Misfolded proteins destined for the cell surface are recognized and degraded by the ERAD [ER (endoplasmic reticulum) associated degradation] pathway. TS (temperature-sensitive) mutants at the permissive temperature escape ERAD and reach the cell surface. In this present paper, we examined a TS mutant of the CFTR [CF (cystic fibrosis) transmembrane conductance regulator], CFTR ΔF508, and analysed its cell-surface trafficking after rescue [rΔF508 (rescued ΔF508) CFTR]. We show that rΔF508 CFTR endocytosis is 6-fold more rapid (∼30% per 2.5 min) than WT (wild-type, ∼5% per 2.5 min) CFTR at 37 °C in polarized airway epithelial cells (CFBE41o). We also investigated rΔF508 CFTR endocytosis under two further conditions: in culture at the permissive temperature (27 °C) and following treatment with pharmacological chaperones. At low temperature, rΔF508 CFTR endocytosis slowed to WT rates (20% per 10 min), indicating that the cell-surface trafficking defect of rΔF508 CFTR is TS. Furthermore, rΔF508 CFTR is stabilized at the lower temperature; its half-life increases from <2 h at 37 °C to >8 h at 27 °C. Pharmacological chaperone treatment at 37 °C corrected the rΔF508 CFTR internalization defect, slowing endocytosis from ∼30% per 2.5 min to ∼5% per 2.5 min, and doubled ΔF508 surface half-life from 2 to 4 h. These effects are ΔF508 CFTR-specific, as pharmacological chaperones did not affect WT CFTR or transferrin receptor internalization rates. The results indicate that small molecular correctors may reproduce the effect of incubation at the permissive temperature, not only by rescuing ΔF508 CFTR from ERAD, but also by enhancing its cell-surface stability.

INTRODUCTION

The CFTR [CF (cystic fibrosis) transmembrane conductance regulator] is a cAMP-activated chloride channel expressed in a variety of tissues [14]. CFTR functions in the apical membrane of polarized epithelial cells and undergoes endocytosis via clathrin-coated vesicles [511]. Mutations in the CFTR gene lead to CF, the most common hereditary lethal disease among Caucasians [12,13]. There are more than 1500 mutations listed in the CFTR mutation database (http://www.genet.sickkids.on.ca/cftr), but a 3 bp deletion resulting in loss of a phenylalanine residue at position 508 (ΔF508) is the most prevalent disease-causing mutation.

ΔF508 CFTR is a well-known example of a clinically relevant TS (temperature-sensitive) processing mutant. At 37 °C, the restrictive temperature, the ΔF508 CFTR protein is rapidly degraded by ERAD [ER (endoplasmic reticulum) associated degradation], preventing ΔF508 CFTR expression at the cell surface and resulting in the CF phenotype [1416]. At 27 °C, the permissive temperature, some of the ΔF508 CFTR protein escapes ERAD and is delivered to the cell membrane, where it is called rΔF508 (rescued ΔF508) CFTR. Because rΔF508 CFTR partially retains its chloride channel activity [17], several methods have been introduced to promote ΔF508 CFTR escape from ERAD and deliver it to the cell membrane [1824], but to date, the most efficient method for the rescue of ΔF508 CFTR is permissive temperature cell culture [25]. Although first observed 15 years ago, it remains unclear how culture at 27 °C facilitates ΔF508 CFTR escape from ER quality control. It is well established, however, that returning cells to the restrictive temperature after low temperature rescue results in rapid internalization and degradation of rΔF508 CFTR [26,27]. It is not known whether rΔF508 CFTR displays the same cell-surface instability, or how function of rΔF508 CFTR is affected, if left at the permissive temperature.

In addition to low-temperature culture, chemical compounds such as glycerol [28], DMSO [29] and organic solutes [24] have also been shown to facilitate ΔF508 CFTR escape from ERAD. These compounds exert their effects by enhancing the efficiency of ΔF508 CFTR folding or by increasing differentiation and polarity of the host cell [29]. Recently, a handful of small molecular correctors were identified by high-throughput screening based on their ability to promote rΔF508 CFTR expression [20,22,30,31]. In most cases, the mechanism by which these compounds facilitate ERAD escape is not known. Furthermore, their effects at the cell surface have not been tested.

Although the effects of low temperature culture on ΔF508 CFTR folding in the ER has been studied for years, it is not known whether the surface defects exhibited by rΔF508 CFTR are also TS, and therefore possibly related to the ER folding defect. Additionally, it has not been determined whether treatment of rΔF508 CFTR with chemical compounds known to promote rescue can affect ΔF508 CFTR cell-surface properties, such as surface stability. Answers to these questions are essential to understand the altered trafficking, decreased stability and compromised function of the rΔF508 CFTR protein. In this present study, we provide more detailed information on the effects of permissive temperature culture and of two small molecule correctors on rΔF508 CFTR cell-surface trafficking. We used two different CFTR-expressing model cell lines, HeLa and CFBE41o cells, in order to identify cell-type- and polarization-specific differences in the cell-surface trafficking of WT (wild-type) CFTR and rΔF508 CFTR, and to determine whether methods known to facilitate ΔF508 CFTR exit from the ER also stabilize rΔF508 CFTR at the cell surface.

EXPERIMENTAL

Cell lines and cell culture

Cells were maintained in a 37 °C humidified incubator in 5% CO2. HeLaΔF (where ΔF indicates a cell line expressing ΔF508 CFTR), HeLaWT, CFBE41oΔF and CFBE41oWT cell lines were developed and cultured as described previously [26]. HeLa cells were grown in Eagle's modification of MEM (minimal essential medium; Invitrogen) supplemented with 10% (v/v) FBS (fetal bovine serum). Calu-3 cells were obtained from A.T.C.C. and were maintained in Eagle's modification of MEM supplemented with 10% (v/v) FBS, 2 mM glutamine, 1 mM pyruvate and 0.1 mM non-essential amino acids. CFBE41o cell cultures were maintained in DMEM (Dulbecco's modified Eagle's medium) Ham's F12 medium (50:50, v/v) (Invitrogen) with 10% (v/v) FBS. For experiments requiring polarized cells, Calu-3 CFBE41oΔF and CFBE41oWT cells were seeded on to 12 mm diameter Transwell filters (Costar, Corning). Under these conditions, the cells formed polarized monolayers with transepithelial resistances of >1000 Ω/cm2, as measured by a Millicell electrical resistance system (Millipore).

