Organic Solutes Rescue the Functional Defect in ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator*

The most common defect in cystic fibrosis, deletion of phenylalanine from position 508 of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR), decreases the trafficking of this protein to the cell surface membrane. Previous studies have shown that low temperature and high concentrations of glycerol or trimethylamine N-oxide can partially counteract the processing defect of ΔF508 CFTR. The present study investigates whether physiologically relevant concentrations of organic solutes, accumulated by cotransporter proteins, can rescue the misprocessing of ΔF508 CFTR. Myoinositol alone or myoinositol, betaine, and taurine given sequentially increased the processing of core-glycosylated, endoplasmic reticulum-arrested ΔF508 CFTR into the fully glycosylated form of CFTR in IB3 cells or NIH 3T3 cells stably expressing ΔF508 CFTR. Pulse-chase experiments using transiently transfected COS7 cells demonstrated that organic solutes also increased the processing of the core-glycosylated form of green fluorescent protein-ΔF508 CFTR. Moreover, the prolonged half-life of the complex-glycosylated form of GFP-ΔF508 CFTR suggests that this treatment stabilized the mature form of the protein. In vitro studies of purified NBD1 stability and aggregation showed that myoinositol stabilized both the ΔF508 and wild type CFTR and inhibited ΔF508 misfolding. Most significantly, treatment of CF bronchial airway cells with these transportable organic solutes restores cAMP-stimulated single channel activity of both CFTR and outwardly rectifying chloride channel in the cell surface membrane and also restores a forskolin-stimulated macroscopic 36Cl- efflux. We conclude that organic solutes can repair CFTR functions by enhancing the processing of ΔF508 CFTR to the plasma membrane by stabilizing the complex-glycosylated form of ΔF508 CFTR.

data suggesting that organic solutes can be used in much lower concentrations than previously suspected, by utilizing the activity of endogenous organic solute cotransporters that actively accumulate these solutes. These accumulated solutes not only correct the chloride channel function of CFTR but also promote ORCC function.

MATERIALS AND METHODS
Antibodies-Antibody 169, a rabbit anti-human CFTR R domain polyclonal antibody generated to peptide IEEDSDEPLERRLSLVPDSEQGE, was a gift from Dr. William Guggino (The Johns Hopkins University). This antibody recognized both the core glycosylated band (band B, ϳ160 kDa) and the fully glycosylated form of CFTR (band C, ϳ180 kDa) in T84 cells, HBE cells, and NIH 3T3 cells transfected with WT CFTR. In IB3 cells or NIH 3T3 cells transfected with ⌬F508 CFTR, the major band detected was the core glycosylated B band. Human CFTR C-terminal monoclonal antibody from R&D Systems (Minneapolis, MN) (MAB25031) was used to confirm the results in many experiments and in all experiments gave bands of the same molecular weight as antibody 169. The monoclonal antibodies to the Na,K-ATPase ␣ 1 subunit and ␤-actin were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY) (05-369) and Sigma (A5441), respectively. The monoclonal antibody to glyceraldehyde-3-phosphate dehydrogenase was a gift from Dr. Michael Sirover (Temple University). The horseradish peroxidase-conjugated anti-mouse antibody was from Amersham Biosciences. The polyclonal GFP antibody was from BD Biosciences (8372-2).
Cell Culture-The IB3-1 (IB3) cell line is an SV40-transformed line derived from the bronchial epithelium of a cystic fibrosis patient with the ⌬F508/W1282X genotype (X ϭ stop mutation). The W1282X mutation produces an unstable mRNA (16). IB3 cells were grown in T75 culture flasks in LHC-8 media (Biofluids, Rockville, MD) supplemented with 5% fetal bovine serum (Invitrogen), 100 units/ml penicillin/streptomycin and 2.5 g/ml fungizone (Invitrogen). T84 cells, a human colonic epithelia cell line obtained from ATCC, were grown in 1:1 Dulbecco's modified Eagle's medium/F-12 (Invitrogen) supplemented with 10% fetal bovine serum and 100 units/ml penicillin/streptomycin. NIH 3T3 cells stably transfected with wide-type CFTR or ⌬F508 CFTR (gift from Dr. M. Welsh) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 100 units/ml penicillin/streptomycin. Cells were routinely maintained in 5% CO 2 incubators at 37°C. When cells were treated, organic solutes were added to the routine growth medium. The addition of 10 mM solute marginally increased the osmolality of growth culture medium from 300 mosmol/kg H 2 O to 310 mosmol/kg H 2 O, as measured by a vapor pressure osmometer (Wescor Inc., Logan UT). COS7 cells were obtained from ATCC and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Organic solutes were administered to cells 24 h post-transfection.
Immunoprecipitation and Phosphorylation of CFTR-Biochemical analysis of CFTR expression and glycosylation was performed by immunoprecipitation with anti-CFTR antibodies followed by in vitro phosphorylation using protein kinase A and [␥-32 P]ATP (Fig. 1). The procedures were described previously (3,7,17). Briefly, cells grown under indicated conditions were rinsed twice with phosphate-buffered saline (Sigma) and scraped into lysis buffer (20 mM HEPES, pH 7.0, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40) containing 1 mM ␤-glycerol phosphate, 1 mM L-phenylalanine, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and the following protease inhibitors: pepstatin A (2 g/ml), leupeptin (10 g/ml), aprotinin (10 g/ml), elastinal (4 g/ml), benzamidine (0.5 mg/ml), 1 mM phenylmethanesulfonyl fluoride, 0.4 mM iodoacetic acid, 2.5 mM phenanthroline and 0.1 mM N-tosyl-L-phenylalanine chloromethyl ketone (all from Sigma). The cells were then homogenized in lysis buffer, placed on ice for 60 min, and then centrifuged for 30 s at 10,000 ϫ g in an Eppendorf bench top centrifuge. The protein in the supernatant was quantified with the BCA protein assay kit (Pierce) and stored at Ϫ80°C. One milligram of lysate protein in 1 ml of lysis buffer with protease inhibitors was precleared with 2 l of normal rabbit serum (used only for polyclonal antibody 169) and 30 l of Protein A-Sepharose beads (Amersham Biosciences) at 4°C for 2 h and centrifuged at 10,000 ϫ g to remove nonspecific complexes. Subsequently, either 2 l of antibody 169, a rabbit polyclonal anti-human CFTR R domain antibody, or 1 g of mouse monoclonal C-terminal antibody (IgG 2a ) were added and incubated at 4°C overnight. To pull down the antigen-antibody complexes, equivalent amounts of Protein A beads were added to each reaction tube and incubated at 4°C for 60 min. The antigen-antibody-bead complex was pelleted by a brief spin and then washed five times for 10 min each time with 1 ml of lysis buffer. The immunoprecipitates were then washed once with 1 ml of Tris-buffered saline, pH 8.0, and incubated with 5 units of the catalytic subunit of protein kinase A (Sigma) and 10 Ci of [␥-32 P]ATP (PerkinElmer Life Sciences) in 50 l of PKA buffer (50 mM Tris, pH 7.5, 10 mM MgCl 2 , 0.1 mg/ml bovine serum albumin) at 30°C for 1 h. Following two washes with lysis buffer, the immunoprecipitates were resuspended in 40 l of Laemmli sample buffer (Bio-Rad) and incubated at 65°C for 4 min. The sample was spun 2 min at 8,000 ϫ g, and the supernatant was either stored at Ϫ20°C overnight or loaded directly onto a gel. The proteins were separated on 5% SDS-polyacrylamide gels (Bio-Rad) and prepared for autoradiography. Exposure time was 30 -60 min at Ϫ80°C.
