benzbromarone and glibenclamide (structures shown in Figure 1B) almost completely inhibited cross-linking of M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants. In contrast, the correctors and potentiators had little effect on cross-linking. These results suggest that the channel blockers (benzbromarone and glibenclamide) interact at a different region of CFTR from that of correctors or potentiators to cause conformational changes in the TMDs.

Effects of correctors, channel blockers and potentiators on cross-linking

Figure 4
Effects of correctors, channel blockers and potentiators on cross-linking

HEK-293 cells expressing M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant were treated without (None) or with 0.2 mM VRT-325 (325), VRT-640 (640), corr-2b (2b), corr-3a (3a), corr-4a (4a), benzbromarone (Benz), SF-03 or P5; 1 mM glibenclamide (Glib) or 0.02 mM CFinh-17 (172) for 20 min at 20 °C. The cells were then cooled on ice and treated without (−) or with (+) 25 μM M8M for 4 min [M348C(TM6)/T1142C(TM12) mutant], 200 μM M8M for 10 min [T351C(TM6)/T1142C(TM12) mutant] or 12.5 μM M8M for 4 min [W356C(TM6)/W1145C(TM12) mutant] on ice. The reactions were stopped by addition of SDS sample buffer containing 50 mM EDTA and no thiol-reducing agent. Samples were subjected to immunoblot analysis with a rabbit polyclonal antibody against CFTR. The positions of cross-linked (X-link), mature and immature CFTRs are indicated.

Figure 4
Effects of correctors, channel blockers and potentiators on cross-linking

HEK-293 cells expressing M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant were treated without (None) or with 0.2 mM VRT-325 (325), VRT-640 (640), corr-2b (2b), corr-3a (3a), corr-4a (4a), benzbromarone (Benz), SF-03 or P5; 1 mM glibenclamide (Glib) or 0.02 mM CFinh-17 (172) for 20 min at 20 °C. The cells were then cooled on ice and treated without (−) or with (+) 25 μM M8M for 4 min [M348C(TM6)/T1142C(TM12) mutant], 200 μM M8M for 10 min [T351C(TM6)/T1142C(TM12) mutant] or 12.5 μM M8M for 4 min [W356C(TM6)/W1145C(TM12) mutant] on ice. The reactions were stopped by addition of SDS sample buffer containing 50 mM EDTA and no thiol-reducing agent. Samples were subjected to immunoblot analysis with a rabbit polyclonal antibody against CFTR. The positions of cross-linked (X-link), mature and immature CFTRs are indicated.

Effect of correctors on folding of CFTR mutants in the ER

Since the correctors did not inhibit cross-linking between cysteine residues in M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants, these mutants were useful reporter molecules for testing the effect of correctors on CFTR folding in the ER. We had proposed that the mechanism of correctors was that they acted as pharmacological chaperones to promote folding of misprocessed mutants by interacting with the protein in the ER [34,35]. Alternatively, it has been suggested that correctors might function by stabilizing the protein at the cell surface and/or affecting endocytosis [20].

To determine whether correctors affected folding of CFTR in the ER, we needed to prevent CFTR from leaving the ER. Export of CFTR from the ER requires packaging into COPII vesicles [21]. Preventing COPII coat assembly by inhibiting the Sar1 GTPase with the H89 protein kinase inhibitor reduces CFTR export by 90% [21]. Mutations in the Y563KDAD567 exit motif located in NBD1 of CFTR can also block export of CFTR from the ER [21]. Tyr563, Asp565 and Asp567 in the exit motif are evolutionarily conserved [36,37]. Accordingly, we introduced the Y563N or D565A/D567A mutations in the COPII exit motif of wild-type CFTR. The mutants were expressed in HEK-293 cells and whole-cell SDS extracts were subjected to immunoblot analysis. Figure 5(A) shows that, in both mutants, mature product was not detected, and the major product was the immature form of CFTR. Cell-surface labelling was then used to determine whether mature or immature CFTR was at the cell surface. Cells expressing wild-type CFTR or Y563N or D565A/D567A mutant were treated with sulfo-NHS-SS-biotin, and then lysed with detergent. Biotinylated CFTR was precipitated with streptavidin–agarose beads and released from the beads by addition of reducing agent. Samples were then subjected to immunoblot analysis. The results show that only the mature wild-type CFTR was labelled at the cell surface (Figure 5B). The results are consistent with the previous report that COPII mutations cause CFTR retention in the ER [21].

