Departments of 1Biomedical Sciences and 2Medical Physiology and Pharmacology and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211
Submitted 5 August 2003 ; accepted in final form 10 March 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
protein processing; mouse; retrovirus; gene therapy
Defective processing of F508 hCFTR is at least partially reversible. Studies in cells expressing recombinant
F508 hCFTR and tissues cultured from transgenic
F508 murine (m)CFTR mice have shown that a fraction of the mutant protein undergoes biosynthetic maturation when cells are grown at subphysiological temperatures (
26°C) (13). Studies in which
F508 hCFTR-expressing cells were treated with "chemical chaperones" such as glycerol and dimethyl sulfoxide (DMSO) or organic cellular osmolytes such as myo-inositol and betaine have also found increased maturation of
F508 hCFTR, a result that was attributed to stabilization of the mutant protein (1, 3, 22, 52). Overexpression of the mutant protein either by transfection or by treatment with butyrate results in trafficking of
F508 hCFTR to the plasma membrane (4, 40). The above observations have important therapeutic implications because
F508 hCFTR in the plasma membrane retains its capacity as a cyclic nucleotide-regulated anion channel (13). Studies in cell-free systems indicate that stimulated
F508 hCFTR may have channel kinetics similar to wild-type hCFTR (23, 31, 42), whereas on-cell patch studies measure reduced channel kinetic activity compared with wild-type hCFTR (12, 20, 24). Even if
F508 CFTR has reduced function and shorter membrane residence (32), previous studies suggest that only a small amount (
1015%) of
F508 CFTR activity in the membrane is required to restore epithelial function and moderate disease (15, 26). Combined with recent pharmacological studies showing that
F508 hCFTR channel activity can be significantly enhanced by treatment with compounds such as 8-cyclopentyl-1,3-dipropylxanthine (CPX), genistein, or class III phosphodiesterase inhibitors (28), it is clear that correcting
F508 hCFTR trafficking is a viable strategy for treating CF.
Recent studies have shown that expression of polypeptides representing the structural domain of a protein (i.e., "domain mimics") can predictably alter posttranslational processing of the protein. Expression of a minigene encoding the carboxy terminus of human ether-à-go-go-related gene was shown to rescue a carboxy-terminal truncation mutant from misprocessing (29). In another study, a 6-mer peptide mimic of the reactive center loop of 1-antitrypsin was used to block pathological polymerization of the Z variant mutation of the protein (35). With regard to CFTR, expression of a polypeptide representing the NBF1 plus the regulatory "R" domain of hCFTR was shown to inhibit activity of ENaC, the epithelial Na+ channel reputedly regulated by CFTR (45). Because the NBF1 domain of hCFTR is the location of
F508 as well as several other processing mutants (46), the NBF1 domain itself may serve as a "checkpoint" for recognition by the cellular quality control machinery or, alternatively, may function in a critical intramolecular interaction that stabilizes hCFTR structure within the ER. Therefore, we hypothesized that expression of the NBF1 domain may be sufficient to increase
F508 CFTR activity in epithelial cells. The NBF1 domain of CFTR is well conserved among mammalian species (17), and in the mouse, deletion of F508 results in a mCFTR processing defect that recapitulates the fundamental cellular defect of
F508 homozygous CF patients (16, 50). In the present study, both heterologous cell systems expressing recombinant
F508 hCFTR and epithelial monolayers from a
F508 CFTR homozygous mouse were used to test the hypothesis that expression of a NBF1 domain mimic increases trafficking
F508 CFTR and restores cAMP-stimulated anion secretion.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Stable expression of NBF domain mimics.
