Transcomplementation by a Truncation Mutant of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Enhances ΔF508 Processing through a Biomolecular Interaction

Background: Many therapies are being pursued for CF. One includes transcomplementation whereby truncated forms for CFTR can rescue ΔF508 CFTR.

Results: A new construct, Δ27–264 rescues mature C band of wt and ΔF508, and channel currents and binds to ΔF508 CFTR.

Conclusion: Transcomplementation occurs by the direct binding of Δ27–264 to ΔF508 CFTR.

Significance: Therapeutic rescue of ΔF508 CFTR would benefit the majority of CF patients.

Abstract

We previously showed that a truncation mutant of CFTR missing the first four transmembrane segments of TMD1, Δ264 CFTR, binds to key elements in the ER quality control mechanism to increase the amounts of the mature C band of both wt and ΔF508 CFTR through transcomplementation. Here, we created a new construct, Δ27–264 CFTR. Even though Δ27–264 CFTR is rapidly degraded in the proteasome, steady state protein can be detected by Western blot. Δ27–264 CFTR can also increase the amounts of the mature C band of both wt and ΔF508 CFTR through transcomplementation. Electrophysiology experiments show that Δ27–264 CFTR can restore chloride channel currents. Further experiments with the conduction mutant S341A show conclusively that currents are indeed generated by rescued channel function of ΔF508 CFTR. Immunoprecipitation studies show that Δ27–264 binds to ΔF508-CFTR, suggesting a bimolecular interaction. Thus the adeno-associated viral vector, rAAV-Δ27–264 CFTR, is a highly promising CF gene therapy vector, because it increases the amount of mature band C protein both from wt and ΔF508 CFTR, and rescues channel activity of ΔF508 CFTR.

Introduction

The cystic fibrosis transmembrane conductance regulator (CFTR)² is an ATP-binding cassette protein with two sets of transmembrane domains (TMD), two nucleotide binding domains (NBD), and a regulatory (R) domain. Mutations in CFTR are associated with cystic fibrosis (CF) (1). The most common disease causing mutation in CFTR is a missing phenylalanine at position 508 (ΔF508 CFTR) (2). ΔF508 CFTR is retained in the ER, incompletely glycosylated and rapidly degraded in the proteasome (3).

Over the past several decades, the average age of survival of CF patients has steadily increased due primarily to advances in the medical treatment of symptoms of the disease (4). However, a goal of CF research is to correct the basic defects in CFTR caused by a mutant protein.

Many strategies are being pursued to develop potential therapies for CF to restore the function of CFTR. Two new small molecules developed by Vertex and a consortium of CF researchers, VX-770 and VX-809, identified by high throughput screening of compound libraries, have reached clinical trials (5). VX-770 is a potentiator, designed to activate CFTR gating mutants such as G551D (6). A potentiator acts acutely to active CFTR currents if there is protein already present in the plasma membrane. For example, G551D mutant reaches the plasma membrane but has too little channel activity to support normal CFTR function (7). Thus patients with this mutant have severe CF (6). VX-770 has had remarkable success in reducing pulmonary exacerbations, sweat chloride, and promoting weight gain in patients bearing the G551D mutation (8). All of these health improvements indicate restored CFTR function in these patients. VX-809 is a corrector, designed to rescue the trafficking and channel activity (9). Correctors are designed to act over a period of time to rescue the processing of newly synthesized ΔF508 CFTR (5). In an initial study of CF patients with ΔF508 CFTR, VX-809 (10) had a smaller effect on sweat chloride compared with the effect of VX-770 on G551D patients VX-770 (8), and did not show any effects on pulmonary function or rescue of ΔF508 CFTR in rectal biopsies. Although these initial results with VX-809 are promising, they suggest that more work needs to be done to restore ΔF508 CFTR function to therapeutic levels.