Small molecular correctors (pharmacological chaperones)

Small molecular correctors were provided by Cystic Fibrosis Foundation Therapeutics (Bethesda, MD, U.S.A.). Compounds tested were CFcor-325 (VRT-325, 4-cyclohexyloxy-2-{1[4-(4-methoxy-benzensulfonyl)-piperazin-1-yl]-ethyl}-quinazoline) [20,32] and Corr-4a ({2-(5-chloro-2-methoxy-phenylamino)-4′-methyl-[4,5′]-bithiazolyl-2′-yl}-phenyl-methanone) [22]. Both compounds were used at a 10 mM stock concentration in DMSO and a 10 μM working concentration in OPTIMEM medium (Invitrogen) supplemented with 2% (v/v) FBS. The presence of the vehicle (0.1% DMSO) in the medium did not mediate ER escape of ΔF508 CFTR in control samples, or facilitate changes in internalization. In all experiments, control samples contained the DMSO vehicle.

Immunoprecipitation

Cells were lysed in RIPA buffer [1% Nonidet P40, 0.5% sodium deoxycholate, 150 mM NaCl and 50 mM Tris/HCl (pH 8.0)] containing Complete mini protease inhibitor (Roche). CFTR was immunoprecipitated using 1 μg/ml (final concentration) mouse monoclonal anti-CFTR C-terminal antibody (24-1, A.T.C.C. number HB-11947) and 35 μl of Protein A–agarose (Roche) [8]. TR (transferrin receptor) was immunoprecipitated using 1 μg/ml (final concentration) mouse anti-TR (B3/25; Roche) and 35 μl Protein A-agarose [33]. Immunoprecipitations were carried out for 2h at 4 °C.

Internalization assays

CFTR internalization assays were performed as described previously [8,9]. Briefly, surface carbohydrate groups on the cells were oxidized with sodium periodate (NaIO4, 10mM), washed on ice with Mg2+- and Ca2+-supplemented (0.5 mM MgCl2 and 0.9 mM CaCl2) PBS buffer and warmed to 37 °C or 27 °C for 2.5 or 10 min. Oxidized surface carbohydrate groups remaining on the cell surface after a warm-up period were labelled with biotin–LC–hydrazide (1 mg/ml; Pierce), followed by cell lysis in RIPA buffer. CFTR was then immunoprecipitated as described above. CFTR internalization was identified as the percentage loss of biotinylated CFTR during the warm-up period compared with the control samples (no warm-up period).

CFTR cell-surface half-life measurements

Cells were metabolically labelled [8] and biotinylated as described previously [9]. Initial (0 time point) samples were immediately lysed in RIPA buffer, and the rest of the samples were transferred into to a 37 °C incubator for 2, 4 and 8 h. At each time point, cells were lysed in RIPA buffer, CFTR was immunoprecipitated and analysed by SDS/PAGE (6% gels) and Western blotting (see below), and autoradiography was performed following the manufacturer's instructions (PhosphorImager; Amersham Biosciences). Half-lives of the proteins were calculated as described previously [8,34].

Metabolic pulse–chase experiments

For experiments at 37 °C, cells were metabolically labelled as described previously [8]. For metabolic labelling during permissive temperature (27 °C) culture, cells were transferred to 27 °C and the medium replaced with cysteine- and methionine-free MEM (Specialty Media, Phillipsburg, NJ, U.S.A.) for 1 h. Cells were subsequently incubated with 300 μCi/ml [35S]methionine/cysteine (EasyTag™; PerkinElmer) for 18 h at 27 °C. This extended metabolic labelling pulse was necessary to produce a labelled CFTR signal at 27 °C that formed sufficient quantities of mature, glycosylated CFTR (C band) to follow during the chase period. On completion of labelling, the medium was replaced with complete medium without radioisotopes and cells were cultured at either 37 °C or 27 °C for the time points indicated.

Western Blot

Total and biotinylated CFTR or TR were detected as described previously [8]. Briefly, CFTR and TR were immunoprecipitated and analysed by SDS/PAGE (6% gels) and Western blotting. Membranes were blocked overnight in 3% (w/v) BSA and 0.5% Tween 20 in PBS. All antibodies were incubated for 1 h at 25 °C. Total CFTR was detected with polyclonal anti-[CFTR NBD (nucleotide binding domain)2] antibody (1:5000 dilution, H-182; Santa Cruz Biotechnology). Total TR was detected with a polyclonal anti-(TR external domain) antibody (1:5000 dilution; MorphoSys, Raleigh, NC, U.S.A.). Biotinylated CFTR and TR were detected with HRP (horseradish peroxidase)-conjugated avidin (1:5000 dilution; Sigma). Chemiluminescence was induced with high-sensitivity Immobilon Western substrate (Millipore). The membranes were exposed for different time periods (up to 3 min) and a linear range for a standard set of diluted samples was calibrated. Western blots were analysed and densities measured using ImageJ (National Institutes of Health), ScionImage (National Institutes of Health), or IPLab (BD Biosciences) software. Results are means (n≥3).

Ussing chamber analyses

Measurements of Isc (short-circuit current) and Rt (transepithelial resistance) were performed as described previously [35]. Briefly, filters containing monolayers of either CFBE41oΔF or CFBE41oWT cells were mounted in Ussing chambers (Jim's Instruments, Iowa City, IA, U.S.A.) and bathed on both sides with solutions containing 120 mM NaCl, 25 mM NaHCO3, 3.3 mM KH2PO4, 0.83 mM K2HPO4, 1.2 mM CaCl2, 1.2 mM MgCl2, 10 mM Hepes (sodium-free), 10 mM mannitol (apical compartment) and 10 mM glucose (basolateral compartment). Osmolality of all solutions, as measured by a freezing depression osmometer (Wescor, Logan, UT, U.S.A.), was between 290 and 300 mOsm/kg of water. The bath solutions were stirred vigorously by continuous bubbling with 95% O2 and 5% CO2 at 37 °C (pH 7.4). Monolayers were short-circuited to 0 mV, and Isc (μA/cm2) was measured with an epithelial voltage clamp (VCC-600; Physiologic Instruments, San Diego, CA, U.S.A.). A 10 mV pulse of 1 s duration was imposed every 10 s to monitor Rt, which was calculated using Ohm's law. Data were collected using the Acquire and Analyse program (version 1.45; Physiologic Instruments). Results are ΔIsc forskolin (response to forskolin) and ΔIsc glybenclamide (response to glybenclamide). During Ussing chamber analysis, the temperatures (27 °C or 37 °C) were maintained by using a temperature-controlled water bath for at least 30 min before analysis. Following establishment of steady-state values of Isc and Rt, forskolin (10 μM) and/or the indicated inhibitors were added to the apical compartments, and Isc and Rt were measured continuously until new steady-state values were reached.