NotI restriction endonuclease site-flanked cDNA constructs of wild-type NBF1 and F508 NBF1 (coding for amino acids 429591 of full-length hCFTR) with a flag epitope (DYKDDDDK) inserted at the carboxy terminus were prepared as previously described (6). The constructs were subcloned into the Zeocin-selectable vectors pZEOSV or pCDNA3.1 (Invitrogen, Carlsbad, CA). C127 cell lines were transfected with the NBF1 construct plasmids by electroporation and selected in medium containing 200 µg/ml Zeocin. Clones were isolated 2 wk after electroporation. For stable expression of the NBF1 construct in the polarized MGEF and MGEN epithelial cell lines (passages 316), the NotI-flanked construct was subcloned into the retroviral vector pLXSNz [i.e., pLXSN (a gift from Dr. John Olsen, University of North Carolina) that was engineered with Zeocin resistance] to form the proviral component NBF1-LXSNz. As a positive control, hCFTR cDNA was introduced into the plasmid to produce the proviral component hCFTR-LXSNz. Transfection by Ca/PO4 precipitation of NBF1-LXSNz, hCFTR-LXSNz, and pLXSNz (empty vector control) into the virus-packaging cell line PA317 led to the production of replication-defective virus. Virus stock supernatant was used to infect the MGEF and MGEN cells, which were subsequently selected in medium containing 200 µg/ml Zeocin. Transfected cell lines were screened for expression of the NBF1 constructs by RT-PCR essentially as described previously (19) utilizing a gene-specific primer (5'-GGATCCACTGGAGCAGGCAAG-3') in combination with a primer specific for the flag epitope of the NBF1 domain mimic (5'-ATCATCGTCGTCTTTGTAGTC-3').
Whole cell patch clamp. C127 cells were grown on glass coverslips for whole cell patch-clamp recordings as described previously (47). Briefly, a Cl gradient was established with the following solutions: pipette (in mM): 85 aspartic acid, 5 pyruvic acid, 10 EGTA, 10 HEPES, 20 tetraethylammonium-Cl, 5 Tris, 10 MgATP, 2 MgCl2, and 5.5 glucose (pH 7.4 with CsOH); bath (in mM): 145 NaCl, 5 glucose, 5 HEPES, 2 MgCl2, 1 CaCl2, 5 KCl, and 20 sucrose (pH 7.4 with NaOH). An outward current generated by Cl influx was observed at a holding potential of 0 mV on activation of CFTR. The holding current at 0 mV was normalized with the membrane capacitance to yield current density. Current traces in response to voltage pulses (±100 mV at 20-mV increments) were filtered at 1 kHz with a built-in four-pole Bessel filter and digitized (at 2 kHz) through an ITC-16 interface (Instrutech, Great Neck, NY).
Ussing chambers. MGEN and MGEF cells were cultured on a rat tail collagen membrane covering a 4.5-mm-diameter aperture in polycarbonate cups as previously described (10). Bioelectric measurements were made under voltage-clamp conditions with intermittent voltage spikes to calculate transepithelial resistance (with Ohm's law). The preparations were bathed on the luminal and basolateral surfaces with warmed (37°C), oxygenated Krebs bicarbonate Ringer (KBR) solution containing (in mM) 115 NaCl, 4 K2HPO4, 0.4 KH2PO4, 25 NaHCO3, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose at pH 7.4. In ion substitution studies, gluconate and sulfate replaced Cl, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) replaced HCO3, and tetramethylammonium (TMA+) replaced Na+ on an equimolar basis.
Detection of hCFTR and F508 hCFTR glycosylation status by immunoprecipitation-32P labeling.
Detection of hCFTR and its glycosylation status was performed as described by Gregory et al. (18). Cultured cells (1 confluent T-25 flask) were homogenized in 1 ml of lysis buffer [50 mM Tris pH 7.4, 150 mM NaCl, 10 mM MgCl2, 1% (vol/vol) Triton X-100, 1% (wt/vol) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1 mM Pefabloc, 10 µg/ml aprotinin]. After low-speed centrifugation to remove insoluble material (14,000 g), CFTR was immunoprecipitated with 1.5 µg of the monoclonal antibody M3A7 (27), which recognizes an epitope within the NBF2 domain (Genzyme), and the addition of protein A agarose beads. After tumbling (2 h, 4°C), the beads were washed three times in lysis buffer and incubated for in vitro phosphorylation in buffer containing 25 mU protein kinase A, 0.2 mM rATP, and 50 µCi of [
-32P]ATP for 10 min at 30°C. After washing to remove unincorporated [
-32P]ATP, bound phosphorylated protein was eluted from the beads into SDS-PAGE sample buffer at room temperature for 1 h. Total protein was separated by SDS-PAGE on 420% gels, dried, and autoradiographed (17 days). Densitometric measurements were performed on a Kodak Image Station 2000R (Rochester, NY) with 1D Image Analysis software.