Because of the discovery of the CF gene (11), the objective of our work has been to correct defective CFTR function by inserting a new copy of the CF gene via AAV (adeno-associated virus) gene transfer (12). AAV2 gene transfer was also tested in humans (13). It was found to be safe but did not show any clinical benefit. There were two major reasons for the lack of clinical efficacy. First AAV2 was not very efficient in transducing lung cells. Second, a weak promoter in the vector construct did not express enough protein to be effective (14). To overcome the first hurdle, new more efficient serotypes became available that infect lung cells (15). The solution to the second hurdle was accomplished by adding a more powerful CBA (chicken β actin) promoter (16). However, addition of the promoter required the truncation of first four transmembrane segments of CFTR to fit the packaging capacity of AAV (17). In a previous report, we showed that CFTR, missing the first four transmembrane domains of CFTR (rAAV5-Δ264 CFTR), when infected into monkey lungs Δ264 CFTR increased the levels of endogenous wild type CFTR protein (18). We also showed in cotransfection studies that Δ264 CFTR increased wild-type CFTR protein and increased levels of maturation of immature band B to mature C band of ΔF508 CFTR.

Δ264 CFTR is rapidly degraded similar to ΔF508 CFTR. It binds avidly to VCP and HDAC6, two proteins involved in retrograde translocation from ER to cytosol for proteasomal and aggresomal degradation. Thus one mechanism by which transcomplementation rescues ΔF508 CFTR is by associating with key elements in the CFTR quality control mechanism (19). However, the overall mechanism of how transcomplementation rescues ΔF508 CFTR remains poorly understood.

In this study, we created an improved truncation of CFTR (Δ27–264 CFTR). We have shown previously the first 27 amino acids of CFTR were important for channel expression (20). Others have shown that the N-terminal tail of CFTR binds to important trafficking molecules such as Syntaxin 1a (21). Indeed, we show that Δ27–264 CFTR does function to efficiently increase the amounts of both wt and ΔF508 CFTR protein detected in Western blot experiments. In addition to rescuing the mature C band of ΔF508 CFTR, the new truncation mutant also restores chloride currents. We propose that in addition to affecting the quality control mechanism that this truncation mutant binds to ΔF508 CFTR and likely rescues it through a bi-molecular interaction. This work also provides a new strategy for gene therapy vectors for CF based on correction of mutant ΔF508 CFTR protein.

EXPERIMENTAL PROCEDURES

Cell Culture

African green monkey kidney cells (COS7) obtained from American Type Tissue Culture (ATCC) were maintained in DMEM (Dulbecco's Modified Eagle Medium High Glucose 1×), penicillin (100 units/ml), streptomycin (100 μg/ml), and 10% fetal bovine serum. Media and other components were purchased from (Invitrogen). HeLa cells containing DF508 CFTR were cultured in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen), l-glutamate (200 mm), puromycin (5 μg/ml, Sigma), and penicillin/streptomycin (Invitrogen). The CFBE 41o- cell line was derived by Dieter Gruenert from a CF patient (22). CFBE41o⁻ were cultured in Minimum Essential Medium Eagle (MEM, Invitrogen) with 10% FBS, l-glutamate, and penicillin/streptomycin. CFBE-WTCFTR or DF508 cells (CFBE41o⁻ cells stably transfected with wild-type CFTR (23), also provided by Dr. Gruenert) were cultured the same medium only selected with 300 μg/ml hygromycin B.

Plasmids and Constructs

The construct pEGFP wt CFTR mammalian expression vector is from Bruce A. Stanton (23). The ΔF508 CFTR mutation was generated by site directed mutagenesis in pEGFP- wt CFTR using ΔF508 primers. Wt CFTR, ΔF508 CFTR, and Δ27–264 CFTR were subcloned into pCDNA3.1 with CBA and CMV promoters (Invitrogen, Carlsbad, CA). The plasmids were transfected into the cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 48 h of transfection, the cells were harvested and used for immunoprecipitation and immunoblotting.