Western Blotting-Cell lysates were mixed with Laemmli sample buffer and incubated at room temperature for 60 min. After separating on either 5 or 7.5% SDS-polyacrylamide gels, proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad) in Tris/glycine transfer buffer (Bio-Rad) containing 10% methanol. The membrane was blocked with 5% nonfat milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS) for 1 h at room temperature and then was incubated overnight with primary antibodies in the blocking buffer. The primary antibodies were diluted 1:400 for glyceraldehyde-3-phosphate dehydrogenase, 1:2,000 for actin, and 1:20,000 for Na,K-ATPase ␣ subunit. The membrane was washed three times with TTBS for 10 min each wash and incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse antibody diluted 1:10,000. After washing, the blots were visualized using the SuperSignal West Dura substrate (Pierce).
Pulse Chase and Immunoprecipitation-Experiments were performed utilizing COS7 cells transiently transfected with GFP-⌬F508 CFTR or wild type GFP-CFTR and grown in T75 flasks. The GFP expression plasmids were a gift from Dr. Bruce Stanton (Dartmouth Medical School, Hanover, NH). Previous data showed that the addition of GFP to the N terminus did not affect CFTR trafficking or degradation (18,19). Transfections were performed with LipofectAMINE 2000 (Invitrogen) following the manufacturer's instructions. Cells were rinsed three times and then starved for 1 h at 37°C in methionine-and cysteine-free Dulbecco's modified Eagle's medium containing 10% dialyzed fetal bovine serum (Hyclone, Logan, UT). Cells were pulse-labeled with 500 Ci/ml [ 35 S]methionine and [ 35 S]cysteine (ICN Biomedical, Irvine, CA) for 15 min and then chased with prewarmed Dulbecco's modified Eagle's medium supplemented with 10 mM methionine and 4 mM cysteine for the indicated times. At each time point, the cells were placed on ice, rinsed twice with ice-cold PBS, and lysed in lysis buffer supplemented with a protease inhibitor mixture (Roche Applied Science) for 30 min. Wild type GFP-CFTR or GFP-⌬F508 CFTR was immunoprecipitated using a polyclonal antibody to GFP (8372-2; BD Biosciences Clontech, Palo Alto, CA) from cell lysates precleared for 2 h with Protein A-Sepharose fast flow beads (Amersham Biosciences). The immunoprecipitation products were eluted with sample buffer and separated on a 5% SDS-polyacrylamide gel. Following electrophoresis, the gel was fixed (isopropyl alcohol/water/acetic acid, 25:65:10), enhanced with Amplify (Amersham Biosciences) for 30 min, dried for autoradiography, and read on a phosphoimager (ImageQuant 5.0).
In Vitro Folding and Aggregation-The vectors directing expression of His-tagged CFTR NBD1 and NBD1⌬F are similar to the pET 28aderived vectors described previously (20), except they contain residues 388 -655 (NBD 388 ) or 419-655 (NBD 419 ) of human CFTR. The NBD 388 proteins include all of the residues from the crystal structure of the recently solved CFTR NBD except for a C-terminal helix, which, until recently, has been ascribed to the R-domain. 2 The NBD 419 proteins include the entire F1-like core and the ␣-helical subdomains of the domain but lack a ␤-strand from the ␤-sheet subdomain at their extreme N termini as well as the C-terminal helix. The purified NBD proteins were refolded overnight at 4°C in 100 mM Tris, pH 7.9, 375 mM L-arginine, 2 mM EDTA, 1 mM dithiothreitol, 200 mM guanidine HCl at a final concentration of 4 M and subsequently used to determine thermal stability and aggregation properties in the presence and absence of myoinositol. Both of the domains cooperatively fold and recapitulate the effects of the ⌬F508 mutation on folding, 3 although the NBD 419 cannot reach the native state due to the lack of an intact ␤ subdomain, thereby providing a model for a trapped, partially folded NBD1. The purified CFTR NBD1 and NBD1⌬F proteins were used to assess the effects of myoinositol on the thermodynamic stability and folding and aggregation of the NBD1 as previously described (21). In brief, the refolded wild type CFTR and ⌬F508 NBD1 were subjected to temperature melts to ascertain stabilities in the absence or presence of myoinositol. The temperature was ramped from either 25 to 60°C or from 10 to 70°C over 30 min or 2 h, respectively, and monitored turbidimetrically or by light scattering at 400 nm. To test the effect of myoinositol on the NBD1 aggregation, refolded protein was diluted into arginine buffer containing different concentrations of myoinositol at 37°C, and aggregation was monitored turbidimetrically. The partially folded NBD1 molecules associate in a time-dependent manner to form larger aggregates that scatter visible light. Formation of these aggregated species is continuously monitored by detecting scattered 400 nM light 90°to incident on a PTI Quantamaster or turbidimetrically at 400 nm on a Shimadzu UV-2101PC spectrophotometer. The data are representative of experiments performed at least four times, using two protein preparations per experiment.
