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• W2001530819 abstract "The most common mutation in cystic fibrosis (deletion of Phe-508 in the first nucleotide binding domain (ΔF508)) in the cystic fibrosis transmembrane conductance regulator (CFTR) causes retention of the mutant protein in the endoplasmic reticulum. We previously showed that the ΔF508 mutation causes the CFTR protein to be retained in the endoplasmic reticulum in an inactive and structurally altered state. Proper packing of the transmembrane (TM) segments is critical for function because the TM segments form the chloride channel. Here we tested whether the ΔF508 mutation altered packing of the TM segments by disulfide cross-linking analysis between TM6 and TM12 in wild-type and ΔF508 CFTRs. These TM segments were selected because TM6 appears to line the chloride channel, and cross-linking between these TM segments has been observed in the CFTR sister protein, the multidrug resistance P-glycoprotein. We first mapped potential contact points in wild-type CFTR by cysteine mutagenesis and thiol cross-linking analysis. Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wild-type background. The disulfide cross-linking occurs intramolecularly and was reducible by dithiothreitol. Introduction of the ΔF508 mutation into these cysteine mutants, however, abolished cross-linking. The results suggest that the ΔF508 mutation alters interactions between the TM domains. Therefore, a potential target to correct folding defects in the ΔF508 mutant of CFTR is to identify compounds that promote correct folding of the TM domains. The most common mutation in cystic fibrosis (deletion of Phe-508 in the first nucleotide binding domain (ΔF508)) in the cystic fibrosis transmembrane conductance regulator (CFTR) causes retention of the mutant protein in the endoplasmic reticulum. We previously showed that the ΔF508 mutation causes the CFTR protein to be retained in the endoplasmic reticulum in an inactive and structurally altered state. Proper packing of the transmembrane (TM) segments is critical for function because the TM segments form the chloride channel. Here we tested whether the ΔF508 mutation altered packing of the TM segments by disulfide cross-linking analysis between TM6 and TM12 in wild-type and ΔF508 CFTRs. These TM segments were selected because TM6 appears to line the chloride channel, and cross-linking between these TM segments has been observed in the CFTR sister protein, the multidrug resistance P-glycoprotein. We first mapped potential contact points in wild-type CFTR by cysteine mutagenesis and thiol cross-linking analysis. Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wild-type background. The disulfide cross-linking occurs intramolecularly and was reducible by dithiothreitol. Introduction of the ΔF508 mutation into these cysteine mutants, however, abolished cross-linking. The results suggest that the ΔF508 mutation alters interactions between the TM domains. Therefore, a potential target to correct folding defects in the ΔF508 mutant of CFTR is to identify compounds that promote correct folding of the TM domains. Cystic fibrosis (CF) 1The abbreviations used are: CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator; ER, endoplasmic reticulum; TM, transmembrane segment; DTT, dithiothreitol; TMD, transmembrane domain; NBD, nucleotide binding domain; R, regulatory domain; WT, wild type; ER, endoplasmic reticulum; P-gp, P-glycoprotein; CHO, Chinese hamster ovary; HEK cells, human embryonic kidney cells; M5M, 1,5-pentanediyl-bismethanethiosulfonate; M8M, 3,6-dioxaoctane-1,8-diyl-bismethanethiosulfonate; M17M, 3,6,9,12,15-pentaoxaheptadecane-1,17-diyl-bismethanethiosulfonate. is the most common lethal autosomal recessive disease in the Caucasian population. It affects 1 in every 2500 live births, and 1 in every 25 people is a carrier (1Boat T.F. Cheng P.W. Acta Paediatr. Scand. Suppl. 1989; 363 (discussion 29–30): 25-29Crossref PubMed Google Scholar). It is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (cftr). The gene product, CFTR, is a member of the ABC (ATP-binding cassette) transporter family. It is composed of 1480 amino acids and has the putative structure of an N-terminal cytosolic domain, two transmembrane domains (TMDs), two nucleotide binding domains (NBDs), one regulatory domain, a C-terminal cytosolic tail, and two active N-glycosylation sites on the fourth extracellular loop (2Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.-C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5946) Google Scholar). To date there are more than 1300 mutations identified in the gene (CF mutation data base: www.genet.sickkids.on.ca/cftr). The mutations occur throughout the gene and are classified according to their molecular consequences (for review, see Refs. 3Powell K. Zeitlin P.L. Adv. Drug Delivery Rev. 2002; 54: 1395-1408Crossref PubMed Scopus (48) Google Scholar and 4Rowntree R.K. Harris A. Ann. Hum. Genet. 