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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2007 Sep 14;104(39):15370–15375. doi: 10.1073/pnas.0706974104

Processing and function of CFTR-ΔF508 are species-dependent

Lynda S Ostedgaard *, Christopher S Rogers *, Qian Dong †, Christoph O Randak *, Daniel W Vermeer *, Tatiana Rokhlina *, Philip H Karp *, Michael J Welsh *,†,‡,§
PMCID: PMC1976592  PMID: 17873061

Abstract

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. The most common mutation, a deletion of the phenylalanine at position 508 (ΔF508), disrupts processing of the protein. Nearly all human CFTR-ΔF508 is retained in the endoplasmic reticulum and degraded, preventing maturation to the plasma membrane. In addition, the F508 deletion reduces the activity of single CFTR channels. Human CFTR-ΔF508 has been extensively studied to better understand its defects. Here, we adopted a cross-species comparative approach, examining human, pig, and mouse CFTR-ΔF508. As with human CFTR-ΔF508, the ΔF508 mutation reduced the single-channel activity of the pig and mouse channels. However, the mutant pig and mouse proteins were at least partially processed like their wild-type counterparts. Moreover, pig and mouse CFTR-ΔF508 partially restored transepithelial Cl− transport to CF airway epithelia. Our data, combined with earlier work, suggest that there is a gradient in the severity of the CFTR-ΔF508 processing defect, with human more severe than pig or mouse. These findings may explain some previously puzzling observations in CF mice, they have important implications for evaluation of potential therapeutics, and they suggest new strategies for discovering the mechanisms that disrupt processing of human CFTR-ΔF508.

Keywords: airway epithelia, chloride transport, cystic fibrosis, mouse models

Cystic fibrosis (CF) is a common autosomal recessive disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), an epithelial anion channel (1, 2). The most common CF-associated mutation, accounting for ≈70% of CF alleles, is a deletion of phenylalanine 508 (CFTR-ΔF508, also called F508del-CFTR) (3). The ΔF508 mutation causes retention of the majority of mutant protein in the endoplasmic reticulum (ER) and subsequent degradation, preventing its maturation to the plasma membrane (4–7). In addition, deleting F508 alters channel gating, reducing the rate of channel opening (8–10). Reducing the incubation temperature or adding chemical chaperones, such as glycerol or trimethylamine N-oxide, allows some of the mutant protein to escape the ER and traffic to the cell surface where it retains significant, albeit reduced, activity (11–13). These observations sparked efforts to understand the mechanisms responsible for CFTR-ΔF508 retention and to develop pharmacological agents that could correct the CFTR-ΔF508 channel defects (14–17).

Current knowledge about CFTR-ΔF508 has come predominantly from studies of human CFTR. Because cross-species comparisons have often proven valuable in many fields of biology, we hypothesized that studying the processing and function of CFTR-ΔF508 from other species might help us better understand human CFTR-ΔF508. We were further encouraged to test this hypothesis because investigators study mice bearing the ΔF508 mutation (18–20), and they examine the structure of mouse nucleotide-binding domain 1 (NBD1, which contains F508) (21, 22). The goals of that work are to learn how the F508 deletion causes disease and to improve therapeutic approaches. However surprisingly, the consequences of the ΔF508 mutation on mouse CFTR or other species of CFTR remain unclear. Finally, during their limited lifespan, mice with a targeted CFTR gene fail to develop the airway disease typically found in humans (23). Therefore, we are proceeding to generate a CF pig, because the pig lung shares many anatomical, histological, biochemical, and physiologic features with human lungs. Before generating a CFTR-ΔF508 animal, we wished to know how the ΔF508 mutation alters processing and function. Thus, we compared human, pig, and mouse CFTR-ΔF508.

Results

Sequence of Pig CFTR.