Single Channel Patch Clamp Recordings-Single channel patch clamp studies were performed on excised inside-out patches using conventional procedures as described previously (22). Cells were seeded on polylysine-coated coverslips (Sigma) and treated with 1 or 10 mM myoinositol or betaine for 24 h. Recording pipettes were constructed from borosilicate glass capillaries (Garner Glass, Claremont, CA) using a microelectrode puller (David Kopf Instruments, Tujunga, CA). The pipettes were partially filled with a standard pipette solution and usually had a tip resistance of 5-10 megaohms. Recordings were performed at room temperature (20 -22°C). Single channel currents were recorded with a patch clamp amplifier (L/M EPC7; Darmstadt, Germany) filtered at 10 kHz using an eight-pole Bessel filter and stored on computer. Data were redigitized at 2 kHz for analysis using pCLAMP7 (Axon Instruments Inc., Foster City, CA) The bath solution contained 140 mM NaCl, 2 mM MgCl 2 , 1 mM EGTA, 5 mM HEPES, and 0.5 mM CaCl 2 (free Ca 2ϩ 110 nM as measured by Fura-2), pH 7.3, with or without the addition of organic solutes. The pipette solution contained in 140 mM NaC1 or less to balance the osmolality of organic solutes, 2 mM CaCl 2 , and 5 mM HEPES, pH 7.3. All solutions were filtered through 0.2-m filters. Osmolality was measured by a vapor pressure osmometer (Wescor Inc.) to maintain equal osmolality in the preincubation growth medium and internal and external pipette solutions. Chloride channels were activated by the addition of a final concentration of 75 nM of the catalytic subunit of PKA (Promega, Madison, WI) and 1 mM Mg-ATP. To prevent CFTR channel rundown in excised patches, 1 mM Mg-ATP was added to the bath solution. Each patch was observed for at least 10 min, and open probability (P o ) was calculated from a current recording of at least 3-min duration. ORCC and CFTR channel activity were distinguished by their single channel conductance, respectively. In order to further characterize these channels in some experiments, after the currents were recorded 50 M DIDS (Sigma) or 3 mM diphenylamine-2-carboxylate (Sigma) were added to bath solution to inhibit ORCC and CFTR channel activity, respectively.
The number of detected ⌬F508 CFTR or ORCC channels in the total number of patches recorded is presented as the frequency and was analyzed by Fisher's exact test (Table I). The differences in open probability between treated and control groups were analyzed using Student's t test. Data are indicated as the mean Ϯ S.E., where an asterisk represents p Ͻ 0.05, and a double asterisk indicates p Ͻ 0.01.
Chloride Release Assay-The forskolin-stimulated release of 36 Cl Ϫ from IB3 cells was performed using blinded trials as an estimate of cellular chloride efflux as previously described (23). Cells grown to about 90% confluence in six-well plates were treated with various concentrations of myoinositol in culture medium for 24 h. The cells were washed three times with phosphate-buffered saline (Invitrogen). Each well was then filled with 1.5 ml of Ringer's solution containing a total of 5 Ci of 36 Cl Ϫ (sodium salt with a specific activity of 1 Ci/l from PerkinElmer Life Sciences), and the plates were incubated at 37°C for 2-3 h. All 36 Cl Ϫ release assays were performed in a 37°C warm room, which assured constant temperature, with each well serving as its own control (one trial). At time zero, Ringer's solution without cAMP agonists was added and removed immediately to wash away excess extracellular isotope. A fresh aliquot of Ringer's solution was added immediately after this initial wash, and then 36  The cAMP agonist containing Ringer's solution was collected every 15 s for the remaining 2 min of the release assay. At the end of the run, 0.5 N NaOH was added in two aliquots to lyse the cells, and all of the cell lysate was recovered to determine how much 36 C1 Ϫ remained in the cells. The radioactivity of the efflux samples and the cell lysate was measured in a scintillation counter. The amount of 36 Cl Ϫ accumulated in the medium over each 15-s time period was normalized to the initial 36 C1 Ϫ loss in the first 15 s time point of that trial. The rate of release was calculated by comparing each time point to the previous one. The normalized rate of release of 36 Cl Ϫ for the trials is shown as a normalized number Ϯ S.D. The rates of 36 Cl Ϫ release after the application of cAMP agonists under control and experimental conditions were compared, and the differences in slopes were evaluated using analysis of variance. Differences were considered significant at p Ͻ 0.05.