2003; 67: 471-485Crossref PubMed Scopus (272) Google Scholar) such as null-production, processing defect, regulation defect, conductance defect, and promoter defect. Deletion of phenylalanine 508 (ΔF508) is the most common CF mutation as it accounts for more than 90% of the clinical cases. ΔF508 is a processing-defective mutation. CFTR is first synthesized in the endoplasmic reticulum (ER), where it is core-glycosylated at its N-glycosylation sites and folds with the aid of molecular chaperones. Upon proper folding and passing the ER quality control system, the CFTR protein is then exported into the Golgi apparatus where CFTR and its sugar moieties undergo further modification before being targeted onto the cell surface. Processing defective CFTRs such as ΔF508-CFTR do not make it past the ER quality control system. Instead, they are retained in the ER in an inactive form (5Chen E.Y. Bartlett M.C. Clarke D.M. Biochemistry. 2000; 39: 3797-3803Crossref PubMed Scopus (29) Google Scholar) before rapid degradation by cytosolic proteasomes (6Jensen T.J. Loo M.A. Pind S. Williams D.B. Goldberg A.L. Riordan J.R. Cell. 1995; 83: 129-135Abstract Full Text PDF PubMed Scopus (773) Google Scholar, 7Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1131) Google Scholar). The lack of processing and maturation onto the cell surface by processing mutants such as ΔF508 is the most common cause of severe CF. Other mutations located through out the molecule also cause defective maturation (8Chen E.Y. Clarke D.M. BMC Biochem. 2002; 3: 29Crossref PubMed Scopus (5) Google Scholar, 9Seibert F.S. Jia Y. Mathews C.J. Hanrahan J.W. Riordan J.R. Loo T.W. Clarke D.M. Biochemistry. 1997; 36: 11966-11974Crossref PubMed Scopus (66) Google Scholar, 10Seibert F.S. Linsdell P. Loo T.W. Hanrahan J.W. Clarke D.M. Riordan J.R. J. Biol. Chem. 1996; 271: 15139-15145Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 11Seibert F.S. Linsdell P. Loo T.W. Hanrahan J.W. Riordan J.R. Clarke D.M. J. Biol. Chem. 1996; 271: 27493-27499Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Correction of defective processing of CFTR mutants would benefit patients with CF. An important observation is that the ΔF508 mutant has been shown to be functional if it can be induced to mature and be delivered to the cell surface. Maturation of ΔF508 CFTR can be promoted by incubation at low temperature, expression in the presence of high concentrations of glycerol, or by the co-expression in the presence of high levels of the N-terminal of CFTR (12Clarke L.L. Gawenis L.R. Hwang T.C. Walker N.M. Gruis D.B. Price E.M. Am. J. Physiol. Cell Physiol. 2004; 287: 192-199Crossref PubMed Scopus (11) Google Scholar, 13Sato 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, 14Denning 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, 15Estelle C.-B. Jablonsky M Naren. A.P. Jackson P.L. Muccio D.D. Kirl K.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8221-8226Crossref PubMed Scopus (51) Google Scholar). All of these rescue methods, however, are inefficient and are not feasible in a clinical setting. To devise an improved strategy to induce efficient maturation of CFTR-processing mutants, we must first understand the molecular basis of their defects. There is evidence that there are structural differences between wild-type and mutant ΔF508 CFTRs. Mutant ΔF508 as well as immature wild-type CFTR are more susceptible to protease digestion compared with mature protein (5Chen E.Y. Bartlett M.C. Clarke D.M. Biochemistry. 2000; 39: 3797-3803Crossref PubMed Scopus (29) Google Scholar, 16Zhang F. Kartner N. Lukacs G.L. Nat. Struct. Biol. 1998; 5: 180-183Crossref PubMed Scopus (129) Google Scholar). Results from these protease digestion studies suggest that CFTR-processing mutants are not grossly misfolded but are trapped as partially folded intermediates that are structurally similar to immature wild-type protein. Studies on the CFTR sister protein, P-glycoprotein (P-gp), suggest that one structural alteration caused by the presence of the ΔF508 mutation is incomplete packing of the transmembrane (TM) segments located at the interface between TMD1 and TMD2 (17Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2002; 277: 27585-27588Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). P-gp is another ABC transporter that functions as an ATP-dependent pump. It has been used as a model system to learn about how processing mutations affect CFTR because introduction of CF-type mutations into P-gp also cause the mutant proteins to be retained in the ER in a protease-sensitive state (18Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 