We cloned the pig CFTR cDNA and used it to predict the amino acid sequence [supporting information (SI) Fig. 7]. The pig CFTR amino acid sequence is nearly 93% identical to that of human CFTR. For comparison, mouse CFTR shows 78% identity to human CFTR (24). The region immediately surrounding F508 is highly conserved.

Glycosylation of Human, Pig, and Mouse CFTR-ΔF508.

The pattern of human CFTR glycosylation changes as the protein migrates from the ER to the Golgi complex (25). The nascent protein lacking glycosylation is called “band A.” In the ER, CFTR undergoes core glycosylation and migrates more slowly during electrophoresis as “band B”. In the Golgi complex, more extensive glycosylation occurs, which further slows and broadens the electrophoretic migration of the “band C” form. Differences in glycosylation do not appear to affect function (7, 26) but do provide a convenient way to assess the biosynthetic processing of CFTR. When we expressed wild-type human, pig, and mouse CFTR in the monkey kidney cell line COS7, we observed the typical appearance of bands B and C (Fig. 1A). Human CFTR-ΔF508 produced band B but not band C, consistent with defective exit from the ER. This result agrees with many previous reports in several different cell lines (25, 27, 28). Interestingly, in addition to band B, mouse CFTR-ΔF508 generated a significant proportion of band C protein, consistent with a report of partial CFTR-ΔF508 processing in oviduct of a CF mouse model (see below and ref. 29). Pig CFTR-ΔF508 also produced a small amount of band C. These results suggested that some mouse and pig mutant protein may have trafficked to the Golgi complex.

Fig. 1.

Fig. 1.

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Pig and mouse CFTR-ΔF508 produce some mature band C protein. Shown are immunoprecipitated and in vitro-phosphorylated wild-type and ΔF508 CFTR of human, pig, and mouse. (A) Constructs were expressed for 24, 48, and 72 h in COS7 cells. (B and C) Constructs were expressed for 48 h in NIH 3T3 (B) and LLC-PK1 (C) cell lines. H, human; P, pig; M, mouse. Bands B and C are indicated by arrows.

To learn whether the differences among the three species of CFTR-ΔF508 depended on the primate COS7 cell line, we expressed the constructs in the mouse NIH 3T3 fibroblast line and the pig LLC-PK1 kidney cell line (Fig. 1 B and C) as well as human HEK-293T cells (data not shown). In each of these cell lines, human CFTR-ΔF508 generated only the band B form, whereas pig and mouse CFTR-ΔF508 produced both band B and some fully glycosylated protein, consistent with our studies in COS7 cells. We also noted that some of the wild-type and ΔF508 pig CFTR migrated slightly more rapidly than band B of either human or mouse.

To confirm that the high-molecular-mass C forms of pig and mouse CFTR-ΔF508 were due to complex glycosylation, we used endoglycosidase H digestion. Endoglycosidase H removes carbohydrate from proteins that contain only the sugar added in the ER, but it does not delete complex glycosylation added in the Golgi complex (25, 30). Endoglycosidase H treatment shifted the migration of the band B form of all of the proteins to the unglycosylated form (Fig. 2). However, like the band C form of the wild-type CFTRs, the fully glycosylated mouse and pig CFTR-ΔF508 were resistant to endoglycosidase H, confirming that these proteins were glycosylated in the Golgi complex.

Fig. 2.

Fig. 2.

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Fully glycosylated pig and mouse ΔF508 are not endoglycosidase H-sensitive. Shown are immunoprecipitated and in vitro-phosphorylated human, pig, and mouse wild-type and ΔF508 CFTR incubated in the presence (+) or absence (−) of 10 mU of endoglycosidase H. Human CFTR was from electroporated COS7 cells; we expressed pig and mouse CFTR using adenoviral vectors. Last 2 lanes are COS7 cells infected with Ad-GFP. Bands A, B, and C are indicated by arrows.

Expression of Human, Pig, and Mouse CFTR-ΔF508 at the Cell Surface.