Effect of COPII mutations on cross-linking of cysteine mutants

Figure 5
Effect of COPII mutations on cross-linking of cysteine mutants

(A) Whole-cell SDS extracts of HEK-293 cells expressing wild-type CFTR and wild-type CFTR containing Y563N or D565A/D567A mutation were subjected to immunoblot analysis. (B) HEK-293 cells expressing wild-type CFTR or wild-type CFTR containing Y563N or D565A/D567A mutant were surface-labelled with sulfo-NHS-SS-biotin. Labelled CFTR was precipitated with streptavidin–agarose beads, treated with SDS sample buffer containing 2-mercaptoethanol, and samples were subjected to immunoblot analysis. (C) M348C(TM6)/T1142C(TM12)/Y563N, T351C(TM6)/T1142C(TM12)/Y563N or W356C(TM6)/W1145C(TM12)/Y563N mutant in the cysteine-less/V510A CFTR background was expressed in the absence (−) or presence (+) of 15 μM corr-4a. Wild-type (WT) CFTR was used as a control. Whole-cell SDS extracts were then subjected to immunoblot analysis. (D) Membranes were prepared from HEK-293 cells expressing M348C(TM6)/T1142C(TM12)/Y563N, T351C(TM6)/T1142C(TM12)/Y563N or W356C(TM6)/W1145C(TM12)/Y563N mutant in the cysteine-less/V510A background that were grown in the absence (−) or presence (+) 15 μM corr-4a. Samples were then treated with 0.025 mM M8M [M348C(TM6)/T1142C(TM12)/Y563N, W356C(TM6)/W1145C(TM12)/Y563N] or 0.2 mM M8M [T351C(TM6)/T1142C(TM12)/Y563N] for 10 min at 20 °C. The reactions were stopped by addition of SDS sample buffer containing 50 mM EDTA with (+) or without (−) 20 mM dithiothreitol (+DTT). Samples were subjected to immunoblot analysis with a rabbit polyclonal antibody against CFTR. The positions of cross-linked (X-link), mature and immature CFTRs are indicated. (E) The amount of cross-linked CFTR relative to total CFTR (cross-linked plus immature) observed in (D) was quantified by scanning the gel lanes followed by analysis with the NIH Image program. Results are means±S.D. (n=3).

Figure 5
Effect of COPII mutations on cross-linking of cysteine mutants

(A) Whole-cell SDS extracts of HEK-293 cells expressing wild-type CFTR and wild-type CFTR containing Y563N or D565A/D567A mutation were subjected to immunoblot analysis. (B) HEK-293 cells expressing wild-type CFTR or wild-type CFTR containing Y563N or D565A/D567A mutant were surface-labelled with sulfo-NHS-SS-biotin. Labelled CFTR was precipitated with streptavidin–agarose beads, treated with SDS sample buffer containing 2-mercaptoethanol, and samples were subjected to immunoblot analysis. (C) M348C(TM6)/T1142C(TM12)/Y563N, T351C(TM6)/T1142C(TM12)/Y563N or W356C(TM6)/W1145C(TM12)/Y563N mutant in the cysteine-less/V510A CFTR background was expressed in the absence (−) or presence (+) of 15 μM corr-4a. Wild-type (WT) CFTR was used as a control. Whole-cell SDS extracts were then subjected to immunoblot analysis. (D) Membranes were prepared from HEK-293 cells expressing M348C(TM6)/T1142C(TM12)/Y563N, T351C(TM6)/T1142C(TM12)/Y563N or W356C(TM6)/W1145C(TM12)/Y563N mutant in the cysteine-less/V510A background that were grown in the absence (−) or presence (+) 15 μM corr-4a. Samples were then treated with 0.025 mM M8M [M348C(TM6)/T1142C(TM12)/Y563N, W356C(TM6)/W1145C(TM12)/Y563N] or 0.2 mM M8M [T351C(TM6)/T1142C(TM12)/Y563N] for 10 min at 20 °C. The reactions were stopped by addition of SDS sample buffer containing 50 mM EDTA with (+) or without (−) 20 mM dithiothreitol (+DTT). Samples were subjected to immunoblot analysis with a rabbit polyclonal antibody against CFTR. The positions of cross-linked (X-link), mature and immature CFTRs are indicated. (E) The amount of cross-linked CFTR relative to total CFTR (cross-linked plus immature) observed in (D) was quantified by scanning the gel lanes followed by analysis with the NIH Image program. Results are means±S.D. (n=3).