Materials. Forskolin, genistein, and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) were diluted in DMSO to stock concentrations of 10, 10, and 100 mM, respectively. Amiloride was diluted to a stock concentration of 10 mM in distilled water. All reagents were obtained from either Sigma (St. Louis, MO) or Fisher Scientific (Springfield, NJ).
Statistical analysis. Data are expressed as means ± SE. Data between two treatment groups were compared with a two-tailed Student's t-test assuming equal variances. Data from more than two treatment groups were compared with a one-way ANOVA with a post-hoc Student-Newman-Keuls test. A probability value of P < 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Expression of a NBF1 domain mimic in F508/
F508 CFTR murine epithelial monolayers reconstitutes cAMP-stimulated anion secretion.
The studies described above demonstrate that coexpression of the NBF1 domain mimic can increase plasma membrane activity of
F508 hCFTR in an epithelial cell line expressing recombinant protein driven by a nonnative promoter. Because the level of
F508 hCFTR expression can affect trafficking in the cell (4), we asked whether expression of the NBF1 domain mimic would also improve plasma membrane trafficking of endogenously expressed
F508 CFTR in a polarized epithelium. After an extensive search, we were unable to identify a human
F508/
F508 CFTR epithelial cell line that retained the ability to both form polarized epithelial monolayers and express significant levels of the endogenous
F508 hCFTR protein for the retroviral transfection studies. Because previous studies demonstrated that defects in processing and plasma membrane expression of the
F508 mCFTR homolog in murine epithelia recapitulate the defects of
F508 hCFTR (16), an epithelial cell line (MGEF) developed by transformation of gallbladder epithelia from a
F508/
F508 CFTR mutant mouse model was transfected with the NBF1 domain mimic (50).
As shown by the short-circuit current (Isc) trace from an Ussing chamber experiment (Fig. 2A), expression of the NBF1 domain mimic in the MGEF epithelial monolayer not only increased the basal Isc [means for n = 9 monolayers: NBF1 = 18.5 ± 2.5 µA/cm2 vs. zeomycin-resistance gene (Zeo) = 6.4 ± 1.2 µA/cm2; P < 0.05] but, more importantly, yielded a distinct Isc response to forskolin stimulation compared with MGEF cells transduced with only the empty vector control (pLXSNz). Subsequent treatment of the monolayer with the CFTR channel blocker NPPB inhibited both the cAMP-stimulated Isc and a large fraction of the basal Isc. Although the ionic basis of the residual Isc was not investigated, previous studies indicate the presence of a Ca2+-activated anion channel in the apical membrane of the MGE epithelial cells (9). As a negative control, the mCFTR-null cell line MGEN, developed by transformation of gallbladder epithelium from a CFTR knockout mouse, was also transfected with the NBF1 construct for comparison with the NBF1-expressing MGEF cells. Expression of the NBF1 domain mimic in the MGEN cells slightly increased the basal Isc (means for n = 5 monolayers: NBF1 = 4.7 ± 0.4 vs. Zeo = 1.6 ± 0.6 µA/cm2; P < 0.05). However, in contrast to cAMP-induced Isc responses in the NBF1-expressing MGEF cells, expression of the NBF1 domain mimic in the MGEN cells did not restore the Isc response to cAMP stimulation (see cumulative data in Fig. 2B). As a positive control for the transfection and selection procedure, hCFTR was expressed in the CFTR(/) MGEN cell line by the same transduction process used for the NBF1-expressing cell lines. Expression of hCFTR in the MGEN cell monolayers resulted in a significantly greater basal Isc (mean for n = 7 monolayers: hCFTR = 6.1 ± 1.6 µA/cm2; P < 0.05) and, as shown in Fig. 1C, a greater stimulation of Isc after forskolin treatment than MGEN cells transduced with only pLXSNz.