14671-14674Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The folding defects in P-gp, however, can be corrected by expressing the mutants in the presence of drug substrates (19Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Characterization of the ΔF508-type P-gp mutant suggested that deletion of this residue in NBD1 disrupted packing of the TM segments by affecting the interaction between NBD1 and the first cytoplasmic loop that connects TM segments 2 and 3. This in turn disrupted interactions between TMD1 and TMD2 of P-gp. Interactions between the two TMDs also appear to be critical for proper folding of CFTR since they appear to be the main contact points between the two halves of the molecule (20Ostedgaard L.S. Rich D.P. DeBerg L.G. Welsh M.J. Biochemistry. 1997; 36: 1287-1294Crossref PubMed Scopus (59) Google Scholar). In this study we used disulfide cross-linking analysis to map contact points between TMD1 and TMD2 of CFTR and then tested whether packing of the TM segments was altered by the ΔF508 mutation. Construction of Mutants—The CFTR point mutations were constructed in fragments using PCR mutagenesis. The fragments were then fully sequenced before insertion to create full-length cDNA either in pMT21 (for transient expression) or pcDNA5-FRT (for stable expression). TM6 point mutations (M348C, T351C, and W356C) were generated in the XbaI (bp 573) → KpnI (bp 1370) fragment; TM12 point mutations (T1142C and W1145C) were generated in the EcoRV (bp 2996) → EcoRI (bp 3643) fragment; the ΔF508 mutation was generated in the KpnI (bp 1370) → ApaI (bp 2333) fragment. The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590-S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410-S/C1458S) was performed using the following cDNA fragments. Point mutations C76/126S were generated in sequence in the PstI (bp 1) → XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) → KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) → ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) → EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) → XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21. Preparation of Anti-NBD2 Antibody—Antibodies were prepared against human CFTR NBD2 (residues Asn-1195—Leu-1471). Briefly, an EcoRI (bp 3643) → XhoI (bp 4560) fragment containing NBD2 was ligated into the EcoRI and SalI sites of plasmid vector pMAL-c (New England Biolabs). The vector was then digested with EcoRI and StuI, filled in with Klenow enzyme, and religated. This resulted in a fusion of NBD2 with maltose-binding protein. The maltose-binding protein-NBD2 fusion protein was expressed in Escherichia coli, affinity-purified on an amylose column, and then injected subcutaneously into New Zealand White rabbits. Immune serum obtained was stored at –70 °C and used at a 1:5000 dilution. Expression of Mutants—Subconfluent HEK293 cells were transiently transfected with 1 μg/ml cDNA constructs in pMT21 using a calcium phosphate precipitation method adapted from Chen and Okayama (21Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4821) Google Scholar). At 18–24 h post-transfection, the media (sterile-filtered Dulbecco's modified Eagle's medium plus 10% calf serum plus 5% penicillin-streptomycin plus 5% non-essential amino acids, pH 7.2) was replaced with plain media or media containing 10 μg/ml brefeldin A (Alexis). The cells were harvested after another 24–48 h. Stable CHO cell lines were generated using the Flp-In system (Invitrogen). Briefly, using a calcium phosphate precipitation method adapted from Chen and Okayama (21Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4821) Google Scholar), ∼60% confluent Flp-In CHO cells were co-transfected in a 100-mm cell culture plate with a total of 5 μg/ml cDNA constructs in pcDNA5-FRT and pOGO DNA in a 1:9 ratio. At 18 h post-transfection, the media was replaced with plain F-12 media (Invitrogen). After allowing the cells to recover for another 48 h, the cells were transferred to 5 100-mm plates containing selection media (F-12 plus 600 μg/ml hygromycin (BioShop Canada)). The selection media was then changed 48–72 h later to remove the dead cells. Colonies were picked ∼2 weeks post-transfection and transferred to 24-well plates. The selected colonies were then split into duplicates, with one set analyzed for expression using immunoblot analysis. The positive recombinant clones were subsequently maintained in the presence of 600 μg/ml hygromycin. Disulfide Cross-linking Analysis—CFTR-expressing HEK293 cells grown on 100-mm tissue culture plates were harvested and washed twice with phosphate-buffered saline. The cell pellets were subsequently suspended in 200 μl of phosphate-buffered saline. Samples of 20 μl of phosphate-buffered saline (control) or phosphate-buffered saline containing 300 μm cross-linker (1,5-pentanediyl-bismethanethiosulfonate (M5M), 3,6-dioxaoctane-1,8-diyl-bismethanethiosulfonate (M8M), 3,6,9,12,15-pentaoxaheptadecane-1,17-diyl-bismethanethiosulfonate (M17M); Toronto Research Chemicals) were added to 10 μl of cells. After incubation at room temperature for 15 min, the reactions were stopped by the addition of 130 μlof2× Laemmli sample buffer (3% SDS, 10% glycerol, 62.5 mm Tris-HCl, pH 6.8, 1 mm EDTA, 50 μg/ml AEBSF (4-(2-aminoethyl) benzenesulfonyl fluoride), 10 μg/ml aprotinin, 25 μg/ml benzamidine, 1 μg/ml E64, and 0.5 μg/ml leupeptin). To test for the effect of reducing conditions, 1 μlof1 m dithiothreitol (DTT) was added to the reactions immediately after the 15-min incubation with the thiol-reactive cross-linkers to give a final concentration of 33 mm DTT. After 15 min in the presence of DTT the cells were lysed by the addition of 130 μl of 2× Laemmli sample buffer. Samples (30 μl) of the reactions were loaded onto 7.5% SDS-PAGE gels unless otherwise indicated. The gels were then transferred onto nitrocellulose membranes and subjected to Western blot analysis using a rabbit polyclonal anti-CFTR antibody as the primary antibody and a horseradish peroxidase-labeled goat anti-rabbit antibody as the secondary antibody. The signals were detected by chemiluminescence (enhanced chemiluminescence, Pierce). Measurement of cAMP-stimulated Iodide Efflux—Stable CHO cell lines were grown to 90% confluency in triplicates in 6-well cluster tissue culture plates (Costar). Cells were incubated in the presence of 2 mm sodium butyrate 24 h before assay to enhance the levels of cell surface expression. After aspiration of the growth media, the cells were washed 3 times with 2 ml of loading buffer (136 mm NaI, 4 mm KNO3, 2 mm Ca(NO3)2,2mm Mg (NO3)2,11mm glucose, 20 mm HEPES, pH 7.4). The cells were then loaded with NaI by incubation at room temperature with 2 ml of loading buffer for 1 h in the dark. The cells were then washed 13 times at 1-min intervals with 0.5 ml of efflux buffer (136 mm NaNO3, 4 mm KNO3, 2 mm Ca(NO3)2, 2 mm Mg (NO3)2, 11 mm glucose, 20 mm HEPES, pH 7.4). Solutions from the first 10 washes were discarded. The last three samples were collected and used to create a prestimulation base line. Stimulation buffer (0.5 ml of efflux buffer plus 10 μm forskolin (Sigma)) was added to the cells and removed at 1-min intervals for 12 min. The aspirated solutions were collected and stored in the dark until all samples had been collected. The relative voltages of the samples were subsequently measured using an iodide-specific electrode (Analytical Sensors Inc.). Standard iodide curves were generated using NaI dissolved in efflux buffer to determine the iodide concentrations in the samples for data analysis. CFTR is synthesized in the endoplasmic reticulum (ER) (Fig. 1A) as a core-glycosylated immature protein (Fig. 1C, band B) that then matures as it passes through the Golgi where complex carbohydrate is added (Fig. 1C, band C). The protein is then delivered to the cell surface. The processing defective mutant ΔF508, however, is retained in the ER (Fig. 1B) as a core-glycosylated immature protein (Fig. 1C). Our goal was to determine whether the ΔF508 mutation in CFTR altered packing of the TM segments at the interface between domains TMD1 and TMD2 since this is the major contact point between the two halves of the molecule (20Ostedgaard L.S. Rich D.P. DeBerg L.G. Welsh M.J. Biochemistry. 1997; 36: 1287-1294Crossref PubMed Scopus (59) Google Scholar). Each domain is predicted to contain six TM segments, and the chloride channel through the membrane is predicted to lie at the interface of TMD1 and TMD2 (22Akabas M.H. J. Biol. Chem. 2000; 275: 3729-3732Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The first step was to map a contact point between the two domains. Our approach to map a contact point between TMD1 and TMD2 was to employ cysteine mutagenesis and reaction with thiol cross-linkers. We selected a disulfide cross-linking approach because it has successfully been employed to map contact points between the TM domains of P-gp, the CFTR sister protein (17Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2002; 277: 27585-27588Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 23Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2004; 279: 7692-7697Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 24Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2004; 279: 18232-18238Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 25Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27482-27487Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 26Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 27Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Disulfide cross-linking analysis is very useful to examine contact points between the various domains of ABC transporters because the presence of an interdomain disulfide bond causes the protein to migrate slower on non-reducing SDS-PAGE gels (17Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2002; 277: 27585-27588Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 28Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2002; 277: 41303-41306Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 29Urbatsch I.L. Gimi K. Wilke-Mounts S. Lerner-Marmarosh N. Rousseau M.E. Gros P. Senior A.E. J. Biol. Chem. 2001; 276: 26980-26987Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 30Sauna Z.E. Peng X.H. Krishnamachary N. Samrawit T. Ambubkar S.V. Mol. Pharmacol. 