Organic Solutes Treatment Increases the Fully Glycosylated
Form of CFTR-It is well established that the normal processing of CFTR from the endoplasmic reticulum (ER) to the plasma membrane entails the addition and modification of oligosaccharides in the Golgi apparatus (3,7,17). The immature form of CFTR, representing CFTR situated in the ER, is a core-glycosylated protein that is sensitive to endoglycosidase H (band B). The fully glycosylated, endoglycosidase H-resistant CFTR protein is about 20 -30 kDa greater in molecular mass than the core glycosylated CFTR and represents the mature form of CFTR (band C) that is localized in the post-ER compartments, including the plasma membrane. Immunoprecipitation from T84 cells using either CFTR antibody provided a molecular mass control in each CFTR immunoprecipitation assay, because this cell line expresses mainly the mature, fully glycosylated CFTR (band C) as well as a small amount of the immature core glycosylated form of CFTR (band B) ( Fig. 1 , and E). The CF cell line, IB3, showed mainly the core-glycosylated band B form of this protein (Fig. 1A, lane 1) consistent with expression of the ⌬F508 CFTR mutant. In some experiments (Fig. 1B, lanes 2 and 5), another higher molecular mass band was present in IB3 control cells probably as a result of gentamicin that was added to some of the culture medium provided by the manufacturer. Gentamicin enhances read-through of the W1282X deletion (24). Recent results from myoinositol-treated renal cells transfected with ⌬F508 CFTR show double CFTR bands, suggesting that there may be other explanations for double bands (15). One molar glycerol treatment for 24 h, used as a positive control for correcting the processing defect of the ⌬F508 CFTR (9), enhanced the processing of ⌬F508 CFTR in IB3 cells as shown by an increase in the band C (Fig. 1B, lane 6). However, the glycerol treatment was not always successful (Fig. 1B, lane 3), probably due to toxicity that caused detachment of the majority of the IB3 cells from the culture flasks by the end of the incubation period. Processing of ⌬F508 CFTR in IB3 cells was increased by a 24-h application of 10 mM myoinositol in the absence of a significant increase in osmolality (310 mosmol/kg H 2 O in myoinositol media versus 300 mosmol/kg H 2 O in control media), as shown by the increase in the amount of band C as well as the band C to band B ratio (Fig. 1, A (lane 3), B (lane 7), and E). Similarly, a sequential treatment of 10 mM myoinositol followed by 10 mM betaine and then 10 mM taurine and ending with a treatment with 10 mM myoinositol over a 48-h period resulted in an increase in the amount of band C and an increase in the band C to band B ratio (Fig. 1B, lane 4). These results demonstrate that treatment with organic solutes can rescue the processing defect of ⌬F508 CFTR in the CF bronchial airway IB3 cell line as assessed by the glycosylation state. Because IB3 cells have a W1282X allele that could complicate the data because of potential gentamicin-mediated read-through, we used another cell line. Immunoprecipitation was performed in NIH 3T3 cells permanently transfected with only ⌬F508 CFTR. The results of immunoprecipitation in these cells also showed a measurable increase in the mature form of CFTR as well as an increase in the band C to band B ratio after the same myoinositol or sequential treatments (Fig. 1, C-E). Because organic solutes caused the same outcome in both the pure ⌬F508 CFTR-containing cells and the mixed allele IB3 cells, it is most likely that the effect is caused by improved protein processing rather than read-through. The treatment with myoinositol alone or with multiple organic solutes caused statistically significant in-creases in C to B band ratios with a trend toward an increase in the B band (Fig. 1E).
The effects of organic solutes on the steady-state GFP-⌬F508 CFTR or wild type GFP-CFTR expression were initially assessed by immunoprecipitation with a polyclonal GFP antibody and in vitro phosphorylation to label CFTR. Cells were sequen-  1). B, immunoprecipitation using antibody 169 followed by in vitro phosphorylation from IB3 cells treated with 10 mM myoinositol or 1 M glycerol for 24 h or with a sequential incubation (Seq) of 10 mM myoinositol, 10 mM taurine, 10 mM betaine, and 10 mM myoinositol for 8 -16 h each. Lane 1 showing the CFTR product from T84 cells, as a control for the molecular weight of the fully glycosylated CFTR, was exposed for less time than those for IB3 cells. As shown in lane 3 (Gly), the 1 M glycerol treatment was not always successful in these cells, probably due to high toxicity, which caused many of the cells to detach from the culture flask. As shown in lanes 4 and 7, sequential treatment or myoinositol increased the expression of band C as well as band B when compared with the control IB3 cells (lanes 2 and 5). Although there could be enhanced read-through of the W1282X allele of the CFTR gene in these cells, the major effect of treatment with organic solutes seemed to be an increase in the ratio of the band C compared with the amount of band B. C and D, immunoprecipitation using either the polyclonal antibody (C) or the monoclonal antibody (D) to human CFTR followed by in vitro phosphorylation, in the same cell lysates of NIH 3T3 cells permanently transfected with ⌬F508 CFTR. As seen in the right two lanes, 10 mM myoinositol treatment or sequential treatment increased both the mature (band C) and the immature (band B) forms. These data demonstrate that organic solutes increase the ratio of band C to band B in ⌬F508 CFTR expressing NIH 3T3. E, quantification of mature CFTR (C band) and immature CFTR (B band) for T84 cells, IB3 cells, and permanently transfected NIH 3T3 cells in the absence or presence of organic solutes. IB3 cells were treated with 10 mM myoinositol for 24 h. NIH 3T3 cells were treated with myoinositol, betaine, taurine, and myoinositol sequentially for a total period of 48 h. Because the blot for the T84 cells was exposed for less time than that for the IB3 cells and NIH 3T3 cells, the absolute numbers cannot be compared directly. Data are shown as mean Ϯ S.E., n ϭ 6 for T84 cells, n ϭ 4 for IB3 cells, n ϭ 3 for 3T3 cells. The asterisk represents p Ͻ 0.05 compared with the corresponding control group as determined by Student's t test. F, effect of organic solute treatment on the steady-state expression of GFP-⌬F508 CFTR or GFP-wild type CFTR. COS7 cells transfected with GFP-⌬F508 CFTR or GFP-WT CFTR were treated with 10 mM myoinositol, betaine, and taurine for a 48-h period. Immunoprecipitation with a polyclonal GFP antibody and in vitro phosphorylation were used to analyze the steady-state level of core-glycosylated (immature form) or complex-glycosylated (mature form) CFTR. In the GFP-⌬F508 CFTR-transfected COS7 cells (left two lanes), the GFP antibody recognized the immature band or band B at about 170 kDa (GFP adds ϳ30 kDa to the molecular mass of ⌬F508 CFTR) in the control group. After organic solute treatment, there appeared a diffuse mature band (band C, at about 210 kDa) with a dominant immature band. In the GFP-WT CFTR-transfected COS7 cells (right two lanes), the GFP antibody recognized a dominant mature band and a fainter immature band in the control group. After the organic solute treatment, a much more dominant mature band appeared along with an increased amount of immature band. In both the GFP-⌬F-and GFP-WT-transfected cells, the ratios of band C to band B were increased. Western blot of Na,K-ATPase ␣ 1 subunit and ␤-actin on the same cell lysate used for the immunoprecipitation are shown below the CFTR panel. The same amount of cell lysate was loaded in each lane. There was no difference in either the transport protein, the Na,K-ATPase, or in ␤-actin after the organic solute treatment compared with the control group. tially treated for 12 h with 10 mM myoinositol followed by 12 h of 10 mM betaine, then 12 h of 10 mM taurine, and finally 12 h of 10 mM myoinositol for a 48-h treatment course. In the GFP-⌬F508 CFTR-transfected COS7 cells (Fig. 1F, left two lanes), the GFP antibody recognized the core-glycosylated band (immature band, band B, ϳ170 kDa; GFP adds ϳ30 kDa to the molecular mass of ⌬F508 CFTR) in the control group. However, after treatment with organic solutes, there appeared a diffuse complex-glycosylated band (mature band, band C, ϳ210 kDa) with a dominant immature band below that. In the wild type GFP-CFTR-transfected COS7 cells (Fig. 1F, right two lanes), there were two apparent bands. One main band was about 210 kDa (complex-glycosylated, mature band), and the other fainter band was about 170 kDa (core-glycosylated, immature band) in the control group. After treatment with organic solutes, more mature band was accumulated. In both the GFP-⌬F508 CFTR and GFP WT-transfected cells, the ratios of mature band to immature band were increased.