|
cAMP-stimulated Isc is maintained in successive passages of the NBF1-expressing MGEF epithelial cell line. Previous studies showed that expression of recombinant CFTR can induce downregulation of the cftr gene and adversely affect cell proliferation (41). Therefore, we compared the stability of the Isc response in the NBF1-expressing MGEF cell line with that in the hCFTR-expressing MGEN cell line by measuring the cAMP-stimulated Isc response of monolayers from successive passages of the two cell lines. As shown in Fig. 3, the NBF1-expressing MGEF cell monolayers maintained an almost identical magnitude of cAMP-stimulated Isc response for over 30 passages (passaged every 47 days). In contrast, the magnitude of the cAMP-stimulated Isc progressively decreased in hCFTR-transfected MGEN cells with successive passages and was essentially absent after passage 9.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Restoration of cAMP-stimulated anion secretion in the F508 CFTR-expressing epithelial cells yielded bioelectrical responses with characteristics consistent with activation of a
F508 CFTR conductance. In studies of
F508 hCFTR cells expressing NBF1, genistein-forskolin treatment increased whole cell Cl currents to
50% of those exhibited by C127 cells stably expressing wild-type CFTR. This difference, whether due to reduced open probability or to numbers of membrane channels, is similar to that observed when
F508 hCFTR cells are exposed to reduced ambient temperature or after increased expression of the mutant CFTR (4, 13, 16, 40). In studies of the polarized MGEF monolayers expressing NBF1, the macroscopic Isc response to forskolin treatment activated at a slower rate compared with MGEN cells expressing wild-type hCFTR (data not shown), which might reflect a characteristic of the
F508 CFTR protein that attenuates its protein kinase A phosphorylation-dependent activation (47). However, in contrast to the C127 cells, the absolute magnitude of the cAMP-induced
Isc in the NBF1-expressing MGEF monolayers was approximately the same as in the MGEN monolayers expressing recombinant hCFTR. This variance may reflect differences in the activities of the recombinant vs. the endogenous CFTR channel or result from inherent differences in
F508 CFTR properties between the two species (30). Interestingly, the basal Isc of the NBF1-expressing MGEF and MGEN cells was elevated compared with the Zeocin-expressing control monolayers. This change is consistent with previous demonstrations that expression of NBF1 yields a transmembrane configuration of the peptide and an increase in the basal halide permeability in mammalian cell lines (7, 19). However, similar to our findings with the NBF1-expressing MGEN cells, expression of only the NBF1 domain did not result in cAMP-regulated anion permeability. In the present study, restoration of cAMP-regulated anion secretion required NBF1 coexpression in
F508 CFTR-expressing epithelial cells.
Expression of cytosolic domain mimics has previously been used to alter specific intra- and intermolecular interactions within cells. Pioneering work by Lefkowitz and coworkers (33) used domain mimics to disrupt interactions between plasma membrane receptors and G proteins coupled to phospholipase C activation. More recently, a domain mimic expressed via a minigene was used to rescue misprocessing of a carboxy-terminal truncation mutant of human ether-à-go-go-related protein (29) and a domain peptide was used to selectively mask portions of mutant 1-antitrypsin to prevent posttranslational polymerization within hepatocytes (35). With regard to CFTR, exogenous copies of the R domain have been shown to interact with full-length CFTR and modify gating behavior of its channel activity (34). Domain mimics of CFTR and the integral membrane protein syntaxin 1A were used to modify channel activity and demonstrate a direct protein-protein interaction (37). Furthermore, peptide mimics have been used to demonstrate binding interactions between NBF1 and the R domain of CFTR, which could be partially disrupted by introduction of the
F508 mutation (6).