To determine whether the human, pig, and mouse CFTR-ΔF508 were localized at the apical membrane of airway, we expressed the proteins in well differentiated human CF airway epithelia and examined them with confocal immunocytochemistry. Consistent with earlier studies (31), wild-type human CFTR was localized to the apical membrane, and human CFTR-ΔF508 appeared to be expressed diffusely throughout the cell (Fig. 3). As expected from the biochemical studies, both pig and mouse wild-type CFTR were localized to the apical membrane. However, in contrast to human CFTR-ΔF508, both the pig and mouse mutants were also detected in the apical portion of the airway cells.

Fig. 3.

Fig. 3.

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Human, pig, and mouse wild-type CFTR and pig and mouse CFTR-ΔF508 are expressed on the apical surface of differentiated airway epithelia. Immunostaining of differentiated human CF airway epithelia expressing human, pig, and mouse wild-type and ΔF508 CFTR. Data are X-Y (A, B, E, F, I, and J) and X-Z (C, D, G, H, K, and L) confocal images. CFTR immunostaining is in green, and ZO-1 (tight junction) is in red. Apical membrane is shown by arrow, and filter (at the basal membrane) is indicated by dotted line. In B, faint staining of CFTR-ΔF508 is visible beneath the apical surface. (Scale bars, 10 μm.)

Single-Channel Gating of Human, Pig, and Mouse CFTR-ΔF508.

Most, although not all, studies indicate that human CFTR-ΔF508 manifests a channel-gating defect that reduces activity (8, 9, 11, 32–34). To learn whether the ΔF508 mutation compromises the channel activity in pig and mouse CFTR, we studied excised, inside-out patches of membrane containing CFTR channels. We readily detected channels in patches taken from cells expressing pig and mouse CFTR-ΔF508 grown at 37°C, consistent with the conclusion that some pig and mouse CFTR-ΔF508 are able to reach the cell membrane under physiological conditions. This contrasts with human CFTR-ΔF508, which must be incubated at lowered temperatures to produce significant amounts of cell-surface protein (11, 35). Phosphorylation by the catalytic domain of cAMP-dependent protein kinase (PKA) and cytosolic ATP were required for activity of all versions studied. The single-channel conductances for the wild-type channels were human (8.3 pS) > pig (6.7 pS) > mouse (4.3 pS), and they were not significantly altered by the ΔF508 mutation (Fig. 4 A and B, and ref. 10). Lansdell et al. (36) reported that heavily filtering currents recorded from mouse CFTR revealed a subconductance state that was ≈10% the amplitude of the full conductance. With heavy filtering, we also observed the subconductance in both wild-type and ΔF508 channels (Fig. 4A).

Fig. 4.

Fig. 4.

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Single channel currents from human, pig, and mouse wild-type and ΔF508 CFTR. (A) Representative current traces from excised, inside-out patches of HeLa cells containing single channels of human, pig, and mouse wild-type and ΔF508 CFTR are shown. Holding voltages were human at −80 mV, pig at −100 mV, and mouse wild-type at −50 mV and mouse ΔF508 at −80 mV. Human tracings were from cells incubated at reduced temperature and then studied at 37°C and are taken from Teem et al. (10); pig and mouse channels were from cells incubated at 37°C and studied at ≈25°C. Expanded tracings at the bottom show subconductance in mouse wild-type and ΔF508 CFTR. (B) Properties of wild-type and ΔF508-CFTR. Data are mean ± SEM for single-channel conductance (g), open-state probability (Po), burst duration (BD), and interburst interval (IBI). n = 4–5 membrane patches for each. Asterisks indicate P < 0.05 compared with wild-type CFTR by using Mann–Whitney rank sum test. Note that values for human CFTR and CFTR-ΔF508 were taken from Teem et al. (10).