2004; 65: 675-684Crossref PubMed Scopus (81) Google Scholar). Therefore, it is relatively easy to assay for the presence of a disulfide bond. A potential problem with performing disulfide cross-linking analysis with CFTR, however, is that it contains 18 endogenous cysteines. Mutation of such a large number of cysteines to serines or alanines could alter the structure and properties of the protein. In preliminary studies we found that mutation of all 18 cysteines to serines caused defective maturation (Fig. 1C). It appeared, however, that it would be possible to conduct cross-linking studies in a wild-type CFTR background because no evidence of cross-linking was observed when it was treated with various methanethiosulfonate cross-linkers (see below). Promiscuous cross-linking agents such as copper phenanthroline, however, could not be used because they induced cross-linking in wild-type CFTR (data not shown). To examine inter-TMD interactions in CFTR using disulfide cross-linking, we first had to mutate individual residues in the TMDs to cysteines. We chose TM6 because there is strong evidence that TM6 plays a crucial role in the formation of the pore for chloride conductance (31Gupta J. Lindsell P. Mol. Membr. Biol. 2003; 20: 45-52Crossref PubMed Scopus (16) Google Scholar, 32Guinamard R. Akabas M.H. Biochemistry. 1999; 38: 5528-5537Crossref PubMed Scopus (35) Google Scholar). There is, however, little work done on the other TMs of CFTR. Studies on the TM packing of P-gp, the sister protein of CFTR, however, show interactions between TM6 and TM12 (25Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27482-27487Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 26Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Thus, residues in the cytosolic halves of TM6 and TM12 of CFTR were individually mutated to cysteine using site-directed mutagenesis (see Fig. 2A). The cytosolic halves of the TMs were chosen for this study based on the conical-shaped model proposed by Akabas (22Akabas M.H. J. Biol. Chem. 2000; 275: 3729-3732Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Based on the extensive cysteine-scanning mutagenesis studies on the TMs done by Akabas and co-workers (32Guinamard R. Akabas M.H. Biochemistry. 1999; 38: 5528-5537Crossref PubMed Scopus (35) Google Scholar, 33Akabas M.H. Kaufmann C. Cook T.A. Archdeacon P. J. Biol. Chem. 1994; 269: 14865-14868Abstract Full Text PDF PubMed Google Scholar, 34Cheung M. Akabas M.H. J. Gen. Physiol. 1997; 109: 289-299Crossref PubMed Scopus (85) Google Scholar, 35Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 36Akabas M.H. Biochemistry. 1998; 37: 12233-12240Crossref PubMed Scopus (37) Google Scholar) they proposed that the pore of the CFTR chloride channel is conical-shaped, with the cytosolic side of the TMs close together. Single substitution of the TM residues with cysteine had little deleterious effect on the maturation of CFTR as most single cysteine mutants of TMs 6 and 12 had similar levels of maturation as that of WT CFTR (data not shown). Subsequently, paired-cysteine CFTR mutants containing a cysteine in TM6 and another in TM12 were constructed and tested for cross-linking with methanethiosulfonate cross-linkers (Fig. 3A). These methanethiosulfonate cross-linkers have thiol-reactive groups at both ends that are linked by spacer arms of various lengths (27Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Alkylthiosulfonates react selectively with cysteines in protein, resulting in a disulfide attachment of the spacer arm and release of a sulfinic acid byproduct (37Kenyon G.L. Bruice T.W. Methods Enzymol. 