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• W2079521511 abstract "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. 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. The most common mutation leading to cystic fibrosis (CF), 1The abbreviations used are: CFcystic fibrosisDIDS4,4′-diisothiocyanostilbene-2,2′-disulfonic acidCFTRcystic fibrosis transmembrane conductance regulatorORCCoutwardly rectifying chloride channelWTwild typeGFPgreen fluorescent proteinPKAprotein kinase AERendoplasmic reticulum. a deletion of phenylalanine at position 508 in the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR), causes decreased trafficking of CFTR to the surface membrane in the CF sweat duct (1Kartner N. Augustinas O. Jensen T.J. Naismith A.L. Riordan J.R. Nat. Genet. 1992; 1: 321-327Crossref PubMed Scopus (328) Google Scholar), CF primary airway cells (2Denning G.M. Ostedgaard L.S. Welsh M.J. J. Cell Biol. 1992; 118: 551-559Crossref PubMed Scopus (158) Google Scholar), and heterologous expression systems (3Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1423) Google Scholar). The mutation exerts its influence at an early step in folding because the misprocessing of ΔF508 CFTR is evident prior to completion of translation (4Sato S. Ward C.L. Kopito R.R. J. Biol. Chem. 1998; 273: 7189-7192Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). However, the small fraction of ΔF508 CFTR protein that arrives at the plasma membrane retains function as a cAMP-responsive chloride channel, although with a decreased open probability compared with wild-type CFTR (5Dalemans W. Barbry P. Champigny G. Jallat S. Dott K. Dreyer D. Crystal R.G. Pavirani A. Lecocq J.P. Lazdunski M. Nature. 1991; 354: 526-528Crossref PubMed Scopus (569) Google Scholar, 6Drumm M.L. Wilkinson D.J. Smit L.S. Worrell R.T. Strong T.V. Frizzell R.A. Dawson D.C. Collins F.S. Science. 1991; 254: 1797-1799Crossref PubMed Scopus (421) Google Scholar). cystic fibrosis 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid cystic fibrosis transmembrane conductance regulator outwardly rectifying chloride channel wild type green fluorescent protein protein kinase A endoplasmic reticulum. The trafficking defect of ΔF508 CFTR can be reversed by lowering the growth temperature (25-27 °C) of ΔF508 CFTR-expressing cells. Low temperature, in a time-dependent manner, leads to the appearance of the mature, fully glycosylated, high molecular weight form of CFTR, as well as a restoration of cAMP-dependant chloride channel activity (7Denning G.M. Anderson M.P. Amara J.F. Marshall J. Smith A.E. Welsh M.J. Nature. 1992; 358: 761-764Crossref PubMed Scopus (1061) Google Scholar). High concentrations of glycerol (1 m) or 100 mm trimethylamine N-oxide also increase the plasma membrane expression of ΔF508 CFTR in NIH 3T3 cells and HEK 293 cells (8Brown C.R. Hong-Brown L.Q. Biwersi J. Verkman A.S. Welch W.J. Cell Stress Chaperones. 1996; 1: 117-125Crossref PubMed Scopus (362) Google Scholar, 9Sato S. Ward C.L. Krouse M.E. Wine J.J. Kopito R.R. J. Biol. Chem. 1996; 271: 635-638Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). It is well documented that sugars (10Taylor L.S. York P. Williams A.C. Edwards H.G. Mehta V. Jackson G.S. Badcoe I.G. Clarke A.R. Biochim. Biophys. Acta. 1995; 1253: 39-46Crossref PubMed Scopus (44) Google Scholar), the amino acids betaine and taurine, and polyols like glycerol, erythritol, xylitol, sorbitol, and inositol (11Back J.F. Oakenfull D. Smith M.B. Biochemistry. 1979; 18: 5191-5196Crossref PubMed Scopus (755) Google Scholar, 12Gekko K. J. Biochem. (Tokyo). 1981; 90: 1633-1641Crossref PubMed Scopus (153) Google Scholar, 13Gekko K. Ito H. J. Biochem. (Tokyo). 1990; 107: 572-577Crossref PubMed Scopus (49) Google Scholar) stabilize many proteins against denaturation due to thermal stress, or chemical denaturants such as guanidinium. It is currently thought that these organic solutes stabilize proteins by promoting selective hydration of the polypeptide (14Wang A. Bolen D.W. Biochemistry. 1997; 36: 9101-9108Crossref PubMed Scopus (414) Google Scholar). Not surprisingly, the major renal organic solutes such as 300 mm sorbitol, myoinositol, and taurine recently have been shown to increase processing in renal medulla cells transfected with ΔF508 CFTR (15Howard M. Fischer H. Roux J. Santos B.C. Gullans S.R. Yancey P.H. Welch W.J. J. Biol. Chem. 2003; 278: 35159-35167Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Together, these observations suggest the potential utility of treating airways with organic solutes or “chemical chaperones” for CF therapy. Promoting the folding of the mutant protein may lead to trafficking of some of the mislocalized, mutant protein to the plasma membrane, where it may rescue sufficient membrane chloride conductance in CF cells to counteract the genetic defect. However, in addition to its function as a chloride channel, CFTR performs vital regulation of other transport processes in the apical membrane of epithelia, which, when lost, may also contribute to the pathology. We expected that if organic solutes indeed rescued the folding and restored the chloride channel function of CFTR, this treatment should also rescue the interactions that CFTR normally has with other ion channels such as the outwardly rectifying chloride channel (ORCC). One caveat of the previous studies using organic solutes to refold ΔF508 CFTR is the high concentrations of organic solutes that were required to restore channel function. Here, we present 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. 