Given the findings of the present study and previous observations with expression of recombinant F508 CFTR (4, 40), at least two mechanisms can be postulated that would result in increased processing of
F508 CFTR beyond the ER. First, expression of recombinant NBF1 may overwhelm the quality control system that is involved in retention and degradation of the mutant protein. A similar process could be postulated for the plasma membrane appearance of
F508 hCFTR when the mutant protein is overexpressed in heterologous cell systems (4, 40). This mechanism would predict competitive binding for, perhaps specific, chaperone proteins or proteases, thereby allowing escape of mutant protein from the ER. However, some evidence weakens the case for this proposed mechanism. Our measures of protein expression in the C127 cells indicated that clones expressing wild-type NBF1 were more likely to have greater expression of the fully processed
F508 CFTR compared with clones expressing the
F508 NBF1 peptide (Fig. 1). This seems contrary to the prediction that
F508 NBF1 would be more efficacious in competing for quality control proteins. Furthermore, although it is possible that the concentration of the recombinant NBF1 at the quality control binding site(s) far exceeds that needed to discern differences between the two domain mimics, our estimates of NBF1 peptide concentration (<5 ng/µl in cell pellets, as based on detection of the flag epitope with M2 monoclonal antibody) are well below the millimolar concentrations that are typically necessary to inhibit proteases with chemical inhibitors (25, 49). The second potential mechanism by which NBF1 expression may lead to increased processing of
F508 CFTR is that coexpression of additional NBF1 peptides may bind immature
F508 CFTR and stabilize its structure in the ER. For example, peptide interaction with the R domain of
F508 CFTR may mask a site on the misfolded protein that is recognized by the quality control machinery, or additional copies of NBF1 may temporarily interact with
F508 CFTR to catalyze a folding reaction that results in properly folded protein. Given recent evidence that mature CFTR protein may dimerize in the plasma membrane (51), it seems plausible that cooperation between different CFTR monomers may be necessary for correct folding and that this interaction may be mimicked by the NBF1 domain peptides.
Although the precise mechanism resulting in increased expression of F508 CFTR at the plasma membrane using coexpression of NBF1 peptides remains speculative, the significant aspect of the present study is that it suggests a refinement for therapeutic approaches to correcting
F508 CFTR processing. One major drawback to the use of viral vectors such as adeno-associated virus 2 for gene therapy is that the packaging capacity of the virus is limited, which, with a large gene like CFTR, leaves little capacity for promoter sequences (14). Thus modification of the strategy to instead transfect only the NBF1 domain is attractive because the small size of the construct (
0.5 kb) would improve packaging efficiency. Furthermore, two biological advantages of using NBF1 domain transduction as therapy can be envisioned. First, the expression of
F508 CFTR is driven by its endogenous promoter, which may avoid the consequences of CFTR overexpression on the epithelium, e.g., reduced cell proliferation potential (41). A second advantage is that the NBF1 peptide is less likely to be toxic than the full-length channel protein when inadvertently expressed in nontarget tissues. The NBF1 construct fused to a protein transduction domain (43) may also be useful for peptide therapy, especially if the smallest effective subunit of NBF1 can be defined. Thus, on the basis of the observations in the present study, the therapeutic approach to correcting the basic protein-processing defect of
F508 CFTR should be refined to focus on NBF1 domain mimics that alter trafficking of the mutant protein.1
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
Present address of D. B. Gruis: Pioneer Hi-Bred International, Inc., 400 Locust St., Suite 800, PO Box 14453, Des Moines, IA 50306-3453.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 An abstract presented at the 16th Annual North American Cystic Fibrosis Conference reported that coexpression of F508 CFTR with an amino-terminal fragment of CFTR (amino acids 1633) induced the maturation of
F508 CFTR in Cos7 cells (Cormet-Boyaka EA and Kirk KL. Trans-complementation of
508-CFTR by an amino terminal fragment of CFTR. Pediatr Pulmonol Suppl 24: 183, 2002).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bradbury NA, Jilling T, Berta G, Sorscher EJ, Bridges RJ, and Kirk KL. Regulation of plasma membrane recycling by CFTR. Science 256: 530531, 1992.[ISI][Medline]
3. Cheng SH, Fang S, Zabner J, Marshall J, Piraino S, Schiavi SC, Jefferson DM, Welsh MJ, and Smith AE. Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation. J Biol Chem 271: 635638, 1996.