Treatment

After transfection, cells were treated either with proteasome inhibitors, MG132 (Calbiochem), PS341/Bortezomib (Millennium Pharmaceuticals), or the lysosomal inhibitor, E 64 (Calbiochem) for 16 h. Cycloheximide (CXH, Sigma) was used at 25 μg/ml for various times as indicated.

Immunoprecipitation

Cells were harvested and processed as described previously (24). Briefly, cells were solubilized in lysis buffer (50 mm NaCl, 150 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, and in complete protease inhibitors (Roche, Indianapolis, IL). The cell lysates were spun at 14,000 × g for 15 min at 4 °C to pellet insoluble material. 10 μl of anti-GFP antibodies (Roche) were added to the lysate and allowed to incubate for 30 min. 50 μl of A/G-agarose beads (Santa Cruz Biotechnology) were added and incubated with gentle rocking for 4 h at 4 °C. Beads were washed four times with lysis buffer, and centrifuged to remove the lysis buffer. The beads were suspended in Laemmli sample buffer (50 μl) containing β-mercaptoethanol, vortexed for 1 min, and resolved by 5% SDS-PAGE. CFTR was detected as described below.

Immunoblotting

Cells were harvested and processed as described previously (24). Briefly, cells were solubilized in lysis buffer (50 mm NaCl, 150 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, and in complete protease inhibitors (Roche, Indianapolis, Illinois). The cell lysates were spun at 14,000 × g for 15 min at 4 °C to pellet insoluble material. The supernatants were subjected to 5% SDS-PAGE and Western blotting followed by enhanced chemiluminescence (ECL; Amersham Biosciences-Pharmacia, Piscataway, NJ). The chemiluminescence signal on the PVDF membrane was directly captured by FujiFilm LAS-1000 plus system with a cooled CCD camera. CFTR was detected with monoclonal anti-human CFTR (217) antibody (provided by Dr. J. Riordan Department of Biochemistry and Biophysics and Cystic Fibrosis Center of North Carolina at Chapel Hill, NC). GAPDH (glyceraldehyde-3-phosphate dehydrogenase), used as a loading control, was detected with monoclonal GAPDH antibody (1:10,000; US Biological).

Short-Circuit Current Measurements

Short-circuit current (I_SC) measurements were conducted using a six-channel Easy-Mount chamber system (Physiologic Instruments, San Diego, CA). Cells stably expressing ΔF508-CFTR (gift of Eric Sorscher, UAB Birmingham) were grown on Snapwell filters (Corning Costar, Acton, MA; 3407). Cells were infected with either 50 or 100 μl of AAV5 (8–9 × 10¹³ vector genomes/ml produced by the CF Vector Core at the University of Florida using standard techniques (25)) for 3 days and Isc measured 1 week later postinfection. I_SC was measured with a VCCMC6 multichannel voltage-current clamp amplifier (Physiologic Instruments). Data were acquired on an 1.71-GHz PC running Windows XP (Microsoft, Redmond, WA) and equipped with DI-720 (DATAQ Instruments, Akron, OH), with Acquire and Analyze version 2.3.159 (Physiologic Instruments) software. The cell monolayers were bathed on both sides with solution containing 120 mm NaCl, 5 mm KCl, 2 mm MgCl₂, 2 mm CaCl₂, 10 mm d-glucose, and 10 mm HEPES (pH 7.3 with NaOH). The solution was maintained at 37 °C, and bubbled gently with air. Amiloride (10 μm) was added to the mucosal solution, and after stabilization, forskolin (10 μm forskolin with 5 μm IBMX) was added to the both chambers, followed by the CFTR channel inhibitor (26) CFTRinh-172 (10 μm).