In the presence of PKA and 1 mM ATP, the open state probability (Po) of wild-type CFTR varied in the order, pig (0.39) ≈ human (0.37) > mouse (0.08) (Fig. 4B); the values for human were taken from our earlier study (10). In assessing mouse Po, we did not take into account the subconductance state; as reported by Lansdell et al. (36), it was very difficult to study because of its small single-channel conductance. The ΔF508 mutation reduced the Po of human CFTR to 27% (10), pig CFTR to 46%, and mouse CFTR to 50% of the corresponding wild-type channel (Fig. 4 A and B). The cause of the reduced Po was a decrease in the rate of channel opening without a significant alteration of burst duration (Fig. 4B). Thus, in all three species, the ΔF508 mutation altered gating by a similar mechanism.

Transepithelial Cl− Current Generated by Human, Mouse, and Pig CFTR-ΔF508.

Assays of Cl− channel function are more sensitive at detecting CFTR in the cell surface than are biochemical or immunostaining assays. Therefore, because both pig and mouse CFTR-ΔF508 were partially processed through the Golgi complex and likely targeted to the apical membrane and because they both retained partial Cl− channel activity, we predicted that they would generate transepithelial Cl− currents when expressed in well differentiated CF airway epithelia (37). To assay transepithelial Cl− transport, we mounted epithelia in modified Ussing chambers and measured transepithelial Cl− current. We first inhibited Na+ current with amiloride, which hyperpolarizes the apical membrane voltage, increasing the driving force for Cl− secretion through CFTR. Then, we increased CFTR activity by elevating cellular levels of cAMP with forskolin and IBMX. Finally, we reduced transepithelial Cl− current by inhibiting the Na+-K+-Cl− cotransporter with basolateral bumetanide; the resulting change in current provides a good measure of the Cl− current (37). We chose bumetanide rather than CFTR inhibitors, because they can have different efficacy on CFTR from different species (38).

Expressing wild-type human CFTR produced significant transepithelial Cl− current (Figs. 5 and 6), as described (39). The same was true for wild-type pig and mouse CFTR. As expected, human CFTR-ΔF508 produced only a small amount of current, 6.9 ± 1.56% of wild-type current. However, relative to their wild-type controls, pig (25.6 ± 3.38%) and mouse (17.1 ± 4.94%) CFTR-ΔF508 produced substantially more current than human CFTR-ΔF508 (Fig. 6B). To determine whether these results were limited to expression in human epithelia, we repeated the study using airway epithelia derived from CF mice; the results were qualitatively similar (Fig. 6 A and B). Thus, pig and mouse CFTR-ΔF508 generated relatively larger transepithelial Cl− currents than human CFTR-ΔF508 in CF airway epithelia from two different species. These results indicate that some pig and mouse CFTR-ΔF508 were present and active in the apical membrane of airway epithelia.

Fig. 5.

Fig. 5.

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Transepithelial currents in human CF airway epithelia expressing human, pig, and mouse CFTR and CFTR-ΔF508. Examples of current traces of human, pig, and mouse wild-type CFTR and CFTR-ΔF508 expressed in differentiated human CF airway are shown. Agents were present during times indicated by bars. DIDS, 4,4′-diisothiocyanoto-stilbene-2,2′-disulfonic acid.

Fig. 6.

Fig. 6.

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Bumetanide-sensitive cAMP-stimulated current in differentiated CF airway epithelia. (A) Currents in human and mouse airway epithelia expressing human, pig, and mouse wild-type CFTR and CFTR-ΔF508 CFTR after subtraction of currents from GFP-expressing control epithelia. (B) Bumetanide-inhibited current in CF epithelia expressing CFTR-ΔF508 as a percentage of bumentanide-inhibited current in CF epithelia expressing wild-type CFTR of each species.