1977; 47: 407-430Crossref PubMed Scopus (171) Google Scholar, 38Bruice T.W. Kenyon G.L. J. Protein Chem. 1982; 1: 47-58Crossref Scopus (94) Google Scholar). The methanethiosulfonate compounds are generally more reactive than other thiol-specific compounds such as maleimides or iodoacetates (38Bruice T.W. Kenyon G.L. J. Protein Chem. 1982; 1: 47-58Crossref Scopus (94) Google Scholar). For our cross-linking assays, HEK293 cells transiently transfected with individual constructs of CFTR cDNA were harvested and treated with or without the thiol-reactive cross-linkers, M5M, M8M, or M17M. The cells were incubated with the cross-linkers for 15 min at room temperature, then lysed with the 2× SDS sample buffer and subjected to immunoblot analysis. Cross-linking of CFTR would cause the protein to migrate slower on SDS-PAGE gels. As shown in Fig. 3B, the addition of the thiol-reactive cross-linkers to cells expressing WT CFTR does not lead to cross-linking. Three positive cross-linking mutants, M348C/T1142C, T351C/T1142C, and W356C/W1145C were identified (see Fig. 3B, band X) and selected for further study. These were useful mutants because none of the mutations appeared to affect maturation of the protein. Fig. 2B shows the expression of WT CFTR, the single cysteine mutants M348C, T351C, W356C, T1142C, and W1145C, and the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C. All of the mutants yielded relatively high levels of mature CFTR proteins (band C) relative to the core-glycosylated form of the protein (band B). The cross-linking patterns of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C showed differences when treated with different cross-linkers. Mutant M348C/T1142C, for example, showed cross-linking with M5M and M8M but not with M17M. Mutant T351C/T1142C, on the other hand, shows extensive cross-linking with M8M but not with M5M or M17M. It is interesting to note that both M348C and T351C in TM6 showed cross-linking to T1142C in TM12. Residue Met-348 is three residues away from Thr-351, which would put them on the same face of an α-helix. Therefore, it is not surprising that the substituted cysteines at both of these positions would cross-link to the same residue, T1142C. The positive mutant, W356C/W1145C, showed cross-linking with all three cross-linkers (Fig. 3B). Most of the other mutants tested did not show cross-linking with M5M, M8M, and M17M. An example of a mutant that did not show cross-linking, T351C/L1143C, is shown in Fig. 3B. Because the cross-linkable mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C also contained the 18 endogenous cysteines, it was important to test whether any of the single M348C, T351C, W356C, T1142C, or W1145C mutants showed evidence of cross-linking with endogenous cysteines. Accordingly, cells expressing each of the single cysteine mutants were treated with M5M, M8M, or M17M, and samples were subjected to immunoblot analysis. Fig. 3B, right panel, shows that none of the single cysteine mutants showed cross-linking. Reduced levels of the T352C and W356C mutants, however, were observed with M8M, and this may be due to aggregation. Aggregation of CFTR protein is normally observed in cells (39Kopito R.R. Trends Cell Biol. 2000; 10: 524-530Abstract Full Text Full Text PDF PubMed Scopus (1602) Google Scholar) and caused some problems in these studies. In the immunoblot analysis of cross-linking only 10 μl of the assay samples were loaded in the negative control lanes (i.e. no cross-linker added) compared with the 30 μl that was loaded in the cross-linker lanes. This is because the cross-linkers, particularly M5M and M8M, also caused nonspecific cross-linking that leads to aggregation of the CFTR protein as shown in Fig. 4. Fig. 4 shows an overexposed immunoblot of the effect of cross-linkers on WT and ΔF508 CFTR. The presence of the M5M and M8M cross-linkers led to accumulation of aggregates in the stacking gel. This is similar to the nonspecific aggregates we observed when we treated the cells with oxidants such as copper phenanthroline. The aggregates are likely due to reaction of the thiol-reactive cross-linkers with endogenous cysteines between CFTR molecules or between CFTR and other proteins. In addition, the studies were complicated by the presence of aggregates even in the absence of cross-linker (Fig. 4, lanes 0). It is also worth noting that it is possible that the efficiency of intramolecular cross-linking would be reduced if a separate cross-linker labels each of the introduced cysteines. Attachment of separate cross-linker labels to each cysteine or incomplete cross-linking (cross-linker only attached to one of the cysteines) may also promote aggregation when the reaction is stopped. It is also possible that formation of an intramolecular disulfide bond between TM6 and TM12 blocks aggregation. Despite the problems with aggregation, cross-l" @default.
• W2001530819 created "2016-06-24" @default.
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• W2001530819 title "The ΔF508 Mutation Disrupts Packing of the Transmembrane Segments of the Cystic Fibrosis Transmembrane Conductance Regulator" @default.
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• W2001530819 doi "https://doi.org/10.1074/jbc.m407887200" @default.
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