A domain mimic increases {Delta}F508 CFTR trafficking and restores cAMP-stimulated anion secretion in cystic fibrosis epithelia

Lane L. Clarke,1 Lara R. Gawenis,1 Tzyh-Chang Hwang,2 Nancy M. Walker,1 Darren B. Gruis,1 and Elmer M. Price1

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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The major disease-causing mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine 508 ({Delta}F508), which adversely affects processing and plasma membrane targeting of CFTR. Under conditions predicted to stabilize protein folding, {Delta}F508 CFTR is capable of trafficking to the plasma membrane and retains cAMP-regulated anion channel activity. Overexpression is one factor that increases CFTR trafficking; therefore, we hypothesized that expression of a domain mimic of the first nucleotide-binding fold (NBF1) of CFTR, i.e., the site of F508, may be sufficient to overwhelm the quality control process or otherwise stabilize {Delta}F508 CFTR and thereby restore cAMP-stimulated anion secretion. In epithelial cells expressing recombinant {Delta}F508 human (h)CFTR, expression of wild-type NBF1 increased the amount of both core-glycosylated and mature protein to a greater extent than expression of {Delta}F508 NBF1. Expression of wild-type NBF1 in the {Delta}F508 hCFTR cells increased whole cell Cl current density to ~50% of that in cells expressing wild-type hCFTR. Expression of NBF1 in polarized epithelial monolayers from a {Delta}F508/{Delta}F508 cystic fibrosis mouse (MGEF) restored cAMP-stimulated transepithelial anion secretion but not in monolayers from a CFTR-null mouse (MGEN). Restoration of anion secretion was sustained in NBF1-expressing MGEF for >30 passages, whereas MGEN corrected with hCFTR progressively lost anion secretion capability. We conclude that expression of a NBF1 domain mimic may be useful for correction of the {Delta}F508 CFTR protein trafficking defect in cystic fibrosis epithelia.

protein processing; mouse; retrovirus; gene therapy


THE MAJOR DISEASE-CAUSING mutation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein is a deletion of phenylalanine at position 508 ({Delta}F508) in the predicted cytoplasmic domain known as the first nucleotide binding fold (NBF1). Approximately 70% of CF patients are homozygous for the {Delta}F508 cftr, and >90% carry at least one {Delta}F508 cftr allele (compound heterozygotes) (11). Nascent wild-type human (h)CFTR is translated and core-glycosylated at the endoplasmic reticulum (ER). A fraction of the core-glycosylated hCFTR protein (25–40%; Refs. 36 and 48) is transported to the Golgi apparatus, where glycosylation is further modified to form mature hCFTR that is targeted to the plasma membrane (5). There, the wild-type hCFTR functions as a cyclic nucleotide-regulated anion channel and serves a number of transport functions, including regulation of other ion transport proteins and membrane recycling phenomena (2, 8). In contrast to wild-type hCFTR, nearly all of {Delta}F508 hCFTR is retained in the ER in its core-glycosylated form and sorted to the ubiquitin-proteasome protein degradation pathway instead of the Golgi apparatus (5, 25). It is believed that improper folding of the mutant protein is recognized by proteins comprising a cellular "quality control" mechanism that retains misfolded proteins for degradation (44). Chaperone proteins are also involved in the processing of hCFTR and at least two proteins, HSP70 and calnexin, bind {Delta}F508 hCFTR with greater avidity than wild-type hCFTR (39). The process of recognition by the quality control machinery and the conformation of the misfolded {Delta}F508 hCFTR have not been elucidated. In epithelia homozygous for the {Delta}F508 mutation, retention and degradation of the mutant hCFTR essentially eliminates the capacity for cyclic nucleotide-regulated anion secretion, and it is believed that this deficit is fundamental to the pathogenesis of the disease (38).