Discussion

The ΔF508 mutation confers at least three defects on human CFTR; it reduces channel activity, it impairs processing, and it reduces the protein's stability at the cell surface (4–10, 35, 40, 41). Our data suggest that the ΔF508 mutation inhibited gating of CFTR channels from all three species by the same mechanism, a reduced opening rate. In contrast, the characteristic processing defect observed in human CFTR-ΔF508 was less severe in pig and mouse proteins. This conclusion was supported by the findings that they showed complex glycosylation, they were readily detected in excised patches from cells grown at 37°C, immunocytochemistry localized some of the protein to the apical membrane of airway epithelia, and they corrected the Cl− transport defect in CF airway epithelia more than did human CFTR-ΔF508. This was unexpected, because impaired ER escape is thought to be a major factor reducing CFTR-ΔF508 function in humans. However, even with human CFTR-ΔF508, variations in the amount of mutant protein delivered to the cell surface have been reported, especially in studies of epithelia (42–47). Based on our data and previous work by others, it appears that there is a gradient in the severity of the ΔF508 processing defect, with human worse than pig and pig somewhat worse than mouse. These results also suggest that the processing defect and the functional defect in CFTR-ΔF508 arise from different causes. The ability to separate these two defects by using different species suggests that further studies may help pinpoint their causes as well as the reduced cell surface stability, which we have not investigated. These results also raise several interesting questions and they invite some speculation.

Do the Processing and Functional Effects of the ΔF508 Mutation Explain Cl− Channel Function in CFTR-ΔF508 Mice?

Three groups have engineered a ΔF508 mutation into the mouse CFTR genome (18–20). One strain (Cftrtm1Eur) had normal transcript levels (19). These mice showed residual transepithelial Cl− transport, and they did not manifest the severe intestinal phenotype usually observed in CFTR null mice that produce no CFTR. Our data suggest that correct processing of some mouse CFTR-ΔF508 produced functional cell-surface channels that generated transepithelial Cl− transport and minimized intestinal disease in these mice. Consistent with this conclusion, a previous report on Cftrtm1Eur mice found that the oviduct produced a small amount of band C protein (29). How much CFTR function is required to prevent intestinal disease is uncertain, but Dorin et al. (48) estimated that 5% of wild-type function was sufficient.

In the other two mouse strains with the ΔF508 mutation (Cftrtm2Cam and Cftrtm1Kth) (18, 20), the levels of CFTR-ΔF508 transcripts were decreased compared with those from the wild-type allele (49). Both of these strains lacked evidence of Cl− transport, and they died from intestinal disease similar to that in CFTR null animals. In these animals, it seems likely that impaired processing and function of the CFTR-ΔF508 protein, combined with decreased transcript numbers, reduced CFTR below the level required to prevent disease.

Can Our Findings Explain Some Earlier Puzzling Observations in CFTR-ΔF508 Mice?

An earlier study showed that administering curcumin to CFTR-ΔF508 mice, but not to CFTR null mice, partially corrected the Cl− transport defect in nasal epithelia and prevented the lethal intestinal phenotype (50). Subsequent studies suggested that curcumin increased the activity of human CFTR-ΔF508 channels but had minimal effects on the defective processing of human CFTR-ΔF508 (51–53). Our finding that mouse CFTR-ΔF508 is partially processed correctly suggests that the beneficial effect of curcumin in CFTR-ΔF508 mice may have occurred because it increased activity of CFTR-ΔF508 channels that were present in the apical membrane. Another study found that the combination of the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor milrinone increased Cl− transport across the nasal epithelia of CFTR-ΔF508 mice but not CFTR null mice (54). Because increasing cellular levels of cAMP has not been shown to rescue the defective processing of human CFTR-ΔF508, we speculate that the increase in cellular levels of cAMP may have increased phosphorylation of CFTR-ΔF508 already resident in the nasal epithelium. In support of this, Wang et al. (33) have shown that the Po of human CFTR-ΔF508 can be increased by prolonged phosphorylation. Several additional studies have shown beneficial effects of compounds on Cl− transport in CFTR-ΔF508 mice without necessarily demonstrating an effect on the processing of CFTR-ΔF508. Thus, we speculate that some of these responses might have occurred through activation of CFTR-ΔF508 that resides in the apical membrane.

What Are the Implications for Developing CF Therapeutics?