Whole Cell Patch Clamp

CHO cells (ECACC) were cultured in Ham's F-12 media (Lonza Corp.), supplemented with 10% FBS (Invitrogen), at 37 °C. CHO cells were transfected with a plasmid containing various human CFTR constructs using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. A GFP plasmid was co-transfected at 1/10 the concentration for non-GFP-containing CFTR constructs to mark cells for targeting. Whole cell recordings were performed using an Axopatch 200B amplifier (Axon Instruments), visualized using a Nikon Eclipse Fluorescent inverted microscope (Nikon, Japan), and analyzed using PClamp 9.2 software (Axon Instruments). Cells were bathed in (mm): 140 NaCl, 2 CaCl₂, 1 MgCl₂, 80 d-mannitol, and 10 Hepes, pH 7.4. The pipette solution consisted of (mm): 135 CsCl, 2 MgCl₂, 2 ATP, 10 Hepes, and 0.001 free Ca²⁺, pH 7.4. The hypertonic bath solution was used to prevent the activation of any swelling activated Cl⁻ conductance. Forskolin and cpt-cAMP were purchased from Sigma.

RESULTS

Δ27–264 CFTR

Previously, we studied the truncation mutant, Δ264 CFTR, which as mentioned above is missing the first 4 transmembrane segments of TMD1. However, because a number of molecules involved in CFTR processing and trafficking bind to the N terminus of CFTR (27), and because of our past experience with the N terminus increasing CFTR protein expression (20), we reasoned that adding the first 26 amino acids of the extreme N terminus of CFTR would improve the protein expression of Δ264 CFTR (Fig. 1). Fig. 1 shows that wt-CFTR resolves into two bands a full glycosylated mature band C and an immature partially glycosylated band B. Note that the protein expression of Δ27–264 CFTR is clearly detectable in a Western blot compared with that of Δ264 CFTR which is barely detectable. On the other hand, the protein expression of Δ27–264 CFTR is still much less than that of wild-type CFTR.

FIGURE 1.

Δ27–264 CFTR (right panel) has a molecular weight lower than that of wild type CFTR (left panel) but slightly higher than Δ264. COS7 cells were transfected with 4 μg of CB-Δ264CFTR, CB-Δ27–264CFTR, and 4 μg of pCDNA3.1 -wt CFTR. After 48 h, cells were lysed, and the total lysate was analyzed by Western blot using anti-human CFTR antibodies (antibody 217). Loading was evaluated with GAPDH antibodies (US Biological) in all experiments even if data are not shown (representative of five experiments). Band intensity is 1.6 ± 0.6 (p < 0.05) greater for Δ27–264 compared with Δ27–264 CFTR.

To evaluate the degradation of Δ27–264 CFTR, we treated cells with the inhibitor, MG132, a nonspecific inhibitor of proteasomal degradation and PS341, a more specific inhibitor. Fig. 2 shows that in the presence of MG132 or PS341, the protein expression of Δ27–264 CFTR increases. This is similar to what we showed previously for Δ264 CFTR (19). Whereas, when cells are treated with the lysosomal inhibitor, E64, Δ27–264 CFTR protein expression is not affected. These data suggest that like Δ264-CFTR, Δ27–264 CFTR is degraded primarily in the proteasome.

FIGURE 2.

A, proteasome inhibition. Cells were transfected with Δ27–264 cDNA and treated with MG132 or PS341. Δ27–264 CFTR protein expression is increased dramatically by proteasome inhibition (upper two panels). B, lysosome inhibition. COS7 cells were transfected with Δ27–264 CFTR and treated with lysosome inhibitor (E64) for 16 h. There is very little change in band density among all the treatment groups and Δ264 CFTR in the absence of the inhibitor (representative of n = 4 experiments).

To evaluate how fast Δ27–264 CFTR protein is degraded, we treated transfected cells with cycloheximide. As shown in Fig. 3, Δ27–264 CFTR rapidly disappears in cells treated with cycloheximide suggesting that it is rapidly degraded similar to ΔF508 CFTR (see Ref. 19). This is similar to that observed for Δ264 CFTR (19).