Defective processing of {Delta}F508 hCFTR is at least partially reversible. Studies in cells expressing recombinant {Delta}F508 hCFTR and tissues cultured from transgenic {Delta}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 {Delta}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 {Delta}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 {Delta}F508 hCFTR to the plasma membrane (4, 40). The above observations have important therapeutic implications because {Delta}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 {Delta}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 {Delta}F508 CFTR has reduced function and shorter membrane residence (32), previous studies suggest that only a small amount (~10–15%) of {Delta}F508 CFTR activity in the membrane is required to restore epithelial function and moderate disease (15, 26). Combined with recent pharmacological studies showing that {Delta}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 {Delta}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 {alpha}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 {Delta}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 {Delta}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 {Delta}F508 homozygous CF patients (16, 50). In the present study, both heterologous cell systems expressing recombinant {Delta}F508 hCFTR and epithelial monolayers from a {Delta}F508 CFTR homozygous mouse were used to test the hypothesis that expression of a NBF1 domain mimic increases trafficking {Delta}F508 CFTR and restores cAMP-stimulated anion secretion.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Mouse mammary epithelial cell lines (C127) expressing wild-type hCFTR, {Delta}F508 hCFTR, or empty plasmid were obtained courtesy of Genzyme (Cambridge, MA) and maintained in plastic culture flasks by using Dulbecco's modified Eagle's medium (DMEM) with high glucose supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 200 µg/ml geneticin. SV40-LT antigen-immortalized mouse gallbladder epithelial cells were derived from a mCFTR knockout (cftr–/–) mouse (i.e., the MGEN cell line) and a {Delta}F508 mCFTR ({Delta}F508cftr/{Delta}F508cftr) mouse (i.e., the MGEF cell line). Both mouse strains for these purposes were maintained on a C57BL/6J background. Both cell lines were grown on Transwell-COL permeable supports (Corning Costar, Cambridge, MA) in Ham's F-12 medium containing 1 µg/ml insulin, 7.5 µg/ml transferrin, 1 µM hydrocortisone, 30 nM triiodothyronine, 2.5 ng/ml epidermal growth factor, and 10 ng/ml endothelial cell growth substance and supplemented (1:1) with 3T3 fibroblast-conditioned DMEM containing 2% (vol/vol) FBS, as previously described (9).

Stable expression of NBF domain mimics. NotI restriction endonuclease site-flanked cDNA constructs of wild-type NBF1 and {Delta}F508 NBF1 (coding for amino acids 429–591 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 3–16), 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 {Delta}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 [{gamma}-32P]ATP for 10 min at 30°C. After washing to remove unincorporated [{gamma}-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 4–20% gels, dried, and autoradiographed (1–7 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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Expression of NBF1 domain mimics increases {Delta}F508 hCFTR protein trafficking and reconstitutes cAMP-stimulated whole cell Cl currents. On the basis of the observation that overexpression increases CFTR trafficking (4), we hypothesized that expression of NBF1 domain mimics in {Delta}F508 hCFTR-expressing cells may produce excess NBF1 polypeptides that would either competitively inhibit quality control proteins that retain and degrade {Delta}F508 hCFTR during processing or reconstitute domain interactions that may permit a fraction of {Delta}F508 hCFTR to be fully processed. C127 cells expressing {Delta}F508 hCFTR were transfected with either {Delta}F508 NBF1 or wild-type NBF1 domains and assayed for hCFTR expression by immunoprecipitation-32P labeling. With this method, the mature, fully processed CFTR (band C) electrophoreses at ~180 kDa whereas the immature, unprocessed CFTR (band B) runs at ~160 kDa. As shown in Fig. 1A, most wild-type hCFTR expressed in the C127 cells (lane 1) is processed to the high-molecular-mass band C form, indicative of glycosylation chain modifications occurring at the trans-Golgi network. The apparent dominance of band C hCFTR is typical for these cells with the immunoprecipitation-32P labeling technique. Clonal cell lines (Fig. 1A, lanes 2–5) coexpressing {Delta}F508 hCFTR and the {Delta}F508 NBF1 domain mimic uniformly expressed the nascent or core-glycosylated forms of unprocessed hCFTR, i.e., band B, but expressed very small amounts of the fully glycosylated hCFTR, i.e., band C. The density of band B shown in Fig. 1A, lanes 2–5, is typical of the {Delta}F508 C127 cells that have not been transfected (n > 20; data not shown). Surprisingly, clonal cell lines coexpressing {Delta}F508 hCFTR and the wild-type NBF1 domain mimic (Fig. 1A, lanes 6–8) expressed greater amounts of both fully processed and core-glycosylated {Delta}F508 hCFTR, suggesting that the wild-type NBF1 domain mimic stabilizes {Delta}F508 CFTR protein throughout processing and membrane trafficking.