The Y563N mutation was then introduced into M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant in the cysteine-less/V510A background. The mutants were each expressed in HEK-293 cells to test whether the Y563N mutation would block maturation when they were expressed in the absence or presence of corr-4a. Corr-4a was initially selected because it was relatively more efficient than other specific correctors in promoting maturation of CFTR-processing mutants [15]. Whole-cell SDS extracts were then subjected to immunoblot analysis. Figure 5(C) shows that mature CFTR was not detected even after expression in the presence of corr-4a. These results indicate that the COPII exit motif mutations in cysteine-less/V510A CFTR still blocked maturation even when expressed in the presence of a corrector.

Membranes were then prepared from cells transfected with M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant cDNA in the cysteine-less/V510A/Y563N background and grown in the presence or absence of corr-4a. Samples were then treated with M8M cross-linker followed by immunoblot analysis. Figures 5(D) and 5(E) show that cross-linked product was barely detected (2–4% of total CFTR; cross-linked plus immature) in membranes from cells grown in the absence of corr-4a. The CFTR product migrating with lower mobility in SDS/PAGE gels represented cross-linked CFTR because it was sensitive to the presence of the thiol-reducing agent, dithiothreitol (Figure 5D, +DTT). The amount of cross-linked CFTR, however, was greatly increased (to 22–35% of total) when the mutants were expressed in the presence of corr-4a (Figures 5D and 5E). Similar results were observed when the mutants were expressed in the presence of corrector VRT-325 (results not shown). These results suggest that the correctors interact with CFTR in the ER to promote folding.

To confirm that the corrector was modulating folding in the ER, we expressed wild-type CFTR and T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A mutant in the absence or presence of brefeldin A before cross-linking with M8M cross-linker. Figure 6(A) shows that, when wild-type CFTR was expressed in the absence of brefeldin A, both mature and immature forms of CFTR were detected. In the presence of brefeldin A, however, only the immature form of CFTR was detected. These results confirm that brefeldin A inhibits protein transport from the ER to the Golgi [38]. Cross-linking of T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A mutant expressed in the presence of brefeldin A with or without corr-4a showed that there was more cross-linked product when corr-4a was present (Figure 6B). These results again suggest that corr-4a promotes folding of the mutant CFTR in the ER.

Effect of brefeldin A on maturation of wild-type CFTR and cross-linking analysis of T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A mutant

Figure 6
Effect of brefeldin A on maturation of wild-type CFTR and cross-linking analysis of T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A mutant

HEK-293 cells were transfected with wild-type or T351C(TM6)/T1142C(TM12)/Y563N/cysteineless/V510A mutant CFTR cDNAs. (A) After 16 h, the cells expressing wild-type CFTR were incubated with fresh medium in the absence (−) or presence (+) of 10 μg/ml brefeldin A. Whole-cell SDS extracts were then subjected to immunoblot analysis. (B) After 16 h, the medium in cells expressing T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A mutant was replaced with fresh medium containing 10 μg/ml brefeldin A with (+) or without (−) 15 μM corr-4a. After another 24 h, membranes were prepared and cross-linked with 0.2 mM M8M cross-linker for 10 min at 20 °C. The reactions were stopped by the addition of SDS sample buffer containing 50 mM EDTA and no thiol-reducing agent. Samples were subjected to immunoblot analysis with a rabbit polyclonal antibody against CFTR. The positions of cross-linked (X-link), mature and immature CFTRs are indicated.