FIGURE 3.

Degradation of Δ27–264 CFTR assayed by inhibition of protein synthesis. COS7 cells were transfected with Δ27–264 CFTR and treated with cycloheximide (25 μg/ml) for the indicated times. The differences in the rate of decay between Δ27–264 CFTR is rapid and comparable to what we observed with ΔF508-CFTR (19).

Δ27–264 CFTR Increases Expression of wt CFTR and Rescues ΔF508 CFTR Trafficking and Stability

We tested whether this new truncation mutant is capable of transcomplementation. When Δ27–264 CFTR cDNA is transfected into CFBE cells stably transfected with wt CFTR, there is an increase in mature band C protein (Fig. 4A). To study whether Δ27–264 CFTR affects the maturation of ΔF508 CFTR, Δ27–264 CFTR was transfected into Hela cells stably expressing ΔF508 CFTR (gift of J. P. Clancy (28)) (Fig. 4B). Δ27–264 CFTR increased the mature C band of ΔF508 CFTR in these cells.

FIGURE 4.

Transcomplementation by Δ27–264 CFTR. Δ27–264 CFTR significantly increases expression of both C and B bands of ΔF508 CFTR. Experiments were conducted on: A, CF bronchial epithelial cells stably transfected with wild-type CFTR (CFBE41o-N6.2kbWT) (see Ref. 41 provided by Dieter Gruenert); B, HeLa cells stably transfected with ΔF508 CFTR (provided by J. P. Clancy).

To test whether Δ27–264 CFTR reduces the degradation of ΔF508 CFTR, we treated Hela cells stably expressing ΔF508 CFTR with cycloheximide and monitored the mature C band of ΔF508 CFTR (Fig. 5). Note that when cells are transfected with Δ27–264 CFTR, the rescued mature C band of ΔF508 CFTR remains relatively stable over the 6-h experimental period. This indicates that transcomplementation has rescued ΔF508 CFTR from endoplasmic reticulum-associated degradation (ERAD). As shown in Fig. 6, Δ27–264 CFTR is much more effective in CFBE cells at lower amounts of transfected cDNA compared with Δ264 CFTR at both rescuing the mature C band of ΔF508 and keeping it from being degraded.

FIGURE 5.

Degradation of ΔF508 CFTR assayed by inhibition of protein synthesis. A, Hela cells containing ΔF508 CFTR were treated with cycloheximide (25 μg/ml) and the disappearance of ΔF508 CFTR monitored for the times indicated. B, Hela cells containing ΔF508 CFTR were transfected with Δ27–264 CFTR and treated with cycloheximide for the indicated times. Note that the rate of disappearance of B band and residual C band of ΔF508 CFTR in A is rapid, indicating robust degradation of both bands. Importantly, when ΔF508 CFTR is transcomplemented with Δ27–264 CFTR, the rescued C band of ΔF508 CFTR remains remarkably stable over the 6-h experimental period. This is comparable to what we observed with wt-CFTR (19). Thus, ΔF508 CFTR is more rapidly degraded compared with transcomplemented ΔF508 CFTR suggesting that transcomplementation rescues ΔF508 CFTR in such a way that it is not recognized as a mutant protein by ERAD (n = 2).

FIGURE 6.

Degradation of ΔF508 CFTR: A, ΔF508 is rapidly degraded when cells are transfected with 0.5 μg of Δ264. B, in contrast with the same amount of Δ27–264, rescued C band is now present consistent with transcomplementation and both C and B bands remain relatively stable for 6 h (n = 2, see Fig. 5 for details).

Transepithelial Transport

A key measure of rescue of ΔF508-CFTR is restoration of transepithelial currents. To assess whether transcomplementation could restore transport CFBE cells stably expressing ΔF508-CFTR were infected with AAV5 containing either Δ264 or Δ27–264 CFTR. Fig. 7 shows that both are able to restore transepithelial currents generated by CFTR. The data again show that Δ27–264 CFTR is effective at lower doses than Δ264 CFTR.