There is a substantial effort in academia and industry to develop compounds that correct the processing defect of CFTR-ΔF508, moving it from the ER to the apical membrane where it could generate some channel activity (16, 55). Our data suggest that the evaluation of such agents could be influenced by the species of CFTR-ΔF508. For example, CFTR-ΔF508 mice may have a significant amount of CFTR at the cell surface even in the absence of therapeutics. In addition, the use of CFTR-ΔF508 mice to test compounds that potentiate the activity of CFTR could provide information about their efficacy.

Why Are Pig and Mouse CFTR-ΔF508 Less Misprocessed Than Human ΔF508?

Our results showing that mouse and pig CFTR-ΔF508 are processed at least partially like wild-type CFTR were obtained with several methods, the studies were done in multiple cell types, and all three species were studied in parallel. As a result, it is difficult to attribute the species-dependent differences to a specific cell type and, thus, to the presence or absence of a specific chaperone or other cellular protein. We therefore conclude that structural differences between the human ΔF508 protein and that of the other two species cause the differences.

To begin to obtain clues about structural differences, we looked for regions where human residues differ from both pig and mouse sequences; there are 78 such amino acids. In the linear amino acid sequence of CFTR, the residues surrounding F508 are conserved across all three species. In addition, the crystal structures of mouse and human NBD1 are similar and resemble the structure of NBDs from other ABC transporters with two exceptions (21, 22); CFTR NBD1 contains additional sequence within the N terminus (405–437) not present in other ABC transporters and sequence at the C terminus (654–670) that lies beyond the C terminus of canonical ABC transporters. It is in these areas where we see the greatest divergence in the sequence of human, pig, and mouse NBD1. These two areas may deserve additional attention in understanding the consequences of the ΔF508 mutation and the interactions between NBD1 and the rest of the protein (7, 56). In addition, previous studies have shown that mutating four arginine-framed motifs (AFM) in CFTR-ΔF508 partially restored processing in human CFTR-ΔF508 (57, 58). Interestingly, two AFMs are missing in the mouse sequence, although mouse CFTR has an additional AFM not present in pig or human. Pig CFTR retains the four human AFMs plus an additional AFM. Thus, although changes in the AFMs could potentially contribute to the processing differences, they are not likely solely responsible.

Although these sequence differences do not tell us why there is a difference in processing between human CFTR-ΔF508 and the pig and mouse mutant channels, or in channel function between wild-type and ΔF508 of all three species, they do suggest strategies for discovering the reasons.

Our findings open avenues for understanding the biosynthesis of CFTR-ΔF508. Many different strategies and approaches have been used to discover the cellular and molecular bases for the human CFTR-ΔF508 defect; inclusion of pig and/or mouse CFTR-ΔF508 in those experiments might provide an important control that could include or exclude alternative explanations. Our results also suggest that development of another animal model, such as a pig with the CFTR-ΔF508 mutation, might yield important insight into the mechanisms of CFTR-ΔF508 dysfunction. In addition, discovering the reason for species-dependent differences in CFTR-ΔF508 processing would seem to hold promise for suggesting novel therapeutic approaches. Finally, our findings suggest that a comparative approach might also be of value in investigating how disease-associated mutations cause dysfunction of other proteins.

Materials and Methods

Please see SI Methods for a more detailed description of the methods.

Vectors and Expression.

Plasmids encoding human wild-type and ΔF508 CFTR have been described (10, 59). Pig CFTR cDNA was amplified from domestic pig (Sus scrofa). Mouse CFTR cDNA was a kind gift from Christopher Boyd (University of Edinburgh, Edinburgh, U.K.) and Brandon Wainwright (University of Queensland, Queensland, Australia). We subcloned all three CFTR cDNAs into pcDNA3.1 (Invitrogen, Carlsbad, CA) and recombinant adenoviruses. For recombinant adenovirus of mouse CFTR, we had to remove intron 11, which had been inserted previously to stabilize the vector (36).