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Fig. 1. Immunoblot analysis and whole cell Cl current densities of phenylalanine 508-deleted ({Delta}F508) human (h) cystic fibrosis transmembrane conductance regulator (CFTR)-transfected C127 cells coexpressing first nucleotide-binding fold (NBF1) domain mimics. A: lane 1, cells expressing wild-type (WT) hCFTR; lanes 2–5, {Delta}F508 hCFTR cell lines coexpressing {Delta}F508 ({Delta}F) NBF1; lanes 6–8, {Delta}F508 hCFTR cell lines coexpressing wild-type NBF1. B: relative contribution of band C to pixel intensity profile/lane for WT hCFTR, {Delta}F508 hCFTR cell lines coexpressing {Delta}F508 NBF1 (n = 4) and {Delta}F508 hCFTR cell lines coexpressing wild-type NBF1 (n = 3) from data in A. Relative intensity was calculated as the pixel intensity of band C/combined pixel intensity for bands B + C (after correction for background in each lane). *Significantly different from {Delta}F NBF1. C: whole cell patch-clamp measurements of Cl current density (in pA/pF to normalize for differences in cell size) of C127 epithelial cell transfectants. Data were collected after cell treatment with a cocktail containing 2 µM forskolin and 100 µM genistein. WT and {Delta}F508 C127 cell lines expressing full-length WT and {Delta}F508 hCFTR, respectively; {Delta}F508/27°C, C127 cells expressing {Delta}F508 hCFTR that were maintained at 27°C for 48 h before experimentation; NBF1, wild-type NBF1 domain mimic. Bars labeled with different letters are significantly different.

 
Whole cell patch-clamp analysis was used to investigate Cl current density in the cell clones showing a maximal increase in {Delta}F508 hCFTR maturation after coexpression of the wild-type NBF1 domain mimic. Because the open probabilities of wild-type hCFTR and {Delta}F508 hCFTR are greatly enhanced in the presence of forskolin plus genistein (23), the whole cell Cl current density was used to estimate the activity of functional channels in the plasma membrane. As positive controls, cells expressing wild-type hCFTR and cells expressing {Delta}F508 hCFTR but exposed to processing-permissive temperature conditions (27°C) for 48 h were also assayed for comparison. Earlier studies using cells expressing recombinant {Delta}F508 hCFTR showed that temperature-induced band C expression correlates positively with whole cell Cl current and I efflux (13, 21). As shown in Fig. 1C, C127 cells expressing wild-type hCFTR demonstrate a whole cell Cl current density that is severalfold greater than that in cells expressing {Delta}F508 hCFTR. Cells expressing {Delta}F508 hCFTR grown at a temperature permissive for {Delta}F508 hCFTR trafficking to the plasma membrane had a Cl current density that was ~50% of wild-type hCFTR-expressing cells. Most importantly, clonal {Delta}F508 hCFTR cell lines expressing wild-type NBF1 showed an increased Cl current density that was similar in magnitude to that in the temperature-corrected {Delta}F508 hCFTR cells.

Expression of a NBF1 domain mimic in {Delta}F508/{Delta}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 {Delta}F508 hCFTR in an epithelial cell line expressing recombinant protein driven by a nonnative promoter. Because the level of {Delta}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 {Delta}F508 CFTR in a polarized epithelium. After an extensive search, we were unable to identify a human {Delta}F508/{Delta}F508 CFTR epithelial cell line that retained the ability to both form polarized epithelial monolayers and express significant levels of the endogenous {Delta}F508 hCFTR protein for the retroviral transfection studies. Because previous studies demonstrated that defects in processing and plasma membrane expression of the {Delta}F508 mCFTR homolog in murine epithelia recapitulate the defects of {Delta}F508 hCFTR (16), an epithelial cell line (MGEF) developed by transformation of gallbladder epithelia from a {Delta}F508/{Delta}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.