FIGURE 7.

Transcomplementation rescues ΔF508 CFTR currents. As we have shown using other assays, both Δ264 and Δ27–264 CFTR can rescue ΔF508 however, Δ27–264 CFTR measured by several different assays within this study is effective at lower doses. CFBE cells stably expressing ΔF508 (see “Experimental Procedures”). In the graph, 10 μm forskolin was added at the first arrow, and 10 μm CFTRinh-172 was added at the second arrow to demonstrate that the currents were indeed generated by CFTR. Small currents were generated because Isc was measured using the same solutions in both chambers. (n = 2). These currents are comparable to those measured from CFBE cells stably expressing wt-CFTR used as a positive equipment control on the day of the experiment. As shown in Fig. 8, no currents are measured from CFBE cells containing ΔF508 CFTR in the absence of transcomplementation.

CFTR Chloride Currents

In a previous study, our laboratory transfected Δ264 CFTR into IB3–1 bronchial epithelial cells and measured single channel currents with open probability and conductance similar to wild-type CFTR (17). In that study, we concluded that Δ264 CFTR was most likely producing these near normal channel currents. However, IB3–1 cells are bronchial epithelial cells, which do contain ΔF508 CFTR (29). Our recent studies on transcomplementation (19) raise the possibility that Δ264 CFTR through transcomplementation is rescuing ΔF508 CFTR, and the latter is generating the currents. To address this, whole cell patch clamp studies were conducted in Chinese hamster ovary, CHO, cells that do not have any detectable endogenous CFTR or CFTR-generated chloride currents. Only when both ΔF508 CFTR and Δ27–264 CFTR are cotransfected do currents appear (Fig. 8). Interestingly, the currents generated approach those of wild-type CFTR.

FIGURE 8.

Functional study of Δ27–264 CFTR in CHO cells. A, whole cell currents were recorded from CHO cells overexpressing wt CFTR GFP, ΔF508 GFP, Δ27–264 CFTR, or both Δ27–264 CFTR and ΔF508 GFP. Currents were elicited by 15 mV voltage steps from −100 mV to 125 mV. CFTR currents were activated by the inclusion of 500 μm cpt-cAMP and 40 μm forskolin. Though not shown, both the wtCFTR GFP currents and the Δ27–264 CFTR/ΔF508 GFP currents were inhibited by 10 μm CFTR-Inhib172. B, summary current/voltage plots of whole cell currents of CFTR. Conclusion: Transcomplementation with Δ27–264 CFTR rescues currents (solid squares) compared with open squares.

To explore more definitely which form of CFTR is generating the currents, we utilized the conduction mutant S341A. This mutation is located in transmembrane 6 of TMD1 of CFTR. This transmembrane segment has been studied extensively and has been suggested to be part of the conduction pore for chloride movement through CFTR (30). S341A has been shown to alter the ion selectivity of CFTR (30). Fig. 9 shows that the S341A mutation in wild type CFTR dramatically reduces the whole cell currents without affecting protein expression. With this result in hand, we then tested the double mutant ΔF508/S341A CFTR (Fig 10). This double mutant by itself does not generate any currents consistent with our results on ΔF508 CFTR. Interestingly, when Δ27–264 CFTR containing the wild-type CFTR conduction pore and the double mutant ΔF508/S341A CFTR are cotransfected there are again hardly any currents detected despite the presence of ample amounts of protein. This suggests conclusively that in cotransfection experiments with ΔF508 CFTR and Δ27–264 CFTR that the currents we observe are being generated by rescued ΔF508 CFTR and not from Δ27–264 CFTR.

FIGURE 9.

Functional study of S341A CFTR in CHO cells. S341A CFTR-expressing CHO cells have smaller whole cell currents than CHO cells expressing W+ CFTR (A and B). C, summary I/V data; ±S.E. (S341A CFTR, red circles, n = 4; W+CFTR, black squares, n = 3).