For protein-processing studies, COS7 cells were electroporated (59); 3T3 and LLC-PK1 cells were transfected with plasmid and Lipofectamine 2000. For deglycosylation studies, COS7 cells were electroporated with human CFTR or infected with adenovirus encoding pig or mouse CFTR. For patch–clamp studies, HeLa cells were infected with adenovirus encoding mouse CFTR or transfected by using a hybrid vaccinia virus system encoding pig CFTR (10). Expression in human and mouse airway epithelia was with recombinant adenoviruses. Murine tracheal epithelia were cultured from ΔF508/ΔF508 transgenic mice (Cftrtm1Kth) or CFTR null mice expressing the intestinal FABP-CFTR (Cftrtm1Unc/FABP-CFTR) (18, 60, 61). In the absence of gene transfer, there were no cAMP-stimulated Cl− currents in either mouse genotype. CF human airway epithelia were obtained as described (37).

Biochemical Studies.

COS7 cells were solubilized in lysis buffer with 1% TX-100 and proteinase inhibitors (59). CFTR was immunoprecipitated with M3A7 antibody (Upstate Biotechnology, Lake Placid, NY) and then in vitro-phosphorylated as described (59). Note that the consensus phosphorylation sites and N-glycosylation sites are conserved in all three species (SI. Fig. 7). Processing studies in NIH 3T3 and LLC-PK1 cells were carried out similarly to those in COS7 cells. For deglycosylation studies, membranes were isolated (62) from COS7 cells and solubilized in LB plus 1% Nonidet P-40 (Pierce, Rockford, IL). Supernatants were divided, immunoprecipitated, and resuspended with or without endoglycosidase H (62). After incubation, precipitates were in vitro-phosphorylated as described above.

Immunocytochemistry.

Three days after gene transfer, epithelia were fixed, permeabilized, and incubated with a mixture of anti-CFTR antibodies (M3A7, MM13-4 (Upstate Biotechnology) and 13–1 (R&D Systems, Minneapolis, MN) and anti-ZO-1 (Zymed, San Francisco, CA) primary antibodies, followed by Alexa Fluor-conjugated secondary antibodies (Molecular Probes, Eugene, OR) and examined by confocal laser scanning microscopy (39).

Electrophysiology.

For Ussing chamber studies, transepithelial Cl− current was measured 3 days after gene transfer by using a Cl− concentration gradient as described (39). For patch–clamp studies CFTR currents were studied in excised, inside-out membrane patches of HeLa cells as described (10, 63). Channels were activated with the catalytic subunit of PKA and Mg-ATP; PKA was present in all cytosolic solutions that contained ATP. Holding voltage was −50 to −100 mV. Experiments were performed at 23°C to 26°C. Data acquisition, processing, and analysis were performed as described (63). Data are mean ± SEM unless otherwise stated. P < 0.05 was considered statistically significant.

Supplementary Material

Supporting Information
pnas_0706974104_index.html (10.9KB, html)

Acknowledgments

We thank the University of Iowa In Vitro Models and Cell Culture Core [supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL51670, Cystic Fibrosis Foundation (CFF) Grants R458-CR02 and ENGLH9850, and National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK54759], and the University of Iowa Vector Core [supported by the Roy J. Carver Charitable Trust; NHLBI Grant HL51670, CFF Grant R458-CR02, and NIDDK Grant DK54759]. This work was supported by NHLBI Grant HL61234 (to M.J.W.) and CFF Grant OSTEDG06G0 (to L.S.O.). C.S.R. was supported by NIH Training Grant HL07638, C.O.R. was supported by Cystic Fibrosis Foundation Fellowship RANDAK05FO, and M.J.W. is an Investigator of the Howard Hughes Medical Institute.

Abbreviations

CF

cystic fibrosis

CFTR

CF transmembrane conductance regulator

ER

endoplasmic reticulum.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0706974104/DC1.

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