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Fig. 2. Short-circuit current (Isc) measurements of {Delta}F508/{Delta}F508 MGEF or murine (m)CFTR-null MGEN epithelial monolayers. A: Isc recording of MGEF cells expressing either the selectable zeomycin-resistance gene (Zeo) or NBF1 + Zeo (NBF1). Repetitive upward current deflections result from applied voltage (2 mV) used to measure transepithelial resistance. Treatments (arrows) were sequential additions to the luminal bath of 10 µM forskolin (Forsk) and 100 µM 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB). B: comparison of the maximal Isc change ({Delta}Isc) after 10 µM forskolin treatment for MGEF or MGEN cell lines transfected with Zeo, NBF1 + Zeo (NBF1), or wild-type hCFTR. *Significantly different from MGEN + Zeo and MGEF + Zeo (n = 5–9). C: ion substitution studies of the {Delta}Isc induced by treatment with 10 µM forskolin of MGEF cells expressing NBF1 + Zeo (NBF1). KBR, Krebs bicarbonate Ringer solution. *Significantly different from KBR (n = 4–9).

 
Because the cAMP-stimulated Isc in the NBF1-expressing {Delta}F508 homozygous MGEF monolayers could result from either electrogenic cation (Na+) absorption or electrogenic anion (Cl + HCO3) secretion, ion substitution studies were used to investigate the ionic basis of the cAMP-stimulated Isc in the NBF1-expressing MGEF cell line. As shown in Fig. 2C, substitution of Na+ with the impermeant cation TMA+ in the mucosal bath did not significantly affect the cAMP-stimulated Isc response compared with the response in standard KBR solution. Subsequent addition of 10 µM amiloride to the mucosal solution of NBF1-expressing MGEF cells bathed in the standard KBR solution also did not affect the magnitude of the cAMP-stimulated Isc [change in ({Delta})Isc = –0.1 ± 0.3 µA/cm2; n = 3]. These findings indicated that the cAMP-induced Isc response in the transduced cell line was not due to electrogenic Na+ absorption. In contrast, when both Cl and HCO3 were substituted with impermeant anions in the mucosal and serosal bathing solutions, the cAMP-stimulated Isc of the NBF1-expressing MGEF cell monolayers was nearly eliminated. Together with the evidence that the cAMP-stimulated Isc response in the NBF1-expressing MGEF cells is sensitive to NPPB (see Fig. 2A), the findings are consistent with the conclusion that the stimulated Isc represents {Delta}F508 CFTR-mediated anion secretion.

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 4–7 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.



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Fig. 3. Comparison of the maximal {Delta}Isc resulting from treatment with 10 µM forskolin for the MGEN cell line expressing hCFTR and the MGEF cell line expressing NBF1 for successive passages. Cell lines were passaged approximately once per week. Each data point represents an average of experiments from 2–5 monolayers.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Misfolded CFTR protein is discriminated by a stringent quality control process, which retains the protein within the ER and directs most to a cytosolic ubiquitination/proteosomal pathway (44). Only 20% of wild-type CFTR and very little of {Delta}F508 CFTR completes the process to yield functional channels at the plasma membrane (48). Neither the action by which the {Delta}F508 mutation disrupts the yield of correctly folded CFTR nor the molecular features of misfolded protein that are recognized as abnormal are known. The present study was based on the premise that the NBF1 domain, i.e., the location of {Delta}F508 and several other mutations that affect CFTR processing (46), may be the key molecular component involved in successful processing of CFTR. By expressing a wild-type NBF1 domain mimic, increased amounts of fully glycosylated {Delta}F508 CFTR were observed and cAMP-stimulated anion secretion was restored in epithelial cells expressing recombinant {Delta}F508 hCFTR. Furthermore, expression of the wild-type NBF1 also increased cAMP-stimulated anion secretion in polarized epithelial monolayers expressing endogenous {Delta}F508 mCFTR. Together these results indicate that one function of the NBF1 domain mimic is to increase {Delta}F508 hCFTR trafficking to the plasma membrane.