FIGURE 10.

Dual expression of Δ508 CFTR and Δ27–264 CFTR results in ΔF508 CFTR-mediated whole cell currents. Expression of Δ508/S341A CFTR along with Δ27–264 CFTR results in robust expression, with slightly elevated B and C bands as compared with Δ508 CFTR/Δ27–264 CFTR expression (A), but with reduced whole cell currents (B) as compared with currents from cells expressing Δ508 CFTR/Δ27–264 CFTR (B), consistent with the previously described conductance defect in S341A CFTR (34). C, summary I/V data for CHO cells expressing Δ508/S341A CFTR and Δ27–264 CFTR (n = 7; red squares) and Δ508 CFTR and Δ27–264 CFTR (n = 5; black circles); ± S.E.

Interactions between ΔF508 CFTR and Δ27–264 CFTR

Our previous results showed that Δ264 CFTR by binding to key elements in ER-associated quality control caused the rescue of ΔF508 CFTR from the ER to the cell surface (19). The observation in this study that transcomplementation with the new truncated form partially restores whole cells current generated by ΔF508 CFTR suggests perhaps that both ΔF508 and Δ27–264 CFTR interact with one another. To test this, coimmunoprecipitation experiments were performed. Fig. 11 shows that pulling down GFP-labeled ΔF508 CFTR with an anti-GFP antibody and immunoblotting with anti-CFTR antibody does show that Δ27–264 CFTR can bind to ΔF508 CFTR. We also show that binding increases in the presence of MG132. Fig. 12 shows that Δ264 CFTR also binds with ΔF508 but the binding is detectable only at 12-fold high concentrations of transfected cDNA compared with Δ27–264 again showing that addition of the N-terminal amino acids does improve the ability to rescue the trafficking of ΔF508 CFTR.

FIGURE 11.

Δ27–264 CFTR binds to ΔF508-CFTR. COS7 cells were transfected with Δ27–264 CFTR or ΔF508 CFTR containing GFP or cotransfected with both plasmids. ΔF508 CFTR was pulled down with anti-GFP antibodies, and the gels were blotted with anti-CFTR antibodies (representative of five experiments).

FIGURE 12.

Δ27–264 binds to ΔF508 CFTR. Note that binding of Δ264 to ΔF508 CFTR is detected at 6 μg compared with 0.5 μg for 27–264 shown in Fig. 9 (n = 3). See Fig. 11 for details.

DISCUSSION

Δ27–264 CFTR Has Properties Similar to Δ264 CFTR

In this study we created a new construct by adding the first 27 amino acids from wild-type CFTR to the N terminus of our previously published construct, Δ264 CFTR(19). Δ27–264 CFTR has properties similar to Δ264 CFTR. It is rapidly degraded in the proteasome. Importantly similar to Δ264 CFTR, Δ27–264 can increase the steady levels of the mature C band on wild type CFTR and increase the processing of immature B band of ΔF508 CFTR by the process of transcomplementation.

Δ27–264 CFTR Is Not Functional but Rescues the Channel Activity of ΔF508 CFTR

ΔF508 CFTR is a channel whose protein is rapidly degraded and does not reach the plasma membrane Riordan (31) and whose channel function is severely reduced compared with wild type CFTR (see Fig. 6). Importantly, we show here that transcomplementation of ΔF508 CFTR by Δ27–264 CFTR rescues both mature C band and channel activity of ΔF508 CFTR. We showed in a previous study of Δ264 CFTR our earlier truncation mutant transfected into IB3–1 bronchial epithelial cells that single channel currents occurred with open probability and conductance similar to wild-type CFTR (17) but the exact origin of the currents was inconclusive. Cormet-Boyaka et al. have recently measured single channel properties of currents generated in cells cotransfected with a small fragment of CFTR 1–633 and ΔF508 CFTR (32). What is remarkable about their results is that the currents rescued by 1–633 CFTR had a single channel open probability near to that of wt-CFTR similar to our earlier results. However, given that it has been shown (33) that small fragments of CFTR encompassing only the first 6 transmembrane domains could form ion channels with properties similar to wt-CFTR, the rescued currents could have originated from the truncation mutant. Thus the exact origin of the rescued currents was not verified.