Figure 6
Effect of brefeldin A on maturation of wild-type CFTR and cross-linking analysis of T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A mutant

HEK-293 cells were transfected with wild-type or T351C(TM6)/T1142C(TM12)/Y563N/cysteineless/V510A mutant CFTR cDNAs. (A) After 16 h, the cells expressing wild-type CFTR were incubated with fresh medium in the absence (−) or presence (+) of 10 μg/ml brefeldin A. Whole-cell SDS extracts were then subjected to immunoblot analysis. (B) After 16 h, the medium in cells expressing T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A mutant was replaced with fresh medium containing 10 μg/ml brefeldin A with (+) or without (−) 15 μM corr-4a. After another 24 h, membranes were prepared and cross-linked with 0.2 mM M8M cross-linker for 10 min at 20 °C. The reactions were stopped by the addition of SDS sample buffer containing 50 mM EDTA and no thiol-reducing agent. Samples were subjected to immunoblot analysis with a rabbit polyclonal antibody against CFTR. The positions of cross-linked (X-link), mature and immature CFTRs are indicated.

The ΔF508 mutation causes misfolding of CFTR and the mutant protein to be retained in the ER [5]. Expression in the presence of corr-4a, however, promotes maturation of ΔF508 CFTR [15]. To test whether corr-4a promoted folding of the ΔF508 mutant in the ER, the ΔF508 mutation was introduced into the double-cysteine mutants M348C(TM6)/T1142C-(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) in the Y563N/cysteine-less/V510A CFTR background. The mutants were expressed in the presence or absence of corr-4a. Membranes were prepared, cross-linked with M8M cross-linker and subjected to immunoblot analysis. In the absence of corr-4a, cross-linked product was not detected in any of the mutants (<1% of total; Figure 7). Expression in the presence of corr-4a, however, increased the yield of cross-linked product of ΔF508/M348C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A, ΔF508/T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A and ΔF508/W356C(TM6)/W1145C(TM12)/Y563N/cysteine-less/V510A mutants to 5, 11 and 10% respectively (Figure 7). The presence of the ΔF508 mutation, however, caused an approx. 3-fold decrease in the amount of cross-linked product when compared with mutants without the ΔF508 mutation (Figure 5E). We have observed previously that the presence of the ΔF508 mutation greatly reduced maturation of cysteine-less CFTR even when it was expressed at 27 °C [17].

Effects of ΔF508 mutation on cross-linking of COPII cysteine mutants

Figure 7
Effects of ΔF508 mutation on cross-linking of COPII cysteine mutants

HEK-293 cells expressing CFTR ΔF508/M348C(TM6)/T1142C(TM12)/Y563N/cysteineless/V510A, ΔF508/T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A or ΔF508/W356C(TM6)/W1145C(TM12)/Y563N/cysteine-less/V510A mutant were grown in the absence (−) or presence (+) of 15 μM corr-4a. Membranes were prepared, and cross-linking with M8M cross-linker was performed. Samples were subjected to immunoblot analysis (A) and the amount of cross-linked product (B) was determined as described in the legend to Figure 5. Results are means±S.D. (n=3). The positions of cross-linked (X-link) and immature CFTRs are indicated.

Figure 7
Effects of ΔF508 mutation on cross-linking of COPII cysteine mutants

HEK-293 cells expressing CFTR ΔF508/M348C(TM6)/T1142C(TM12)/Y563N/cysteineless/V510A, ΔF508/T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A or ΔF508/W356C(TM6)/W1145C(TM12)/Y563N/cysteine-less/V510A mutant were grown in the absence (−) or presence (+) of 15 μM corr-4a. Membranes were prepared, and cross-linking with M8M cross-linker was performed. Samples were subjected to immunoblot analysis (A) and the amount of cross-linked product (B) was determined as described in the legend to Figure 5. Results are means±S.D. (n=3). The positions of cross-linked (X-link) and immature CFTRs are indicated.