Statistical analysis

Results are means±S.D. and statistical significances were calculated by using the two-tailed Student's t test.

RESULTS

ΔF508 CFTR rescue by permissive temperature in HeLaΔF cells

In order to study the cell-surface trafficking of rΔF508 CFTR, we first established whether permissive temperature culture of HeLaΔF cells could generate sufficient amounts of fully glycosylated ΔF508 CFTR at the cell surface in order to measure endocytosis rates. Following culture at 27 °C for 48 h, CFTR was immunoprecipitated from whole-cell lysates and analysed for the presence of mature, glycosylated band C (rΔF508 CFTR). The results in Figure 1(A) demonstrate that a significant amount of fully glycosylated CFTR is formed during 27 °C culture. To confirm that the fully glycosylated rΔF508 CFTR was expressed at the cell surface, cells were surface biotinylated using biotin–LC–hydrazide [9]. Biotinylated CFTR was detected in HeLaΔF cells after 27 °C culture, but not in cells maintained at 37 °C (Figure 1A). Control cells expressing WT CFTR served as a comparison in order to illustrate the level of ΔF508 CFTR rescue.

ΔF508 CFTR rescue and stability in HeLa cells

Figure 1
ΔF508 CFTR rescue and stability in HeLa cells

(A) Low temperature (27 °C) rescue of ΔF508 CFTR in HeLaΔF cells. CFTR was immunoprecipitated from HeLaΔF cells that had been cultured at 27 °C or 37 °C for 48 h or HeLaWT cells cultured at 37 °C, analysed by SDS/PAGE, Western blotted and detected using polyclonal anti-(CFTR NBD2) antibody (Total, left-hand panel). Arrows indicate ER (Band B) and post-ER forms (Band C) of CFTR. The presence of rΔF508 CFTR at the cell surface (Band C) was confirmed by cell-surface biotinylation, immunoprecipitation of CFTR and Western blotting with HRP-conjugated avidin (Surface, right-hand panel). (B) WT CFTR and rΔF508 CFTR half-lives at 37 °C. CFTR half-lives were measured in metabolic pulse–chase experiments (see the Experimental section). HeLaΔF and HeLaWT cells were cultured at 27 °C for 24 h, followed by metabolic labelling for 18 h and chased at 37 °C for up to 18 h. At the time points indicated, cells were subjected to lysis, CFTR immunoprecipitation, SDS/PAGE and phosphorimaging. Representative gels of pulse–chase experiments for WT CFTR and rΔF508 CFTR (ΔF508, top and middle panels respectively) and calculated half-lives (bottom panel) are shown (n=4). (C) Extended half-lives of WT CFTR and rΔF508 CFTR at 27 °C. Pulse–chase experiments were performed in HeLaΔF and HeLaWT cells as described in (B), except that the chase was performed at 27 °C (see the Experimental section). Representative images (top and middle panels) and calculated half-lives (bottom panel) are shown (n=4).

Figure 1
ΔF508 CFTR rescue and stability in HeLa cells

(A) Low temperature (27 °C) rescue of ΔF508 CFTR in HeLaΔF cells. CFTR was immunoprecipitated from HeLaΔF cells that had been cultured at 27 °C or 37 °C for 48 h or HeLaWT cells cultured at 37 °C, analysed by SDS/PAGE, Western blotted and detected using polyclonal anti-(CFTR NBD2) antibody (Total, left-hand panel). Arrows indicate ER (Band B) and post-ER forms (Band C) of CFTR. The presence of rΔF508 CFTR at the cell surface (Band C) was confirmed by cell-surface biotinylation, immunoprecipitation of CFTR and Western blotting with HRP-conjugated avidin (Surface, right-hand panel). (B) WT CFTR and rΔF508 CFTR half-lives at 37 °C. CFTR half-lives were measured in metabolic pulse–chase experiments (see the Experimental section). HeLaΔF and HeLaWT cells were cultured at 27 °C for 24 h, followed by metabolic labelling for 18 h and chased at 37 °C for up to 18 h. At the time points indicated, cells were subjected to lysis, CFTR immunoprecipitation, SDS/PAGE and phosphorimaging. Representative gels of pulse–chase experiments for WT CFTR and rΔF508 CFTR (ΔF508, top and middle panels respectively) and calculated half-lives (bottom panel) are shown (n=4). (C) Extended half-lives of WT CFTR and rΔF508 CFTR at 27 °C. Pulse–chase experiments were performed in HeLaΔF and HeLaWT cells as described in (B), except that the chase was performed at 27 °C (see the Experimental section). Representative images (top and middle panels) and calculated half-lives (bottom panel) are shown (n=4).

To determine the stability of rΔF508 CFTR after permissive temperature rescue, WT CFTR- or rΔF508 CFTR-expressing HeLa cells were raised to the restrictive temperature and the protein half-lives of WT CFTR and rΔF508 CFTR were determined by metabolic pulse–chase analysis. For these experiments, cells were cultured at 27 °C for 24 h and metabolically labelled with [35S]methionine for an additional 18 h at 27 °C (see the Experimental section). The cells were then returned to 37 °C, and the labelled proteins were chased for the time periods specified. The results indicate that the half-life of WT CFTR is 12±1.5 h, whereas the half-life of rΔF508 CFTR is 4±1 h (Figure 1B). The rΔF508 CFTR protein half-life is much shorter than that of WT CFTR (P<0.001), and these results are in agreement with previously published experiments performed in BHK (baby-hamster kidney) and CHO (Chinese-hamster ovary) cells [7,36].

Extended half-life of rΔF508 CFTR at the permissive temperature in HeLaΔF cells

Next, we tested whether extended culture at the permissive temperature would affect the half-lives of WT CFTR and rΔF508 CFTR in HeLa cells. We followed the protocol described above (see the Experimental section), but instead of transferring the cells to 37 °C after metabolic labelling, they were incubated at 27 °C for the time periods indicated. The results show that at 27 °C, the half-lives of WT CFTR and rΔF508 CFTR were 60±11 h and 63±9 h respectively (Figure 1C), demonstrating that the instability of rΔF508 CFTR compared with WT CFTR which was observed at the restrictive temperature is not apparent at the permissive temperature; in other words, the rΔF508 CFTR half-life defect is a TS defect.