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, ∼160 kDa) and the fully glycosylated form of CFTR (band C, ∼180 kDa) in T84 cells, HBE cells, and NIH 3T3 cells transfected with WT CFTR. In IB3 cells or NIH 3T3 cells transfected with ΔF508 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 α1 subunit and β-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 ΔF508/W1282X genotype (X = stop mutation). The W1282X mutation produces an unstable mRNA (16Hamosh A. Rosenstein B.J. Cutting G.R. Hum. Mol. Genet. 1992; 1: 542-544Crossref PubMed Scopus (87) Google Scholar). 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 μg/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 ΔF508 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% CO2 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 H2O to 310 mosmol/kg H2O, 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 [γ-32P]ATP (Fig. 1). The procedures were described previously (3Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1423) Google Scholar, 7Denning G.M. Anderson M.P. Amara J.F. Marshall J. Smith A.E. Welsh M.J. Nature. 1992; 358: 761-764Crossref PubMed Scopus (1061) Google Scholar, 17Gregory R.J. Cheng S.H. Rich D.P. Marshall J. Paul S. Hehir K. Ostedgaard L. Klinger K.W. Welsh M.J. Smith A.E. Nature. 1990; 347: 382-386Crossref PubMed Scopus (272) Google Scholar). 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 β-glycerol phosphate, 1 mml-phenylalanine, 1 mm sodium orthovanadate, 50 mm sodium fluoride, and the following protease inhibitors: pepstatin A (2 μg/ml), leupeptin (10 μg/ml), aprotinin (10 μg/ml), elastinal (4 μg/ml), benzamidine (0.5 mg/ml), 1 mm phenylmethanesulfonyl fluoride, 0.4 mm iodoacetic acid, 2.5 mm phenanthroline and 0.1 mmN-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 × g in an Eppendorf bench top centrifuge. The protein in the supernatant was quantified with the BCA protein assay kit (Pierce) and stored at -80 °C. One milligram of lysate protein in 1 ml of lysis buffer with protease inhibitors was precleared with 2 μl of normal rabbit serum (used only for polyclonal antibody 169) and 30 μl of Protein A-Sepharose beads (Amersham Biosciences) at 4 °C for 2 h and centrifuged at 10,000 × g to remove nonspecific complexes. Subsequently, either 2 μl of antibody 169, a rabbit polyclonal anti-human CFTR R domain antibody, or 1 μg of mouse monoclonal C-terminal antibody (IgG2a) 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 μCi of [γ-32P]ATP (PerkinElmer Life Sciences) in 50 μl of PKA buffer (50 mm Tris, pH 7.5, 10 mm MgCl2, 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 μl of Laemmli sample buffer (Bio-Rad) and incubated at 65 °C for 4 min. The sample was spun 2 min at 8,000 × g, and the supernatant was either stored at -20 °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 -80 °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 α 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-ΔF508 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 (18Moyer B.D. Loffing J. Schwiebert E.M. Loffing-Cueni D. Halpin P.A. Karlson K.H. Ismailov I.I. Guggino W.B. Langford G.M. Stanton B.A. J. Biol. Chem. 1998; 273: 21759-21768Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 19Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1777) Google Scholar). 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 μCi/ml [35S]methionine and [35S]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-ΔF508 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 NBD1ΔF are similar to the pET 28a-derived vectors described previously (20Qu B.H. Thomas P.J. J. Biol. Chem. 1996; 271: 7261-7264Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), except they contain residues 388-655 (NBD388) or 419-655 (NBD419) of human CFTR. The NBD388 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. 