To test the possibility that the effects of organic solutes on ⌬F508 CFTR processing and maturation were not due to nonspecific increases in all proteins, several internal controls were performed. Equivalent amounts of cell lysate were loaded in each lane, and then Western blotting of actin, glyceraldehyde-3-phosphate dehydrogenase (data not shown), and Na,K-ATPase were performed on the same samples utilized in the CFTR immunoprecipitations shown in Fig. 1F. There were no significant differences in the amounts of either actin or glyceraldehyde-3-phosphate dehydrogenase proteins when organic solute-treated cells were compared with nontreated cells (Fig.  1, A-D; data not shown; Fig. 1F). Thus, it suggests that the effects of organic solutes on processing were only evident for the inefficient processing of CFTR. Furthermore, the observation that the amount of a plasma membrane transport protein, the Na,K-ATPase, did not change (Fig. 1, A-D and F; data not shown) strengthens the idea that the effect of organic solutes was specifically on CFTR and did not alter the amount of other transport proteins.
Metabolic Labeling and Pulse-Chase-To test the mechanism by which organic solutes treatment rescues the functional defect of ⌬F508 CFTR, we performed metabolic pulse-chase experiments on GFP-⌬F508 CFTR transiently transfected COS7 cells (Fig. 2). Compared with the control group ( Fig. 2A, left four lanes), both the core-glycosylated and complex-glycosylated forms of GFP-⌬F508 CFTR were increased in the treatment group (Fig. 2A, right four lanes). Interestingly, even at time point 0 h there was more core-glycosylated band in the treatment group than in the control group. This resulted because cells were treated with organic solutes for 48 h prior to the pulse. The major effect of treatment was a significant increase in the ratio of the mature band (C band) to the immature band (B band). The ratios of C band to B band at corresponding time points in the absence or presence of organic solutes are shown in Fig. 2B. In the control group, the C/B ratio was below 0.08, suggesting a finite leak in the C band probably due to high expression levels of CFTR in the transfected cells used in these labeling experiments. After the treatment with organic solutes, not only was the C/B ratio increased 2-5-fold, suggesting that the treatment increased the processing of GFP-⌬F508 CFTR, but also the value increased at 2 and 4 h, because C band degraded more slowly than the B band. The half-life of the complex-glycosylated form of GFP-⌬F508 CFTR was prolonged from 55 min to 110 min after the treatment with organic solutes (Fig. 3A). By contrast, the half-life of the core-glycosylated form of GFP-⌬F508 CFTR, about 60 min, was not dramatically altered by the treatment (Fig. 3B). The half-lives of untreated GFP-⌬F508 CFTR and wild type GFP-CFTR were estimated to be 60 min (Fig. 3B) and 12 h (data not shown), respectively, consistent with the previously reported values for CFTR without a GFP tag in the HEK cells or COS7 cells (9,25), suggesting that the GFP tag did not affect CFTR turnover.
Effect of Myoinositol on the Thermal Stability of NBD1 and ⌬F NBD1 Proteins and the Rate of Formation of "Off Pathway" Conformers-Previous in vitro studies have measured similar thermal and chemical stabilities for both the wild-type and ⌬F508 NBD proteins (20,21). Consistent with these previous studies, the wild-type and ⌬F508 proteins had indistinguishable thermal stabilities in the NBD1 388 (not shown) or NBD1 419 constructs (Fig. 4, A and B, closed circles), and similar chemical stabilities in guanidinium melts (data not shown). A representative experiment (Fig. 4, A and B) shows that 100 mM myoinositol increased both the wild-type and ⌬F508 CFTR NBD1 419 thermal stabilities. A similar increase in thermal stability was seen with the NBD1 388 proteins (not shown). Multiple experiments (n Ն 4, on at least two preparations of each protein) on mutant and wild-type proteins using both constructs revealed that myoinositol shifted the transition mid-points by 3.5°C when temperature was ramped over 2 h to 5.5°C with a 30-min temperature ramp time. Small changes in the slopes, shapes, and amplitudes of the transition regions are seen between the myoinositol-free and myoinositol-treated samples. Intrinsic fluorescence emission spectra of the NBD1 proteins were identical in the presence and absence of myoinositol (data not shown), suggesting that myoinositol is stabilizing but not dramatically altering the conformation of the NBD protein.
Aggregation experiments were also performed on refolded protein to assess the effect of myoinositol on inhibiting the misfolding and aggregation of NBD1. Refolded protein was diluted into buffer containing increasing concentrations of myoinositol and incubated at 37°C, and protein aggregation was monitored by changes in turbidity at 400 nm. Increasing myoinositol concentrations reduced the maximum turbidity in the NBD1 wild-type and ⌬F508 samples and slowed the apparent rate of aggregation (⌬F508 NBD1 419 (not shown)) (Fig. 4C). However, it is not known if this effect is due to increased solubilization of monomeric protein or if it reflects a true stabilization of the domain, which in turn would lead to a decrease in the rate of aggregation.