DISCUSSION

Expression of wild-type or CFTR-processing mutants in the presence of correctors enhances maturation of the protein [1315]. Some correctors, such as corr-2b, corr-4a or VRT-532, are relatively specific for CFTR because they do not promote maturation of HERG (human ether-a-go-go-related gene) channel- or P-gp [P-glycoprotein or ABCB1 (ATP-binding cassette transporter B1)]-processing mutants [15,16]. These correctors might specifically promote folding of CFTR because they bind to the protein [17]. The results of the present study suggest that the corr-4a is able to interact with CFTR in the ER to promote folding between the TMDs. A model of how correctors could interact with CFTR is shown in Figure 8. Wild-type CFTR is first synthesized as an immature protein with incomplete domain–domain interactions (Figure 8A). Folding of wild-type CFTR into a native structure involves interactions between the TMDs and between the NBDs and TMDs (Figure 8A). The presence of a processing mutation such as ΔF508 disrupts interactions between the TMDs (Figure 8B). Deficient TMD interactions are detected by Derlin-1 [39] and RMA1 [RING (really interesting new gene) finger protein with membrane anchor 1] [4], which then target the processing mutant for degradation by the ubiquitin–proteasome pathway. The model for corr-4a interactions with CFTR (Figure 8C) is similar to that proposed for the interaction of drug substrates or modulators with P-gp for rescuing P-gp-processing mutants [34]. Drug substrates or modulators bind at the interface between the TMDs of P-gp and act as a scaffold to allow for the proper interactions between the TMDs [40,41]. Therefore correctors may also bind at the interface between the TMDs of CFTR (Figure 8C). Correctors such as corr-4a are also predicted to interact with the TMDs because they modulate the topology of a CFTR mutant lacking the cytosolic domains [42]. It should be pointed out, however, that the correctors could bind outside the TMDs to promote domain–domain interactions through long-range conformational changes.

Models of the effects of processing mutations and correctors on maturation of CFTR

Figure 8
Models of the effects of processing mutations and correctors on maturation of CFTR

(A) Wild-type CFTR is initially synthesized as an inactive immature protein that undergoes post-translational folding (interactions among various domains) to yield an active enzyme and is then delivered to the cell surface. (B) The presence of a processing mutation (X, ΔF508) disrupts interactions among the domains and caused the protein to be trapped in the ER. (C) Expression of CFTR-processing mutations in the presence of corrector restores interactions among the various domains and promotes proper folding of CFTR.

Figure 8
Models of the effects of processing mutations and correctors on maturation of CFTR

(A) Wild-type CFTR is initially synthesized as an inactive immature protein that undergoes post-translational folding (interactions among various domains) to yield an active enzyme and is then delivered to the cell surface. (B) The presence of a processing mutation (X, ΔF508) disrupts interactions among the domains and caused the protein to be trapped in the ER. (C) Expression of CFTR-processing mutations in the presence of corrector restores interactions among the various domains and promotes proper folding of CFTR.

The channel blockers benzbromarone and glibenclamide, but not CFinh-172, could inhibit cross-linking of the TM6/TM12 cysteine mutants (Figure 4). One reason that the channel blocker CFinh-172 did not inhibit cross-linking is that it is postulated to bind to the extracellular surface of the pore region of CFTR, whereas benzbromarone and glibenclamide are postulated to interact at a more cytosolic side of the pore [43]. Another explanation is that the solubility of CFinh-172 in water is limited, and this property makes it less potent in intact cells [44].