Restoration of cAMP-stimulated anion secretion in the {Delta}F508 CFTR-expressing epithelial cells yielded bioelectrical responses with characteristics consistent with activation of a {Delta}F508 CFTR conductance. In studies of {Delta}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 {Delta}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 {Delta}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 {Delta}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 {Delta}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 {Delta}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 {alpha}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 {Delta}F508 mutation (6).

Given the findings of the present study and previous observations with expression of recombinant {Delta}F508 CFTR (4, 40), at least two mechanisms can be postulated that would result in increased processing of {Delta}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 {Delta}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 {Delta}F508 CFTR compared with clones expressing the {Delta}F508 NBF1 peptide (Fig. 1). This seems contrary to the prediction that {Delta}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 {Delta}F508 CFTR is that coexpression of additional NBF1 peptides may bind immature {Delta}F508 CFTR and stabilize its structure in the ER. For example, peptide interaction with the R domain of {Delta}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 {Delta}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 {Delta}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 {Delta}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 {Delta}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 {Delta}F508 CFTR should be refined to focus on NBF1 domain mimics that alter trafficking of the mutant protein.1


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The study was supported by Cystic Fibrosis Foundation Grants CLARKE99P0 and CLARKE99G0 and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48816 (to L. L. Clarke).


    ACKNOWLEDGMENTS
 
We acknowledge the technical assistance of Julie Schultz and Shenghui Hu.

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
 

Address for reprint requests and other correspondence: L. L. Clarke, 324D Dalton Cardiovascular Research Center, Research Park Drive, Univ. of Missouri-Columbia, Columbia, MO 65211 (E-mail: clarkel{at}missouri.edu).

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 {Delta}F508 CFTR with an amino-terminal fragment of CFTR (amino acids 1–633) induced the maturation of {Delta}F508 CFTR in Cos7 cells (Cormet-Boyaka EA and Kirk KL. Trans-complementation of {Delta}508-CFTR by an amino terminal fragment of CFTR. Pediatr Pulmonol Suppl 24: 183, 2002). Back


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Bebok Z, Venglarik CJ, Panczel Z, Jilling T, Kirk KL, and Sorscher EJ. Activation of {Delta}F508 CFTR in an epithelial monolayer. Am J Physiol Cell Physiol 275: C599–C607, 1998.[Abstract/Free Full Text]

2. Bradbury NA, Jilling T, Berta G, Sorscher EJ, Bridges RJ, and Kirk KL. Regulation of plasma membrane recycling by CFTR. Science 256: 530–531, 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: 635–638, 1996.[Abstract/Free Full Text]

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 {Delta}F508-CFTR by overexpression. Am J Physiol Lung Cell Mol Physiol 268: L615–L624, 1995.[Abstract/Free Full Text]

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: 827–834, 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: 15222–15230, 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: G259–G267, 1996.[Abstract/Free Full Text]

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: C777–C787, 1999.[Abstract/Free Full Text]

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: C644–C655, 1992.[Abstract/Free Full Text]

11. Collins FS. Cystic fibrosis: molecular biology and therapeutic implications. Science 256: 774–779, 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 {Delta}F508 cystic fibrosis mutation. Nature 354: 526–528, 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: 761–764, 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: 2101–2112, 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: 465–472, 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 {Delta}F508 mutation in mouse cystic fibrosis transmembrane conductance regulator results in a temperature-sensitive processing defect in vivo. J Clin Invest 98: 1304–1312, 1996.[Abstract/Free Full Text]

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: 1070–1072, 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: 382–386, 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: 7739–7745, 1997.[CrossRef][ISI][Medline]

20. Haws C, Nepomuceno IB, Krouse ME, Wakelee H, Law T, Xia Y, Nguyen H, and Wine JJ. {Delta}F508-CFTR channels: kinetics, activation by forskolin, and potentiation by xanthines. Am J Physiol Cell Physiol 270: C1544–C1555, 1996.[Abstract/Free Full Text]