Organic Solute Treatment Restores Chloride Channel Activity-The biochemical data suggest that organic solute treatment enhanced the processing and maturation of ⌬F508 CFTR molecules to a post-ER compartment in IB3 cells because after organic solute treatment the amount of fully glycosylated band C increases. To test more directly whether the rescued ⌬F508 CFTR functions as an ion channel in the plasma membrane, we examined chloride channel activity using single channel patch clamp recordings from IB3 cells treated with organic solutes compared with their nontreated controls. Fig. 5 shows the induction of both CFTR and ORCC single channel activity after 24-h pretreatment of IB3 cells with either myoinositol (Fig. 5B) or betaine (Fig. 5C). Consistent with previous reports (22,26), untreated IB3 cells exhibit no detectable chloride channel activity in excised inside-out patches perfused with PKA and ATP at the intracellular face of the patch (0 of 54 patches) (Fig. 5A, Table I). The addition of 0.2 M to 10 mM myoinositol or betaine to the inside face of the patch for about 10 min did not induce ion channel activity, suggesting that myoinositol and betaine have no direct, acute effects on chloride channel activities. When IB3 cells were treated with 1 mM or 10 mM myoinositol or betaine for 24 h, recordings made from excised inside-out patches in the absence of PKA and ATP still did not exhibit chloride channel activity (traces on the left in Fig. 5B). However, upon the addition of PKA and ATP, CFTR and ORCC channel activity was observed, consistent with the presence of functional CFTR and ORCC in the membrane (Fig. 5, B and C). The single channel conductance of the ⌬F508 CFTR was 9.1 Ϯ 0.04 picosiemens (mean Ϯ S.E., n ϭ 23), and the anion selectivity was Br Ϫ Ͼ Cl Ϫ Ͼ I Ϫ . The channel was completely blocked by diphenylamine-2-carboxylate but was insensitive to DIDS (data not shown). These properties are consistent with previously published CFTR channel characteristics (5,26,27). ORCC channel activity was characterized by the outwardly rectifying current-voltage relationship and an anion selectivity of I Ϫ Ͼ Cl Ϫ as previously reported (28). The conductance of ORCC was 22.8 Ϯ 0.52 picosiemens at Ϫ50 mV with a P o of 0.6 Ϯ 0.014 (mean Ϯ S.E., n ϭ 21). The conductance of ORCC was 55.8 Ϯ 1.16 picosiemens (n ϭ 5) when the membrane potential was at ϩ50 mV. This ORCC channel activity was blocked by 50 M DIDS (data not shown). As seen in Table I, there was a statistically significant increase in detected ⌬F508 CFTR channel activity and ORCC activity in IB3 cells treated with either 1 mM or 10 mM myoinositol compared with control FIG. 3. Effect of organic solutes treatment on degradation of GFP ⌬F508 CFTR assessed by metabolic 35 S labeling. A, a natural logarithmic plot of kinetics of complex-glycosylated GFP-⌬F508 CFTR (mature ⌬F508 CFTR) in the absence or presence of organic solutes was obtained by densitometric scanning and normalized to the maximum value of complex-glycosylated GFP-⌬F508 CFTR in the absence or presence of organic solutes, respectively. Data are shown from four independent experiments. B, a natural logarithmic plot of kinetics of core-glycosylated GFP-⌬F508 CFTR (immature ⌬F508 CFTR) in the absence or presence of organic solutes was obtained by densitometric scanning and normalized to the maximum value of core-glycosylated GFP-⌬F508 CFTR in the absence or presence of organic solutes, respectively. Data are shown from four independent experiments. IB3 cells (p Ͻ 0.01). Cells treated with 1 mM betaine had a statistically significant increase in ORCC activity (p Ͻ 0.05), although the influence on CFTR channel activity did not achieve statistical significance. When cells were treated for 24 h with 10 mM betaine, both CFTR and ORCC channel activities increased significantly (p Ͻ 0.01) above that of untreated control IB3 cells. Because CFTR has smaller single channel amplitude and a much smaller open probability than ORCC, it is often difficult to observe CFTR channel activity when the ORCC channels are active, which would result in an underestimate of the number of CFTR channels in the membrane. The P o of ⌬F508 CFTR channels in IB3 cells treated with 1 mM myoinositol was 0.08 Ϯ 0.008 (n ϭ 7), a value that was significantly greater than the P o measured from cells treated with 10 mM myoinositol (0.05 Ϯ 0.006, n ϭ 10, p Ͻ 0.01). The P o of ⌬F508 CFTR channels in IB3 cells treated with 10 mM betaine (0.08 Ϯ 0.012, n ϭ 5) did not differ significantly from the P o of CFTR channels of IB3 cells treated with 1 mM myoinositol. These data demonstrate that treatment of defective airway cells with myoinositol or betaine for 24 h caused a significant amount of ⌬F508 CFTR to reach the cell surface membrane and restore the chloride channel function of both ⌬F508 CFTR and ORCC channels. However, the treatment with organic solutes did not restore the open probability of ⌬F508 CFTR to the level of WT CFTR, which has a P o value of about 0.6 (29).