The channel blockers have been postulated to interact with the TMDs of CFTR [43] and they may interact at site(s) that are different from the corrector-binding sites. Both benzbromarone and glibenclamide blocked cross-linking between cysteine residues in TM6 and TM12, whereas the correctors (VRT-325, VRT-640, corr-2b, corr-3a and corr-4a) did not inhibit cross-linking (Figure 4). In contrast, it was shown in another study that the corr-4a and VRT-532 inhibited cross-linking between cysteine residues in TM6 and TM7 [I340C(TM6)/S877C(TM7)] [17]. An explanation for the different patterns of inhibition of cross-linking is that the channel blockers bind to the pore region [43], whereas the correctors bind outside the pore region, since they do not inhibit channel activity [14,15]. Therefore we have placed the corrector-binding site(s) outside the pore region in our model (Figure 8C). It is also possible, however, that the binding sites for correctors and channel blockers could show some overlap. An example is the channel blocker, benzbromarone, which also inhibited cross-linking between cysteine residues in TM6 and TM7 [I340C(TM6)/S877C(TM7) mutant] [17], as well as between cysteine residues in TM6 and TM12 [M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants] (Figure 4).

Although the channel blockers benzbromarone and glibenclamide probably interact with the TMDs, they did not promote maturation of CFTR-processing mutants (results not shown). They may not promote maturation because they may interact only with TMD1 of CFTR. It has been shown that the TMs lining the pore appear to reside mainly in TMD1 [45,46]. Studies on P-gp suggest that compounds that promote maturation bind at the interface between the TMDs [40,47]. Accordingly, the model shown in Figure 8(C) predicts that corr-4a interacts at the interface between the TMDs of CFTR.

Maturation of CFTR is inefficient in most cell lines, including human airway cells [48]. Approx. 60–80% of wild-type CFTR and 99% of ΔF508 CFTR are degraded soon after synthesis [27]. Wild-type and ΔF508 CFTR appear to have similar structures during the initial stages of folding, but the presence of a processing mutation causes a higher amount of the CFTR molecules to be trapped as an incompletely folded protein in the ER [2]. A potential rate-limiting step in the folding process might be the folding of the TMs between TMD1 and TMD2. It has been shown that interactions between the TMDs occur post-translationally [49]. This delayed interaction between the TMDs might be reflected in the relatively low amount (2–4% of total CFTR) of cross-linked product in M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants when grown in the absence of corr-4a, but is increased to 22–35% when grown in the presence of corr-4a (Figures 5D and 5E). It is interesting that some of the CFTR trapped in the ER when expressed in the absence of corrector was still cross-linked, indicating that this population of CFTR has a native-like structure. The results are consistent with the observation from patch-clamp studies that some of the ΔF508 CFTR retained in the ER is functional [50].

The results of the present study provide clues about features that may be important for the design of more effective corrector molecules. The most effective target in CFTR may be at the interface between the TMDs, and future studies will be required to refine the location(s) of the corrector and channel-blocker-binding sites. The ability of correctors to modulate CFTR folding in the ER indicates that correctors that can reach the ER in higher concentrations would have greater therapeutic value.

This work was supported by grants from the Canadian Institutes of Health Research (MOP-62832) and the Cystic Fibrosis Foundation (CLARKE08G0). We thank the Cystic Fibrosis Foundation Therapeutics, Inc. and Dr Robert Bridges (Rosalind Franklin University, Chicago, IL, U.S.A.) for providing VRT-325, VRT-640, corr-4a, SF-03 and P5. D. M. C. is the incumbent of the Canadian Research Chair in Membrane Biology.

Abbreviations

     
  • CF

    cystic fibrosis

  •  
  • CFTR

    cystic fibrosis transmembrane conductance regulator

  •  
  • COPII

    coatamer protein II

  •  
  • ER

    endoplasmic reticulum

  •  
  • HEK-293

    human embryonic kidney

  •  
  • M8M

    3,6-dioxaoctane-1,8-bismethanethiosulfonate

  •  
  • NBD

    nucleotide-binding domain

  •  
  • P-gp

    P-glycoprotein

  •  
  • sulfo-NHS-SS-biotin

    sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate

  •  
  • TM

    transmembrane segment

  •  
  • TMD

    transmembrane domain

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