4. Cheng SH, Fang SL, Zabner J, Marshall J, Piraino S, Schiavi SC, Jefferson DM, Welsh MJ, and Smith AE. Functional activation of the cystic fibrosis trafficking mutant F508-CFTR by overexpression. Am J Physiol Lung Cell Mol Physiol 268: L615L624, 1995.
5. Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, White GA, O'Riordan CR, and Smith AE. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63: 827834, 1990.[ISI][Medline]
6. Ciaccia AV, Pitterle DM, and Price EM. Domain-domain interactions in the CFTR (Abstract). Pediatr Pulmonol Suppl 10: 180, 1994.
7. Clancy JP, Hong JS, Bebok Z, King SA, Demolombe S, Bedwell DM, and Sorscher EJ. Cystic fibrosis transmembrane conductance regulator (CFTR) nucleotide-binding domain 1 (NBD-1) and CFTR truncated within NBD-1 target to the epithelial plasma membrane and increase anion permeability. Biochemistry 37: 1522215230, 1998.[CrossRef][ISI][Medline]
8. Clarke LL and Harline MC. CFTR is required for cAMP inhibition of intestinal Na+ absorption in a cystic fibrosis mouse model. Am J Physiol Gastrointest Liver Physiol 270: G259G267, 1996.
9. Clarke LL, Harline MC, Ortero MA, Glover GG, Garrad RC, Krugh B, Walker NM, Gonzalez FA, Turner JT, and Weisman GA. Desensitization of P2Y2 receptor-activated transepithelial anion secretion. Am J Physiol Cell Physiol 276: C777C787, 1999.
10. Clarke LL, Paradiso AM, Mason SJ, and Boucher RC. Effects of bradykinin on Na+ and Cl transport in human nasal epithelium. Am J Physiol Cell Physiol 262: C644C655, 1992.
11. Collins FS. Cystic fibrosis: molecular biology and therapeutic implications. Science 256: 774779, 1992.[ISI][Medline]
12. Dalemans W, Barbry P, Champigny G, Jallat S, Dott K, Dreyer D, Crystal RG, Pavirani A, Lecocq JP, and Lazdunski M. Altered chloride ion channel kinetics associated with the F508 cystic fibrosis mutation. Nature 354: 526528, 1991.[CrossRef][ISI][Medline]
13. Denning GM, Anderson MP, Amara J, Marshall J, Smith AE, and Welsh MJ. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 358: 761764, 1992.[CrossRef][ISI][Medline]
14. Dong JY, Fan PD, and Frizzell RA. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther 7: 21012112, 1996.[ISI][Medline]
15. Dorin JR, Stevenson BJ, Fleming S, Alton EW, Dickinson P, and Porteous DJ. Long-term survival of the exon 10 insertional cystic fibrosis mutant mouse is a consequence of low level residual wild-type Cftr gene expression. Mamm Genome 5: 465472, 1994.[ISI][Medline]
16. French PJ, van Doorninck JH, Peters RH, Verbeek E, Ameen NA, Marino CR, De Jonge HR, Bijman J, and Scholte BJ. A F508 mutation in mouse cystic fibrosis transmembrane conductance regulator results in a temperature-sensitive processing defect in vivo. J Clin Invest 98: 13041312, 1996.