Organic Solute Treatment Restores Macroscopic Chloride Efflux-To test whether macroscopic chloride efflux is enhanced by organic solute treatment, we performed experiments measuring the rate of 36 Cl Ϫ loss from IB3 cells. In blinded 36 Cl Ϫ release assays, IB3 cells grown under control conditions did not demonstrate an increase in the rate of 36 Cl Ϫ efflux in response to cAMP agonist treatment (Fig. 6, closed circles; n ϭ 11 trials), consistent with previous reports (22,26). However, the rate of 36 Cl Ϫ efflux in response to cAMP agonists increased significantly in IB3 cells treated for 24 h with 100 M myoinositol (data not shown, n ϭ 10 trials, p Ͻ 0.05), 500 M myoinositol (Fig. 6, upright triangles; n ϭ 9 trials, p Ͻ 0.001), 1 mM myoinositol (Fig. 4, open circles; n ϭ 10 trials, p Ͻ 0.001), 2 mM myoinositol (Fig. 6, open squares; n ϭ 9 trials, p Ͻ 0.001), and 4 mM myoinositol (data not shown; n ϭ 6 trials, p Ͻ 0.001). There was no statistically significant increase in the rate of FIG. 4. Myoinositol stabilization of NBD1. Wild-type NBD1 (A) or ⌬F508 NBD1 419 (B) was refolded overnight at 4°C and subjected to temperature melts to ascertain the relative domain stabilities in the absence (solid circles) or presence (open circles) of 100 mM myoinositol as the temperature was ramped from 10 to 70°C over 2 h. Myoinositol stabilizes both the wild-type and mutant NBD1 domains between 3.5 and 5.0°C when measured at the midpoint of the transition. C, the aggregation of ⌬F508 NBD1 388 is inhibited by myoinositol. Refolded protein was diluted to 2 M into arginine buffer containing 0 (closed circle), 50 (open circle), 100 (closed triangle), or 200 mM (open triangle) myoinositol and incubated at 37°C, and NBD1 aggregation was monitored by changes in turbidity. 36 Cl Ϫ efflux induced by cAMP agonists in cells treated with 10 mM myoinositol (Fig. 4, inverted triangles, n ϭ 8 trials, p Ͼ 0.05), which corresponds to the lesser activation of CFTR channel activity at 1 mM myoinositol compared with 10 mM myoinositol. The effect of myoinositol on 36 Cl Ϫ efflux was maximal at 1 mM. DISCUSSION The studies presented here demonstrate that myoinositol alone, or myoinositol given sequentially with taurine and betaine, can increase the processing of the core-glycosylated, endoplasmic reticulum-arrested ⌬F508 CFTR into the fully glycosylated mature form of CFTR, in both IB3 cells, a CF airway cell line, NIH 3T3 fibroblasts stably transfected with ⌬F508 CFTR, and COS7 cells transiently transfected with GFPtagged ⌬F508 CFTR. Organic solutes like myoinositol, betaine, and taurine are transported into cells via sodium and chloridecoupled cotransporters (i.e. the sodium myoinositol cotransporter (SMIT), the betaine/GABA cotransporter (BGT-1), and the taurine cotransporter (TAUT), respectively). These cotransporters are present in many cells and tissues including kidney, brain, liver, heart, muscle, and placenta (11, 30 -32). Our data indicate that the mRNA for these three cotransporters and the proteins for SMIT and BGT-1 are expressed in primary bron-chial epithelial cells, IB3, CFTE, HBE, NIH 3T3 cells, and COS7 cells (data not shown). These cotransporters provide an entry pathway for organic solutes to be accumulated in airway epithelial cells as they do in other tissues. Both the single channel data and the chloride efflux data demonstrate that the most effective myoinositol or betaine concentrations for repair of CFTR channel function were above the respective organic solute affinities (K d ) of the respective cotransporters (30,31). Furthermore, when the concentration of betaine was at the K d of the cotransporter, there was not a statistically significant effect on CFTR channel activity, yet as the concentration of betaine rose above the K d of the betaine cotransporter, which is between 400 and 900 M, the effectiveness on CFTR channel function was significant (Table I). Thus, treatment with 10 mM betaine was more effective at increasing the channel activity of CFTR than was 1 mM betaine (Table I). However, solute concentrations well above the K d of the cotransporters can also down-regulate the cotransporter protein 4 in a time-dependent manner (over 12 h); therefore, shorter treatment with multiple solutes over a longer period was chosen as an optimal strategy. This appeared to be more effective than treatment with a single 4 S. Guggino, unpublished observations.  or betaine-treated IB3 cells Ratios represent the number of patches that showed ⌬F508 CFTR or ORCC channel activity compared with the total number of patches investigated. The values indicated in parentheses represent the percentage of patches that showed ⌬F508 CFTR or ORCC channel activity. **, p Ͻ 0.01; *, p Ͻ 0.05 as determined by Fisher's exact test. Except for the ⌬F508 CFTR activity in IB3 cells treated with 1 mM betaine (p Ͼ 0.05), the frequencies of detected ⌬F508 CFTR or ORCC activity in other sets of treated IB3 cells were statistically significantly greater than that found in control IB3 cells (p Ͻ 0.01 or p Ͻ 0.05). Although organic solutes like betaine, myoinositol, and taurine are well known for their role in counteracting the effects of hyperosmolality and urea denaturation in the medulla of the kidney (33)(34)(35), these solutes are also present in other cell types that are not normally exposed to hyperosmolality. For example, taurine is most abundant in excitable tissues like the heart and brain that are constantly exposed to elevated levels of oxidants (33,36). Taurine is known to stabilize membranes, modulate intracellular calcium fluxes, and act as an antioxidant (33,36). Taurine and hypotaurine are also present in high concentrations in the male reproductive tract, where they protect sperm from oxidant-dependent denaturation (33). In the lung, taurine is thought to protect against oxidant injury induced by nitrogen dioxide, ozone, and bleomycin (37,38). There are reports that betaine is protective in alcoholic liver disease, where it is thought to protect against oxidative injury (39,40). Since the CF lung is low in surface glutathione (41,42) and has an elevated level of oxidants as a result of the accumulation of neutrophils (43), an additional advantage of these compounds could be protection against oxidative stress.
Cystic fibrosis is one of many disorders that arise from aberrant protein function caused by protein misfolding, misprocessing, and aggregation. This group of diseases, of which nearly 20 have been described, includes some of the currently most perplexing medical problems such as Alzheimer's disease, prion encephalopathies, Parkinson's disease, and cancer (44 -46). Recent studies have shown that several point mutants that cause misfolding and trafficking defects in the aquaporin-2 water channel or the V2 vasopressin receptor, which result in nephrogenic diabetes insipidus, could be rescued using chemical chaperones like glycerol or trimethylamine N-oxide (47,48) or by selective pharmacological ligands such as the nonpeptidic V2 vasopressin receptor antagonists (49). Similarly, synthetic organic compounds have been used as pharmacological ligands to stabilize wild-type p53 against thermal denaturation and also to stabilize a mutant p53 into an active conformation (50). By contrast, because they do not require a specific binding event, we suspect that the naturally occurring organic solutes, used in this study to repair ⌬F508 CFTR in CF airway cells, may represent a generally applicable therapy for diseases resulting from errors in protein conformation, folding, and aggregation.
Some organic solutes are thought to stabilize proteins by enhancing the "hydrophobic effect," preferential hydration of the polypeptide chain leading to a minimization of solvent exposed surface area. Other organic solutes are thought to protect the water-exposed portions of proteins from the kosmotropic effects of highly charged ions (51), which increase the water order and decrease the solubility of a folded protein (salting out) because of increased surface tension. Organic solutes can also act like chaotropic ions that stabilize proteins by decreasing the structure of water and increasing the solubility of the folded protein (salting in). Stabilization of proteins is usually evident as an increase in the denaturation temperature (52) that can be measured as a shift in the transition temperature. To test the mechanism underlying the effects of organic solutes, we performed in vitro denaturation/folding experiments and metabolic pulse-chase experiments in transiently transfected cells. The in vitro experiments demonstrated that organic solutes had significant effects on the thermal stabilities and aggregation properties of both wild-type and ⌬F508 in two distinct NBD1 protein models. Thus, organic solutes appear to promote protein folding and stabilize folded conformations of mutated proteins such as ⌬F508 CFTR.