We next sought to determine why rΔF508 CFTR has a short half-life at the restrictive temperature by examining the internalization properties of WT CFTR and rΔF508 CFTR in HeLa cells and epithelial cells (CFBE41o and Calu-3) under both non-polarized and polarized conditions. These experiments served to test cell-type- and polarization-specific differences in the trafficking of the two proteins.

rΔF508 CFTR endocytosis is accelerated in airway epithelial cells

To determine the cell-surface stability of WT CFTR and rΔF508 CFTR in HeLa cells at 37 °C, we measured their internalization rates using a two-step biotinylation protocol [8]. To measure CFTR endocytosis rates, we oxidized the surface carbohydrate groups of cell surface glycoproteins at the initial (zero) time point (see the Experimental section) and then allowed the proteins to be internalized for 2.5 min, at which time the oxidized glycoproteins remaining at the cell surface were labelled with biotin. An internalization time of 2.5 min was chosen for all assays conducted at 37 °C because, at this temperature, previous internalization time courses in multiple cell lines indicated that periodate-oxidized CFTR modified at the initial time point did not recycle back to the cell surface (results not shown). Since the biotin–LC–hydrazide is membrane impermeable, the only biotin-accessible CFTR is what remains on the cell surface during the warm-up period (see the Experimental section). Therefore changes in the surface pool of CFTR after a 2.5 min warm-up period were reflected in a loss of ‘biotinylatable’ CFTR, and this loss corresponds to the amount of CFTR that had been internalized from the cell surface.

Following biotin labelling, cells were lysed and total CFTR was immunoprecipitated as described above. At each time point, biotinylated CFTR was detected by Western blot with HRP-conjugated avidin. With this protocol, biotin labelling of band B CFTR is not observed unless cells are first permeabilized [9], suggesting that under non-permeabilizing conditions such as those presented herein, this biotinylation method used does not label intracellular proteins. The results of these experiments indicate that both WT CFTR and rΔF508 CFTR endocytosis rates are rapid, with 25±5% and 27±5% of the surface pool internalized in 2.5 min respectively (Figure 2A). This result is consistent with a previous comparison of WT CFTR and rΔF508 CFTR internalization rates in BHK cells [27].

rΔF508 CFTR endocytosis in HeLa and airway epithelial cells.

Figure 2
rΔF508 CFTR endocytosis in HeLa and airway epithelial cells.

(A) Internalization of WT CFTR and rΔF508 CFTR in HeLa cells. CFTR endocytosis was measured using a modified biotinylation assay (see Experimental section). Representative gels of immunoprecipitated (Total) and cell-surface biotinylated WT CFTR and ΔF508 CFTR (Surface) at 0 and 2.5 min of internalization (Int.) are shown. Internalization rates of WT CFTR and rΔF508 CFTR (ΔF508) are plotted as the percentage decrease in CFTR Band C density after a 2.5 min internalization step (n=5). (B) WT CFTR and rΔF508 CFTR internalization in non-polarized and polarized epithelial cells. CFBE41oΔF (ΔF508) and CFBE41oWT (WT) cells grown under non-polarized (plastic dishes, left-hand panels) or polarized (permeable supports, right-hand panels) conditions were tested for CFTR internalization (Int.). Representative gels are shown. Internalization rates are the percentage density decrease in cell surface Band C CFTR after a 2.5 min internalization step compared with 0 min (n=5). (C) WT CFTR internalization in polarized Calu-3 cells. Calu-3 cells grown under polarized conditions (permeable supports) were tested for CFTR internalization. A representative gel showing cell-surface biotinylated WT CFTR (surface) at 0 and 2.5 min of internalization (Int.) is shown (n=4).

Figure 2
rΔF508 CFTR endocytosis in HeLa and airway epithelial cells.

(A) Internalization of WT CFTR and rΔF508 CFTR in HeLa cells. CFTR endocytosis was measured using a modified biotinylation assay (see Experimental section). Representative gels of immunoprecipitated (Total) and cell-surface biotinylated WT CFTR and ΔF508 CFTR (Surface) at 0 and 2.5 min of internalization (Int.) are shown. Internalization rates of WT CFTR and rΔF508 CFTR (ΔF508) are plotted as the percentage decrease in CFTR Band C density after a 2.5 min internalization step (n=5). (B) WT CFTR and rΔF508 CFTR internalization in non-polarized and polarized epithelial cells. CFBE41oΔF (ΔF508) and CFBE41oWT (WT) cells grown under non-polarized (plastic dishes, left-hand panels) or polarized (permeable supports, right-hand panels) conditions were tested for CFTR internalization (Int.). Representative gels are shown. Internalization rates are the percentage density decrease in cell surface Band C CFTR after a 2.5 min internalization step compared with 0 min (n=5). (C) WT CFTR internalization in polarized Calu-3 cells. Calu-3 cells grown under polarized conditions (permeable supports) were tested for CFTR internalization. A representative gel showing cell-surface biotinylated WT CFTR (surface) at 0 and 2.5 min of internalization (Int.) is shown (n=4).

Since in vivo CFTR is expressed on the apical surface in epithelial cells, we next compared the surface stability of WT CFTR and rΔF508 CFTR in both polarized and non-polarized human airway epithelial cells at 37 °C. In CFBE41oWT cells grown on plastic dishes (non-polarized cells), CFTR endocytosis slowed to 14±4% of the surface pool internalized in 2.5 min (P=0.02 compared with HeLaWT). In contrast, 29±5% of rΔF508 CFTR was internalized during the same time period in CFBE41oΔF (Figure 2B), a rate similar to that seen in HeLaΔF cells [P=NS (not significant)]. Furthermore, the difference between WT CFTR and rΔF508 CFTR internalization rates was more pronounced when the cells were grown as polarized monolayers. In polarized cells, WT CFTR internalization dropped significantly to 3.2±2% per 2.5 min (P<0.0001 compared with HeLaWT), whereas rΔF508 CFTR internalization remained at 30±3% per 2.5 min. As a further control, we measured WT CFTR internalization in polarized Calu-3 cells with similar results (5±1% per 2.5 min; P=NS), indicating that the surface pool of WT CFTR is very stable in polarized epithelia (Figure 2C), whereas rΔF508 CFTR is not stable. Because it was clear that WT CFTR trafficking did not appear to be faithfully represented in HeLa cells, and our goal was to identify the defect in rΔF508 CFTR compared with the control WT CFTR protein, we focused our efforts on analysing the rΔF508 CFTR trafficking defect in human airway epithelial cells.

Shortened cell-surface half-life of rΔF508 CFTR in CFBE41oΔF cells

Because polarization affected WT CFTR but not rΔF508 CFTR internalization, we then tested the cell-surface half-lives of both WT CFTR and rΔF508 CFTR in CFBE41o cells using a cell-surface biotinylation-based assay (Figure 3). The results indicate that the surface half-life of biotinylated WT CFTR was 8.5±1 h in non-polarized cells (Figure 3, top left-hand panel) and 8.3±0.3 h in polarized cells (P=NS, Figure 3, top right-hand panel), demonstrating that the stability of the WT CFTR protein was not affected by cell polarization. Examination of rΔF508 CFTR indicated that the surface half-life of the rescued protein was very short (less than 2 h) under both non-polarized and polarized conditions (1.8±0.1 compared with 2.0±0.2 h respectively; P=NS, Figure 3, middle panels). Thus rΔF508 CFTR surface stability is decreased compared with WT CFTR, and polarization did not affect the surface stability of either protein (Figure 3, bottom panels).