17. Gasparini P, Nunes V, Dognini M, and Estivill X. High conservation of sequences involved in cystic fibrosis mutations in five mammalian species. Genomics 10: 10701072, 1991.[ISI][Medline]
18. Gregory RJ, Cheng SH, Rich DP, Marshall J, Paul S, Hehir K, Ostedgaard LS, Klinger KW, Welsch MJ, and Smith AE. Expression and characterization of the cystic fibrosis transmembrane conductance regulator. Nature 347: 382386, 1990.[CrossRef][ISI][Medline]
19. Gruis DB and Price EM. The nucleotide binding folds of the cystic fibrosis transmembrane conductance regulator are extracellularly accessible. Biochemistry 36: 77397745, 1997.[CrossRef][ISI][Medline]
20. Haws C, Nepomuceno IB, Krouse ME, Wakelee H, Law T, Xia Y, Nguyen H, and Wine JJ. F508-CFTR channels: kinetics, activation by forskolin, and potentiation by xanthines. Am J Physiol Cell Physiol 270: C1544C1555, 1996.
21. Heda GD and Marino CR. Surface expression of the cystic fibrosis transmembrane conductance regulator mutant F508 is markedly upregulated by combination treatment with sodium butyrate and low temperature. Biochem Biophys Res Commun 271: 659664, 2000.[CrossRef][ISI][Medline]
22. Howard M, Fischer H, Roux J, Santos BC, Gullans SR, Yancey PH, and Welch WJ. Mammalian osmolytes and S-nitrosoglutathione promote F508 cystic fibrosis transmembrane conductance regulator (CFTR) protein maturation and function. J Biol Chem 278: 3515935167, 2003.
23. Hwang TC, Wang F, Yang ICH, and Reenstra WW. Genistein potentiates wild-type and F508-CFTR channel activity. Am J Physiol Cell Physiol 273: C988C998, 1997.
24. Hwang TC and Sheppard DN. Molecular pharmacology of the CFTR Cl channel. Trends Pharmacol Sci 20: 448453, 1999.[CrossRef][ISI][Medline]
25. Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, and Riordan JR. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83: 129135, 1995.[ISI][Medline]
26. Johnson LG, Olsen JC, Sarkadi B, Moore KL, Swanstrom R, and Boucher RC. Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis. Nat Genet 2: 2125, 1992.[ISI][Medline]
27. Kartner N, Augustinas O, Jensen TJ, Naismith AL, and Riordan JR. Mislocalization of delta F508 CFTR in cystic fibrosis sweat gland. Nat Genet 1: 321327, 1992.[ISI][Medline]
28. Kelley TJ, Al-Nakkash L, Cotton CU, and Drumm ML. Activation of endogenous F508 cystic fibrosis transmembrane conductance regulator by phosphodiesterase inhibition. J Clin Invest 98: 513520, 1996.
29. Kupershmidt S, Yang T, Chanthaphaychith S, Wang Z, Towbin JA, and Roden DM. Defective human ether-à-go-go-related gene trafficking linked to an endoplasmic reticulum retention signal in the C terminus. J Biol Chem 277: 2744227448, 2002.
30. Lansdell KA, Delaney SJ, Lunn DP, Thomson SA, Sheppard DN, and Wainwright BJ. Comparison of the gating behaviour of human and murine cystic fibrosis transmembrane conductance regulator Cl channels expressed in mammalian cells. J Physiol 508: 379392, 1998.
31. Li C, Ramjeesingh M, Reyes E, Jensen TJ, Chang XB, Rommens JM, and Bear CE. The cystic fibrosis mutation (F508) does not influence the chloride channel activity of CFTR. Nat Genet 3: 316, 1993.
32. Lukacs GL, Chang XB, Bear C, Kartner N, Mohamed A, Riordan JR, and Grinstein S. The F508 mutation decreases the stability of cystic fibrosis transmembrane conductance regulator in the plasma membrane. J Biol Chem 268: 2159221598, 1993.
33. Luttrell LM, Ostrowski J, Cotecchia S, Kendall H, and Lefkowitz RJ. Antagonism of catecholamine receptor signaling by expression of cytoplasmic domains of the receptors. Science 259: 14531456, 1993.[ISI][Medline]
34. Ma J, Zhao J, Drumm ML, Xie J, and Davis PB. Function of the R domain in the cystic fibrosis transmembrane conductance regulator chloride channel. J Biol Chem 272: 2813328141, 1997.