21. Heda GD and Marino CR. Surface expression of the cystic fibrosis transmembrane conductance regulator mutant {Delta}F508 is markedly upregulated by combination treatment with sodium butyrate and low temperature. Biochem Biophys Res Commun 271: 659–664, 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 {Delta}F508 cystic fibrosis transmembrane conductance regulator (CFTR) protein maturation and function. J Biol Chem 278: 35159–35167, 2003.[Abstract/Free Full Text]

23. Hwang TC, Wang F, Yang ICH, and Reenstra WW. Genistein potentiates wild-type and {Delta}F508-CFTR channel activity. Am J Physiol Cell Physiol 273: C988–C998, 1997.[Abstract/Free Full Text]

24. Hwang TC and Sheppard DN. Molecular pharmacology of the CFTR Cl channel. Trends Pharmacol Sci 20: 448–453, 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: 129–135, 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: 21–25, 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: 321–327, 1992.[ISI][Medline]

28. Kelley TJ, Al-Nakkash L, Cotton CU, and Drumm ML. Activation of endogenous {Delta}F508 cystic fibrosis transmembrane conductance regulator by phosphodiesterase inhibition. J Clin Invest 98: 513–520, 1996.[Abstract/Free Full Text]

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: 27442–27448, 2002.[Abstract/Free Full Text]

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: 379–392, 1998.[Abstract/Free Full Text]

31. Li C, Ramjeesingh M, Reyes E, Jensen TJ, Chang XB, Rommens JM, and Bear CE. The cystic fibrosis mutation ({Delta}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 {Delta}F508 mutation decreases the stability of cystic fibrosis transmembrane conductance regulator in the plasma membrane. J Biol Chem 268: 21592–21598, 1993.[Abstract/Free Full Text]

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: 1453–1456, 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: 28133–28141, 1997.[Abstract/Free Full Text]

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 {alpha}1-antitrypsin-related cirrhosis. J Biol Chem 277: 6771–6774, 2002.[Abstract/Free Full Text]

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: 2987–2995, 1994.[Abstract/Free Full Text]

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: 10972–10977, 1998.[Abstract/Free Full Text]

38. Pilewski JM and Frizzell RA. Role of CFTR in airway disease. Physiol Rev 79: S215–S255, 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: 12784–12788, 1994.[Abstract/Free Full Text]

40. Rubenstein RC and Zeitlin PL. A pilot clinical trial of oral sodium 4-phenylbutyrate (Buphenyl) in {Delta}F508-homozygous cystic fibrosis patients: partial restoration of nasal epithelial CFTR function. Am J Respir Crit Care Med 157: 484–490, 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: C341–C351, 1996.[Abstract/Free Full Text]

42. Schultz BD, Frizzell RA, and Bridges RJ. Rescue of dysfunctional {Delta}F508-CFTR chloride channel activity by IBMX. J Membr Biol 170: 51–66, 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: 1569–1572, 1999.[Abstract/Free Full Text]

44. Skach W. Defects in processing and trafficking of the cystic fibrosis transmembrane conductance regulator. Kidney Int 57: 825–831, 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: 847–850, 1995.[ISI][Medline]

46. Tsui LC. The spectrum of cystic fibrosis mutations. Trends Genet 8: 392–398, 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: 637–648, 2000.[Abstract/Free Full Text]

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: 25710–25718, 1994.

49. Ward CL, Omura S, and Kopito RR. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83: 121–127, 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 {Delta}F508 allele of cystic fibrosis. J Clin Invest 96: 2051–2064, 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: 7627–7630, 1999.[Abstract/Free Full Text]

52. Zhang XM, Wang XT, Yue H, Leung SW, Thibodeau PH, Thomas PJ, and Guggino SE. Organic solutes rescue the functional defect in {Delta}F508 cystic fibrosis transmembrane conductance regulator. J Biol Chem 278: 51232–51243, 2003.[Abstract/Free Full Text]





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