Cell-surface half-lives of WT CFTR and rΔF508 CFTR in CFBE41o cells

Figure 3
Cell-surface half-lives of WT CFTR and rΔF508 CFTR in CFBE41o cells

Representative gels are shown of WT CFTR (CFBE WT, top panels) and rΔF508 CFTR (CFBE ΔF508, middle panels) half-life measurements performed at 37 °C under non-polarized and polarized conditions. Half-lives were calculated using densitometry followed by analysis as described previously [34], and the results are shown in the bottom panel (n=4).

Figure 3
Cell-surface half-lives of WT CFTR and rΔF508 CFTR in CFBE41o cells

Representative gels are shown of WT CFTR (CFBE WT, top panels) and rΔF508 CFTR (CFBE ΔF508, middle panels) half-life measurements performed at 37 °C under non-polarized and polarized conditions. Half-lives were calculated using densitometry followed by analysis as described previously [34], and the results are shown in the bottom panel (n=4).

Permissive temperature culture stabilizes rΔF508 CFTR in polarized epithelial cells

Since the rΔF508 CFTR trafficking defect in epithelial cells is the result of enhanced endocytosis, we tested whether permissive temperature culture could correct this defect. To answer this question, we compared the effects of 27 °C treatment on WT CFTR and rΔF508 CFTR cell-surface trafficking in polarized CFBE41o cells using cell-surface half-life and internalization experiments. The results show that both WT CFTR and rΔF508 CFTR are extremely stable at the cell surface at 27 °C, with cell-surface half-lives much greater than 8 h (Figure 4, top panels). Because CFTR levels were not monitored beyond 8 h, we cannot calculate to what extent permissive temperature culture stabilized WT CFTR or rΔF508 CFTR. However, since WT CFTR and rΔF508 CFTR surface half-life measurements were similar at 27 °C, these results reveal that 27 °C treatment eliminated the drastic difference in surface half-life between WT CFTR and rΔF508 CFTR observed at 37 °C. Measurement of WT CFTR and rΔF508 CFTR internalization rates at 27 °C revealed a dramatic decrease in both cell lines. In fact, CFTR internalization was not measurable after the 2.5 min warm-up period. After a 10 min warm-up period, WT CFTR and rΔF508 CFTR internalization rates were 20±3% per 10 min (P=0.05) and 22±4% per 10 min (P=0.0004) respectively (Figure 4, middle and bottom panels), which indicated that 27 °C treatment eliminated the difference in internalization rates between WT CFTR and rΔF508 CFTR. Importantly, the results of these studies indicate that both the short surface half-life and the rapid internalization rate of rΔF508 CFTR are TS defects.

Surface stability of rΔF508 CFTR at 27 °C in airway epithelial cells

Figure 4
Surface stability of rΔF508 CFTR at 27 °C in airway epithelial cells

Cell surface stability (top panels) and internalization rates (middle panels) of CFTR were measured at 27 °C (see Experimental section). In polarized CFBE41o cells, both WT CFTR and rΔF508 CFTR are extremely stable at 27 °C. There was no detectable decrease in biotinylated Band C CFTR during the 8 h chase. Representative gels from CFBE41oWT and CFBE41oΔF (ΔF508) are shown (top panels). For internalization rates, total and cell surface CFTR were detected. Representative gels are shown (middle panels). Internalization rates are plotted as the percentage decrease in density of Band C CFTR at 10 min (bottom panel; n=4). Biot., biotinylated CFTR.

Figure 4
Surface stability of rΔF508 CFTR at 27 °C in airway epithelial cells

Cell surface stability (top panels) and internalization rates (middle panels) of CFTR were measured at 27 °C (see Experimental section). In polarized CFBE41o cells, both WT CFTR and rΔF508 CFTR are extremely stable at 27 °C. There was no detectable decrease in biotinylated Band C CFTR during the 8 h chase. Representative gels from CFBE41oWT and CFBE41oΔF (ΔF508) are shown (top panels). For internalization rates, total and cell surface CFTR were detected. Representative gels are shown (middle panels). Internalization rates are plotted as the percentage decrease in density of Band C CFTR at 10 min (bottom panel; n=4). Biot., biotinylated CFTR.

Permissive temperature culture corrects the functional defect associated with rΔF508 CFTR

In addition to the trafficking defect, rΔF508 CFTR fails to respond to cAMP after forskolin stimulation in CFBE41oΔF cells [26]. Since we observed that permissive temperature culture corrects the endocytosis defect in rΔF508 CFTR and restores the protein half-life to levels equivalent to WT CFTR, we then tested whether it might also correct the functional defect [26]. In these experiments, polarized CFTR-expressing CFBE41oΔF monolayers were cultured at 27 °C for 48 h to facilitate rΔF508 CFTR expression on the cell surface. These monolayers were then mounted in Ussing chambers, where cAMP-activated Isc was measured at either 37 °C or 27 °C. Forskolin (10 μM) was added to the apical compartment to enhance intracellular cAMP levels and activate Isc. When the current reached a maximum and stabilized, glybenclamide was added at increasing concentrations to block Isc. In some experiments, a CFTR-specific inhibitor was used to block currents [23], as described previously [35]. Forskolin was used consistently to activate Isc because we found that, in both WT CFTR- and ΔF508 CFTR-expressing cells, it was sufficient to stimulate cAMP-mediated chloride currents. A cocktail designed to maximally stimulate cAMP responses [forskolin, IBMX (3-isobutyl-1-methylxanthine) and bromoadenosine–cAMP] did not increase the current beyond the forskolin-induced current. Likewise, glybenclamide was used consistently because the CFTR-specific inhibitor did not further inhibit the currents, indicating that glybenclamide provided maximal CFTR channel inhibition (results not shown). The magnitude of the resulting Isc was compared with parallel CFBE41oWT controls (see the Experimental section). The results show that at 37 °C, cells expressing WT CFTR channels produce an anion current that is readily stimulated by forskolin and can be inhibited completely by glybenclamide (Figure 5, top left-hand panel), whereas cells expressing rΔF508 CFTR exhibit very weak responses to both treatments (Figure 5, middle left-hand panel), in agreement with our previous findings [26]. When the temperature is maintained at 27 °C, however, rΔF508 CFTR-expressing cells exhibit an anion current similar to cells expressing WT CFTR (Figure 5, top and middle right-hand panels). Direct channel stimulation by 50 μM genistein further enhanced the rΔF508 CFTR current at 27 °C (results not shown), in agreement with a previous observation that at 37 °C, rΔF508 CFTR produced a current in response to genistein [29]. This restoration of the functional defect suggests that the loss of cAMP response has been regained at 27 °C. In consideration with the previous studies, these experiments reveal that both the surface trafficking and functional activity defects of rΔF508 CFTR are TS.

Ussing chamber analysis of rΔF508 CFTR after low temperature correction

Figure 5
Ussing chamber analysis of rΔF508 CFTR after low temperature correction

Polarized CFBE41oWT and CFBE41oΔF (ΔF508) monolayers were cultured at 27 °C for 48 h. Cells were mounted in Ussing chambers and temperature-equilibrated (37 °C or 27 °C as indicated) for 30 min, followed by measurement of baseline steady state Isc and Rt values. Forskolin (FSK, 10 μM) was added to the apical chambers, and Isc and Rt values were monitored until a new baseline was obtained. The indicated concentrations of glybenclamide (GLYB) were then added apically, and Isc and Rt values were monitored. Representative traces (top and middle panels) are shown. Results are ΔIsc (μA/cm2) of FSK (which represents a positive current following forskolin activation) or of GLYB, which represents a negative current following administration of glybenclamide to block channel activity (bottom panels; n≥5). ΔF508 CFTR-expressing monolayers exhibited significantly blunted responses to forskolin and glybenclamide compared with WT CFTR-expressing monolayers at 37 °C (bottom panels). These responses were significantly enhanced when monolayers were maintained at the permissive temperature (27 °C, *P<0.005; +P<0.01), so that there were no significant differences between WT CFTR and rΔF508 CFTR responses at this temperature (bottom panels). For each condition, the number of experimental repeats is indicated in parentheses.

Figure 5
Ussing chamber analysis of rΔF508 CFTR after low temperature correction

Polarized CFBE41oWT and CFBE41oΔF (ΔF508) monolayers were cultured at 27 °C for 48 h. Cells were mounted in Ussing chambers and temperature-equilibrated (37 °C or 27 °C as indicated) for 30 min, followed by measurement of baseline steady state Isc and Rt values. Forskolin (FSK, 10 μM) was added to the apical chambers, and Isc and Rt values were monitored until a new baseline was obtained. The indicated concentrations of glybenclamide (GLYB) were then added apically, and Isc and Rt values were monitored. Representative traces (top and middle panels) are shown. Results are ΔIsc (μA/cm2) of FSK (which represents a positive current following forskolin activation) or of GLYB, which represents a negative current following administration of glybenclamide to block channel activity (bottom panels; n≥5). ΔF508 CFTR-expressing monolayers exhibited significantly blunted responses to forskolin and glybenclamide compared with WT CFTR-expressing monolayers at 37 °C (bottom panels). These responses were significantly enhanced when monolayers were maintained at the permissive temperature (27 °C, *P<0.005; +P<0.01), so that there were no significant differences between WT CFTR and rΔF508 CFTR responses at this temperature (bottom panels). For each condition, the number of experimental repeats is indicated in parentheses.

Pharmacological chaperones correct the internalization defect and increase the surface stability of rΔF508 CFTR

As shown above, maintenance of the TS ΔF508 protein at the permissive temperature corrects multiple trafficking and functional defects, in that 27 °C treatment not only rescues ΔF508 CFTR from ERAD, but also stabilizes the endocytosis and surface stability defects as long as cells are maintained at 27 °C. On the basis of these results, we investigated whether two pharmacological chaperones which facilitate ΔF508 CFTR exit from the ER, a quinazoline compound (CFcor-325) and a bisaminomethylbithiazole compound (Corr-4a) [20,31], have any effect on rΔF508 CFTR or WT CFTR cell-surface trafficking at 37 °C.

First, we studied the effect of the compounds on WT CFTR and rΔF508 CFTR endocytosis in CFBE41o cells. For these experiments, CFBE41oWT or ΔF cells were cultured for 48 h at 27 °C, followed by a 1 h pre-treatment with 10 μM CFcor-325, Corr-4a, or a vehicle control (DMSO) at 37 °C. Internalization assays were then performed at 37 °C. The results indicated that both CFcor-325 and Corr-4a decreased the internalization rate of rΔF508 CFTR from 30% to ∼5% and ∼1% respectively (P<0.005, Figure 6, top and middle panels). Interestingly, the compounds had no effect on WT CFTR endocytosis or TR endocytosis from the apical surface (Figure 6, bottom panels), suggesting that the effect was specific for rΔF508 CFTR.

CFcor-325 and Corr-4a increase the stability of WT CFTR and rΔF508 CFTR

Figure 6
CFcor-325 and Corr-4a increase the stability of WT CFTR and rΔF508 CFTR

Internalization assays for WT CFTR (WT) and rΔF508 CFTR (ΔF508) were performed at 37 °C in CFBE41oΔF and CFBE41oWT cells following low temperature rescue in the presence or absence of CFcor-325 or Corr-4a. Representative gels (top panels) and the percentage of CFTR internalized for each corrector is shown (middle panels; n=4). The percentage of TR internalized, measured under identical conditions, is also shown (bottom panels). WT CFTR and TR internalization rates were tested as controls and ∼5% of WT CFTR and ∼30% of rescued ΔF508 CFTR was internalized in 2.5 min in untreated cells. Both CFcor-325 and Corr-4a treatment significantly decreased rΔF508 CFTR internalization in CFBE41oΔF cells (n=4; P<0.05). No changes in WT CFTR and TR internalization rates were measured.

Figure 6
CFcor-325 and Corr-4a increase the stability of WT CFTR and rΔF508 CFTR

Internalization assays for WT CFTR (WT) and rΔF508 CFTR (ΔF508) were performed at 37 °C in CFBE41oΔF and CFBE41oWT cells following low temperature rescue in the presence or absence of CFcor-325 or Corr-4a. Representative gels (top panels) and the percentage of CFTR internalized for each corrector is shown (middle panels; n=4). The percentage of TR internalized, measured under identical conditions, is also shown (bottom panels). WT CFTR and TR internalization rates were tested as controls and ∼5% of WT CFTR and ∼30% of rescued ΔF508 CFTR was internalized in 2.5 min in untreated cells. Both CFcor-325 and Corr-4a treatment significantly decreased rΔF508 CFTR internalization in CFBE41oΔF cells (n=4; P<0.05). No changes in WT CFTR and TR internalization rates were measured.