2H. Lewis, Structural GenomiX, personal communication. The NBD419 proteins include the entire F1-like core and the α-helical subdomains of the domain but lack a β-strand from the β-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 mml-arginine, 2 mm EDTA, 1 mm dithiothreitol, 200 mm guanidine HCl at a final concentration of 4 μm 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 ΔF508 mutation on folding, 3P. H. Thibodeau and P. Thomas, unpublished data. although the NBD419 cannot reach the native state due to the lack of an intact β subdomain, thereby providing a model for a trapped, partially folded NBD1. The purified CFTR NBD1 and NBD1ΔF proteins were used to assess the effects of myoinositol on the thermodynamic stability and folding and aggregation of the NBD1 as previously described (21Qu B.H. Strickland E.H. Thomas P.J. J. Biol. Chem. 1997; 272: 15739-15744Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). In brief, the refolded wild type CFTR and ΔF508 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 (22Egan M.E. Schwiebert E.M. Guggino W.B. Am. J. Physiol. 1995; 268: C243-C251Crossref PubMed Google Scholar). 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 MgCl2, 1 mm EGTA, 5 mm HEPES, and 0.5 mm CaCl2 (free Ca2+ 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 CaCl2, and 5 mm HEPES, pH 7.3. All solutions were filtered through 0.2-μm 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 (Po) 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 μm 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 ΔF508 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 ± S.E., where an asterisk represents p < 0.05, and a double asterisk indicates p < 0.01.Table IFrequency of chloride channel activity stimulated by PKA and ATP in patches from control, myoinositol-treated (MI), or betaine-treated IB3 cellsControl1 mm MI10 mm MI1 mm betaine10 mm betaineCFTR0/54(0%)7/20(35%)**10/26(38%)**1/12(8%)5/32(16%)**ORCC0/54(0%)7/20(35%)**6/26(23%)**2/12(17%)*6/32(19%)** Open table in a new tab Chloride Release Assay—The forskolin-stimulated release of 36Cl- from IB3 cells was performed using blinded trials as an estimate of cellular chloride efflux as previously described (23Schwiebert E.M. Morales M.M. Devidas S. Egan M.E. Guggino W.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2674-2679Crossref PubMed Scopus (73) Google Scholar). 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 μCi of 36Cl- (sodium salt with a specific activity of 1 μCi/μl from PerkinElmer Life Sciences), and the plates were incubated at 37 °C for 2-3 h. All 36Cl- 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 36Cl- release was measured every 15 s by removing the Ringer's solution and replacing it with fresh Ringer's solution until the 1-min time point was reached. At this time, a Ringer's solution containing forskolin (2.5 μm), 8-bromo-cAMP (250 μm), and 8-(4-chlorophenylthio)-cAMP (250 μm) was added to the dish. 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 36C1- remained in the cells. The radioactivity of the efflux samples and the cell lysate was measured in a scintillation counter. The amount of 36Cl- accumulated in the medium over each 15-s time period was normalized to the initial 36C1- 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 36Cl- for the trials is shown as a normalized number ± S.D. The rates of 36Cl- 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 < 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 (3Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1423) Google Scholar, 7Denning G.M. Anderson M.P. Amara J.F. Marshall J. Smith A.E. Welsh M.J. Nature. 1992; 358: 761-764Crossref PubMed Scopus (1061) Google Scholar, 17Gregory R.J. Cheng S.H. Rich D.P. Marshall J. Paul S. Hehir K. Ostedgaard L. Klinger K.W. Welsh M.J. Smith A.E. Nature. 1990; 347: 382-386Crossref PubMed Scopus (272) Google Scholar). 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, A (lane 2), B (lane 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 ΔF508 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 (24Bedwell D.M. Kaenjak A. Benos D.J. Bebok Z. Bubien J.K. Hong J. Tousson A. Clancy J.P. Sorscher E.J. Nat. Med. 1997; 3: 1280-1284Crossref PubMed Scopus (279) Google Scholar). Recent results from myoinositol-treated renal cells transfected with ΔF508 CFTR show double CFTR bands, suggesting that there may be other explanations for double bands (15Howard M. Fischer H. Roux J. Santos B.C. Gullans S.R. Yancey P.H. Welch W.J. J. Biol. Chem. 2003; 278: 35159-35167Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). One molar glycerol treatment for 24 h, used as a positive control for correcting the processing defect of the ΔF508 CFTR (9Sato S. Ward C.L. Krouse M.E. Wine J.J. Kopito R.R. J. Biol. Chem. 1996; 271: 635-638Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar), enhanced the processing of ΔF508 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" @default.
• W2079521511 created "2016-06-24" @default.
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• W2079521511 date "2003-12-01" @default.
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• W2079521511 title "Organic Solutes Rescue the Functional Defect in ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator" @default.
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