It has been reported that high concentrations of glycerol (1 M) and trimethylammonium oxide (100 mM), like low temperature, result in correction of the protein processing defect of the mutant ⌬F508 CFTR (8,9). These treatments restore the chloride channel function and processing of ⌬F508 CFTR, suggesting that at least some of these molecules adopt the normal conformation of wild type CFTR (8,9). Qu and Thomas (20) concluded that the ⌬F508 CFTR mutation had little effect on the thermodynamic stability of an isolated NBD1 polypeptide compared with wild type because both the mutant and wild type molecules had similar Gibb's energy of denaturation. Instead they found that a ⌬F508 NBD1 peptide had a lower folding yield than the normal NBD protein. They concluded that the phenylalanine 508 residue makes crucial contacts during the folding process that are essential for efficient folding but not domain stability. In this regard, myoinositol increases the thermal stability of an NBD1 model that cannot fold all the way to native state due to the lack of a ␤-strand.
To determine whether the organic solutes stabilize an immature ⌬F508 intermediate and/or the mature ⌬F508 CFTR, we performed the pulse-chase experiments. The results show that organic solutes significantly increased the ratio of band C to band B of GFP-⌬F508 CFTR, indicating that the treatment enhanced the processing of the core-glycosylated form of CFTR. Significantly, the half-life of the complex-glycosylated form of ⌬F508 CFTR was increased from 55 to 110 min, indicating that the organic solutes stabilize the mature ⌬F508 CFTR and retard its turnover. Although the rescued, mature ⌬F508 CFTR has a significantly prolonged half-life, it is still shorter lived than the wild type, suggesting that organic solutes offer only partial correction of the mutant structure as previously reported in the literature (15,53). The half-life of the core-glycosylated form of ⌬F508 CFTR did not appear to change in the presence of organic solutes, suggesting that the immature forms have similar degradation kinetics after organic solute . IB3 cells treated with 500 M myoinositol (n ϭ 9 trials, p Ͻ 0.001), 1 mM myoinositol (n ϭ 10 trials, p Ͻ 0.001), or 2 mM myoinositol (n ϭ 9 trials, p Ͻ 0.001) had a significantly increased chloride efflux rate in the presence of cAMP agonists compared with control cells (n ϭ 11 trials). Cells pretreated with 10 mM myoinositol (n ϭ 8 trials, p Ͼ 0.05) did not show an increased rate of cAMP agonist-stimulated 36 Cl Ϫ efflux. cAMP agonists were added at 60 s (arrow). The error bars show S.D. for control untreated cells and cells treated with 1 mM myoinositol; the other error bars are omitted for clarity. Analysis of the differences in slopes after cAMP was performed by analysis of variance.
treatment. Yet there was a trend or an actual increase in the B band caused by organic solute treatment suggesting that there could be increased synthesis of CFTR. This is rather unlikely, because another transport protein, the Na,K-ATPase, did not show increased abundance after organic solute treatment. It has been noted that significant amounts of misfolded CFTR are sequestered in aggresomes in CFTR overexpression cell lines (19). One limitation of pulse-chase experiments is their inability to evaluate those proteins that form insoluble aggregates. Based on the fact that organic solutes decreased aggregation in the turbidity assay, at least one mechanism by which these solutes work is to chaperone ⌬F508 CFTR into a conformation that will not aggregate but is competent to be released from the endoplasmic reticulum to the later compartments of the secretory pathway. Such a mechanism is one explanation why there is an increase in the amount of B band in the immunoprecipitation experiments. Therefore, the organic solute treatment used in this study probably promotes the processing of ⌬F508 CFTR by releasing CFTR from aggresomes rather than by decreasing degradation of the immature precursor conformation.
It has been reported that only about 25% of the coreglycosylated wild type CFTR matures to the fully glycosylated form (54). Interestingly, pulse-chase experiments indicate that organic solutes have similar effects on wild type CFTR (data not shown), which also matures inefficiently. Organic solutes stabilize both ⌬F508 NBD1 and wild type NBD1 in the denaturation assay, which would also increase the processing of both wild type and ⌬F508 CFTR, effects due to alterations of the solvent environment in which both wild type and mutant proteins reside.
Single channel recordings from IB3 cells treated with either myoinositol or betaine exhibit increased ⌬F508 CFTR single channel activity in excised surface membrane patches compared with patches made from untreated cells that exhibit no CFTR channel activity. Moreover, the activity of the ORCC can be detected in cells treated with either betaine or myoinositol, whereas untreated IB3 cells do not exhibit ORCC single channel fluctuations in excised membrane patches. ORCC channel activity is well known to be modulated by the activity of CFTR (28), and our data suggest that when CFTR is processed to the plasma membrane in the presence of organic solutes added to culture media, the ORCC channel activity is also restored. Moreover, the effect of the extracellular organic solutes on channel activity correlates with oversaturation of the transporters for betaine (31) and myoinositol (55). Significantly, forskolin-stimulated efflux of 36 Cl Ϫ from IB3 cells is measurably enhanced by incubation of cells with myoinositol at concentrations that are at the K d of the cotransporter and reach a maximum when the concentration of the solute approaches saturation of the sodium myoinositol cotransporter. Thus, the treatment of airway cells with these naturally occurring organic solutes may provide a potentially novel therapy to correct the processing defects of ⌬F508 CFTR.
Dietary supplements of betaine have been employed safely and effectively as a therapy in patients with homocystinuria (56,57) and nonalcoholic steatohepatitis (58). Taurine has also been used as a supplement in proprietary milk formulas and employed to treat several diseases, with observed beneficiary effects in patients with heart disease, hepatic disorders, myotonia (36), and cystic fibrosis (51,59). These clinical trials suggest that the potential utilization of systemic organic solutes in treatment of CF or other protein conformational disorders is feasible in human subjects.