In this study, we address this question with the conduction mutant S341A. As mentioned above, this mutation is located in transmembrane 6 of TMD1 of CFTR and most likely represents a critical amino acid in the conduction pore for chloride movement through CFTR(34). S341A has been shown to alter the ion selectivity of CFTR and the sensitivity to chloride channel blockers (30). Our results showed that this mutation by itself does not affect protein processing. Because we could rescue the ΔF508/S341A CFTR protein but not rescue channel currents because of the altered conduction pore, results showed conclusively that the current is indeed generated by rescued ΔF508-CFTR channel gating.

Correcting ΔF508 CFTR

Because Δ27–264 CFTR is missing ICL1 & 2 (intracellular loop) as well as the first four membrane spanning domains, the question then can be raised about what are the critical elements that are necessary for transcomplementation to occur. As mentioned above, Cormet-Boyaka et al. demonstrated transcomplementation of ΔF508 CFTR using a small fragment of CFTR 1–633. This would contain the complete TMD1 and NBD1. Sun et al. utilized a smaller fragment that included only NBD1 and the R domain (35). Given the results from these reports and the data here, the one common element of all these truncated CFTRs is the presence of the NBD1 domain. If NBD1 is the key player then how is it working?

It is known that in isolation, NBD1 can form a crystal dimer (36). Full-length CFTR has also been shown to form a dimer although whether CFTR functions as a dimer or monomer is a matter of controversy (37). We showed here that Δ27–264 CFTR can bind to ΔF508 CFTR. Cormet-Boyaka et al. (32) showed that their truncation mutant can also bind to ΔF508 CFTR. Thus these truncation mutants may function by forming a bimolecular interaction with ΔF508 CFTR. Both of these truncation mutants rescue protein processing and channel function, indicating that transcomplementation with these mutants may be affecting the stability of ΔF508 CFTR in such a way as to rescue channel function. Further evidence for this is seen in Fig. 5, which suggests that transcomplementation with Δ27–264 CFTR rescues ΔF508 CFTR from ERAD.

Our earlier work and that of Sun et al. (35) concluded that the truncation mutants were competing for and displacing ΔF508 CFTR from key elements in the quality control mechanism, in our case, VCP (19) and HDAC6 and in theirs Aha1 (35). Low temperature rescue of ΔF508 CFTR allows the processing of immature band B to mature band C but does not correct channel activity (see Fig. 6). Thus low temperature rescue probably affects the quality control processing ΔF508 CFTR without restoring its inherent defective thermal instability (38). Because transcomplementation can rescue protein processing, and channel activity and reduce ERAD of ΔF508 CFTR, points to a mechanism of action in addition to its effects on quality control.

In summary, our data suggest that transcomplementation of ΔF508 CFTR by Δ27–264 CFTR most likely occurs because Δ27–264 CFTR interacts with proteins in the ERAD pathway and binds to ΔF508 CFTR. Our previous data show that Δ264 CFTR can function as an ion channel at the plasma membrane in Xenopus oocytes (39) and can correct the inflammatory lung disease phenotype the Pseudomonas-beads and Aspergillus fumigatus CFTR knock-out mouse (17, 40). Our new data show that the Δ27–264 CFTR viral vector has a dual benefit. It rescues single channel activity of ΔF508 CFTR, while at the same time it can promote the expression of ΔF508 CFTR mature band C. This dual effect makes the rAAV-Δ264 CFTR a highly promising CF gene therapy vector.