Pharmacological chaperones extend the cell-surface half-life of rΔF508 CFTR

Next, we monitored the effects of CFcor-325 or Corr-4a on the cell-surface half-life of WT CFTR and rΔF508 CFTR. For these experiments, CFBE41oWT or ΔF cells were cultured for 48 h at 27 °C, followed by treatment with 10 μM CFcor-325, Corr-4a or a vehicle control (DMSO) at 37 °C. CFTR cell-surface half-lives were then evaluated in the presence of correctors or vehicle using the surface biotinylation-based assay. The results indicated that treatment with either small molecule corrector stabilized rΔF508 CFTR compared with untreated controls (Figure 7). CFcor-325 extended the half-life of rΔF508 CFTR from 2.5±0.4 h to 4.6±0.9 h (P=0.004), and Corr-4a extended the half-life from 2.6±0.6 h to 4.5±1.2 h (P=0.03), indicating that both compounds stabilized the half-life of rΔF508 CFTR. Significantly, neither compound affected the half-lives of WT CFTR nor TR (Figure 7, bottom panels), suggesting the effects observed are specific for rΔF508 CFTR.

CFcor-325 and Corr-4a extend the cell-surface half-life of rΔF508 CFTR

Figure 7
CFcor-325 and Corr-4a extend the cell-surface half-life of rΔF508 CFTR

Polarized CFBE41oΔF (ΔF508) and CFBE41oWT (WT) cells were cultured at 27 °C for 48 h, returned to 37 °C, and treated with CFcor-325 or Corr-4a for 8 h. Cell-surface CFTR half-lives were measured following cell-surface biotinylation (see Experimental section). Representative gels (top panels) and the mean CFTR half-life for each pharmacological agent and untreated control (middle panels; n=4) are shown. The TR half-life was also measured in the presence or absence of correctors as an additional control (bottom panels; n=4).

Figure 7
CFcor-325 and Corr-4a extend the cell-surface half-life of rΔF508 CFTR

Polarized CFBE41oΔF (ΔF508) and CFBE41oWT (WT) cells were cultured at 27 °C for 48 h, returned to 37 °C, and treated with CFcor-325 or Corr-4a for 8 h. Cell-surface CFTR half-lives were measured following cell-surface biotinylation (see Experimental section). Representative gels (top panels) and the mean CFTR half-life for each pharmacological agent and untreated control (middle panels; n=4) are shown. The TR half-life was also measured in the presence or absence of correctors as an additional control (bottom panels; n=4).

DISCUSSION

The fate of rΔF508 CFTR at the cell surface after low temperature rescue is one of the first examples of how the cellular quality-control mechanisms operate at the plasma membrane and/or early endosomes [27]. Although culture at the permissive temperature allows some of the TS ΔF508 CFTR protein to escape from ERAD, this maturely glycosylated rΔF508 CFTR is rapidly degraded once the temperature is raised to the restrictive temperature, 37 °C. The goal of this study was to follow the cell surface fate of rΔF508 CFTR at the permissive and restrictive temperatures, 27 °C and 37 °C, and compare the results with the WT CFTR protein. Since CFTR is normally expressed in epithelial cells, we also examined the fate of both proteins in polarized epithelia to determine whether any epithelial-specific differences exist between WT CFTR and rΔF508 CFTR surface trafficking. Additionally, we performed functional studies to measure transepithelial chloride currents in response to physiological stimuli, such as cAMP. Finally, on the observation that permissive temperature treatment stabilizes and functionally corrects rΔF508 CFTR at the cell surface, we tested whether pharmacological chaperones that permit escape from ERAD stabilize the rΔF508 CFTR surface pool.

A number of studies have shown that culturing cells at 27 °C is an efficient method of facilitating ΔF508 CFTR delivery to the cell surface [7,10,1820,22,2427,31,36]. However, the fate of the low temperature-cultured rΔF508 CFTR at the cell surface has only been followed at 37 °C [10,26,27]. Here, we show that when the cells are kept at 27 °C, the stability of rΔF508 CFTR is enhanced, and the differences between WT CFTR and rΔF508 CFTR trafficking and half-life that are seen at 37 °C disappear. One potential explanation for this observation is that protein degradation slows down at the permissive temperature, resulting in accumulation of both the WT CFTR and ΔF508 CFTR. This hypothesis is supported by the finding that ubiquitination of rΔF508 CFTR is inhibited at 28 °C [27]. However, another possibility is that at 27 °C, ΔF508 CFTR folds properly and remains in a properly-folded state, resulting not only in exit from the ER, but also in a more stable surface phenotype at this temperature. When cells are returned to 37 °C, the conformation of rΔF508 CFTR reverts to a misfolded stage, as proposed previously [36]. In this misfolded conformation, proteins are more likely to be accessible to ubiquitination and subsequent degradation by cell-surface-associated mechanisms.

The endocytosis defect of rΔF508 CFTR is eliminated at 27 °C, but our results indicate that endocytosis still occurs, albeit more slowly. In a number of cell types, temperatures between 16 °C and 22 °C block degradation of endocytosed proteins by preventing their transport to lysosomes [3739]. ΔF508 CFTR has been shown to accumulate in endocytic-like structures similar to the WT CFTR protein when the cells were shifted to 16 °C [7], consistent with the idea that the early part of the endocytic pathway still operates at this temperature, but delivery to the later stages is blocked. It remains unclear whether transport to the lysosome, lysosomal processing or the initial steps of ubiquitin-dependent endocytosis are still functional at 27 °C, but it is clear from our results here that internalization of rΔF508 CFTR is dramatically slowed down to WT CFTR levels at 27 °C, which is consistent with the latter possibility. Further support for this idea comes from proteasomal inhibition studies using lactacystin in BHK cells expressing rΔF508 CFTR [7]. In these studies, lactacystin treatment dramatically stabilized the surface pool of rΔF508 CFTR [7], consistent with the idea that the free ubiquitin pool is limited during proteasomal inhibition.

One important result from our study is that the surface defect of rΔF508 CFTR is due to an enhanced endocytosis rate compared with WT CFTR. Interestingly, we have also shown here that this defect is only present in polarized epithelial cells, such as CFBE41o cells, and not in HeLa cells. Our results demonstrate that WT CFTR endocytosis is dependent both on cellular background and on polarization. In non-polarized HeLa cells, more than 30% of WT CFTR internalizes within 2.5 min. In CFBE41oWT and Calu-3 cells grown on plastic dishes (non-polarized cells), CFTR internalization slows down to ∼15%. When the cells are grown on permeable supports (polarized), CFTR internalization is only 2–5% in 2.5 min.

It is tempting to speculate that WT CFTR internalization rates are so low in polarized epithelia because the majority of WT CFTR is anchored to the cytoskeleton and does not participate in the internalization process. This notion is consistent with the results of Haggie et al. [40], in which single-particle tracking was used to demonstrate that CFTR is coupled to the actin cytoskeleton via EBP50 (ezrin/radixin/moesin-binding phosphoprotein-50)/ezrin and immobilized at the plasma membrane.

The difference we observed in WT CFTR internalization rates in polarized epithelia differs from our results in HeLa cells and from the work of Lukacs and co-workers in BHK cells [27]. One explanation for these discordant results is that in non-polarized cells, the cell membrane and the cytoskeleton are less organized compared with polarized cells, resulting in inefficient tethering of WT CFTR and an increased mobile pool available for endocytosis.

Importantly, we observed that the trafficking of rΔF508 CFTR did not follow the same kinetics as WT CFTR in polarized epithelial cells, at least at 37 °C. At this temperature, regardless of cell line or polarization, rΔF508 CFTR endocytosis rates remain consistently high (∼30% in 2.5 min). One possible explanation for this observation is that, in contrast to our model for WT CFTR, the majority of rΔF508 CFTR is not tethered properly to the cytoskeleton, resulting in a more mobile surface pool. This inefficient cytoskeletal tethering could result from the inability of the mutant protein to interact with one or more of the cytosolic factors responsible for stabilization of the WT CFTR protein. The idea that rΔF508 CFTR is not in a large macromolecular complex is supported by the observation that rΔF508 CFTR is poorly responsive to cAMP-mediated stimuli in CFBE41oΔF cells at 37 °C [26], suggesting that rΔF508 CFTR has lost the association with the previously identified apical signalling complex [7,26,41]. Alternatively, it is possible that, after rescue from ERAD at the permissive temperature, rΔF508 CFTR reverts to a misfolded state when cells are returned to the restrictive temperature. The resulting conformational alterations in the protein may enhance its ability to interact with the clathrin-based endocytic machinery and/or cell-surface-associated ubiquitination machinery. The consequence is that rΔF508 CFTR may be endocytosed and degraded in the lysosome more actively than the WT CFTR protein at 37 °C.

Because improved surface half-life does not necessarily translate to improved channel activity, it is essential to note that the biochemical rescue mediated by permissive temperature culture is accompanied by a functional correction. We showed that after permissive temperature rescue, continued culture at 27 °C results in rΔF508 CFTR channels which are functionally similar to WT CFTR channels in their ability to respond to physiological stimuli such as forskolin. This functional correction also supports the idea that at the permissive temperature, the rescued protein is associated with the functional complex [26] that allows activation by cAMP. The idea that the channel activity is TS is further supported by a recent report on ΔF508 CFTR with two altered RXR motifs that demonstrated that the single channel activity of rΔF508 CFTR decreased as the temperature was increased from 30 °C to 37 °C [42].

Since extended culture at the permissive temperature slowed the endocytosis rates of rΔF508 CFTR to WT CFTR levels in addition to mediating its escape from ERAD, an obvious question was whether chemical correctors that facilitate ΔF508 CFTR exit from the ER would also provide rΔF508 CFTR cell-surface stability at 37 °C. The results illustrated that two small molecules known to rescue ΔF508 CFTR from ERAD, CFcor-325 and Corr-4a, also stabilized the mutant protein at the cell surface. Importantly, these compounds had no effect on endocytosis or the protein half-life of two other cell-surface molecules, WT CFTR and TR, suggesting that the effects are rΔF508 CFTR-specific. Further support for the idea that the compounds are specific was provided by a recent report by Clarke and co-workers demonstrating that these compounds interact directly with CFTR [43].

A significant result of this study is that both compounds corrected the rΔF508 CFTR internalization defect to WT CFTR rates. Despite this effect, although the surface half-life of the rescued protein increased with these compounds, the corrected half-life remained significantly shorter than WT CFTR. This result indicates that correcting the internalization defect of the rΔF508 CFTR protein may not be sufficient for optimal stabilization, and supports previous findings that impaired recycling is a critical component in the compromised surface stability of rΔF508 CFTR [27].

In summary, in polarized epithelial cells, permissive temperature culture of a TS mutant, ΔF508 CFTR, not only rescues it from ERAD, but also stabilizes it at the cell surface and restores its cAMP responsiveness, suggesting that both the stability and the functional defects at the cell surface are TS, not just the maturation defect. Two pharmacological chaperones, CFcor-325 and Corr-4a, mediate an effect similar to permissive temperature treatment, but the rΔF508 CFTR correction of cell-surface half-life is only partial. Our results indicate that permissive temperature culture of a clinically-relevant TS ER processing mutant can facilitate correction of both cell-surface trafficking and functional defects, and offer hope that the treatments that enhance the release of misfolded proteins from the ER may also benefit protein stability and function at the cell surface. Understanding this process in more detail will provide a broader base of knowledge of how pharmacological chaperones and different classes of compounds can be used to promote protein function and stability of this and other TS mutations.

We thank Dr Robert Bridges (Department of Physiology and Biophysics, Rosalind Franklin University of Medicine and Science, North Chicago, IL, U.S.A.), Dr Melissa Ashlock (Cystic Fibrosis Foundation Therapeutics, Bethesda, MD, U.S.A.) and Cystic Fibrosis Foundation Therapeutics for providing the pharmaceutical correctors. This work was supported by NIH (National Institutes of Health) grants DK60065 (to J. F. C.), HL076587 (to Z. B.), HL075540 (to S. M.), P30 DK072402 (to E. J. S.) and by a Cystic Fibrosis Foundation grant CFF R464-CR07 (to E. J. S.).

Abbreviations

     
  • BHK

    baby-hamster kidney

  •  
  • CF

    cystic fibrosis

  •  
  • CFTR

    CF transmembrane conductance regulator

  •  
  • ER

    endoplasmic reticulum

  •  
  • ERAD

    ER-associated degradation

  •  
  • DF

    indicates cell line expressing ΔF508 CFTR

  •  
  • FBS

    fetal bovine serum

  •  
  • HRP

    horseradish peroxidase

  •  
  • Isc

    short-circuit current

  •  
  • MEM

    minimal essential medium

  •  
  • NS

    not significant

  •  
  • rΔF508

    rescued ΔF508

  •  
  • Rt

    transepithelial resistance

  •  
  • TR

    transferrin receptor

  •  
  • TS

    temperature-sensitive

  •  
  • WT

    wild-type

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Author notes

1

These authors contributed equally to this work.