35. Mahadeva R, Dafforn TR, Carrell RW, and Lomas DA. 6-mer Peptide selectively anneals to a pathogenic serpin conformation and blocks polymerization. Implications for the prevention of Z 1-antitrypsin-related cirrhosis. J Biol Chem 277: 67716774, 2002.
36. Marshall J, Fang S, Ostedgaard LS, O'Riordan CR, Ferrara D, Amara JF, Hoppe H, Scheule RK, Welsh MJ, Smith AE, and Cheng SH. Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro. J Biol Chem 269: 29872995, 1994.
37. Naren AP, Quick MW, Collawn JF, Nelson DJ, and Kirk KL. Syntaxin 1A inhibits CFTR chloride channels by means of domain-specific protein-protein interactions. Proc Natl Acad Sci USA 95: 1097210977, 1998.
38. Pilewski JM and Frizzell RA. Role of CFTR in airway disease. Physiol Rev 79: S215S255, 1999.[Medline]
39. Pind S, Riordan JR, and Williams DB. Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator. J Biol Chem 269: 1278412788, 1994.
40. Rubenstein RC and Zeitlin PL. A pilot clinical trial of oral sodium 4-phenylbutyrate (Buphenyl) in F508-homozygous cystic fibrosis patients: partial restoration of nasal epithelial CFTR function. Am J Respir Crit Care Med 157: 484490, 1998.[ISI][Medline]
41. Schiavi SC, Abdelkader N, Reber S, Pennington S, Narayana R, McPherson JM, Smith AE, Hoppe H IV, and Cheng SH. Biosynthetic and growth abnormalities are associated with high-level expression of CFTR in heterologous cells. Am J Physiol Cell Physiol 270: C341C351, 1996.
42. Schultz BD, Frizzell RA, and Bridges RJ. Rescue of dysfunctional F508-CFTR chloride channel activity by IBMX. J Membr Biol 170: 5166, 1999.[CrossRef][ISI][Medline]
43. Schwarze SR, Ho A, Vocero-Akbani A, and Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285: 15691572, 1999.
44. Skach W. Defects in processing and trafficking of the cystic fibrosis transmembrane conductance regulator. Kidney Int 57: 825831, 2000.[CrossRef][ISI][Medline]
45. Stutts MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC, and Boucher RC. CFTR as a cAMP-dependent regulator of sodium channels. Science 269: 847850, 1995.[ISI][Medline]
46. Tsui LC. The spectrum of cystic fibrosis mutations. Trends Genet 8: 392398, 1992.[ISI][Medline]
47. Wang F, Zeltwanger S, Hu SU, and Hwang TC. Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels. J Physiol 524: 637648, 2000.
48. Ward CL and Kopito RR. Intracellular turnover of cystic fibrosis transmembrane conductance regulator. Inefficient processing and rapid degradation of wild-type and mutant proteins. J Biol Chem 296: 2571025718, 1994.
49. Ward CL, Omura S, and Kopito RR. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83: 121127, 1995.[ISI][Medline]
50. Zeiher BG, Eichwald E, Zabner J, Smith AP, Puga PB, McCray PB, Capecchi MR, Welsh MJ, and Thomas KR. A mouse model for the F508 allele of cystic fibrosis. J Clin Invest 96: 20512064, 1995.[ISI][Medline]
51. Zerhusen B, Zhao JY, Xie JX, Davis PB, and Ma JJ. A single conductance pore for chloride ions formed by two cystic fibrosis transmembrane conductance regulator molecules. J Biol Chem 274: 76277630, 1999.
52. Zhang XM, Wang XT, Yue H, Leung SW, Thibodeau PH, Thomas PJ, and Guggino SE. Organic solutes rescue the functional defect in F508 cystic fibrosis transmembrane conductance regulator. J Biol Chem 278: 5123251243, 2003.
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |