1 Departments of Pharmacology and Toxicology and 2 Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755-3835
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cystic fibrosis (CF) is a
disease that is caused by mutations within the cystic fibrosis
transmembrane conductance regulator (CFTR) gene. The most common
mutation, F508, accounts for 70% of all CF alleles and results in a
protein that is defective in folding and trafficking to the cell
surface. However,
F508-CFTR is functional when properly localized.
We report that a single, noncytotoxic dose of the anthracycline
doxorubicin (Dox, 0.25 µM) significantly increased total cellular
CFTR protein expression, cell surface CFTR protein expression, and
CFTR-associated chloride secretion in cultured T84 epithelial cells.
Dox treatment also increased
F508-CFTR cell surface expression and
F508-CFTR-associated chloride secretion in stably transfected
Madin-Darby canine kidney cells. These results suggest that
anthracycline analogs may be useful for the clinical treatment of CF.
trafficking; chloride secretion
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE CYSTIC FIBROSIS TRANSMEMBRANE conductance regulator (CFTR) is a cAMP- and protein kinase-regulated chloride channel found on the apical membranes of polarized epithelial cells (10). Mutations within this gene lead to the CF phenotype that ultimately results in premature death primarily from a combination of chronic lung infections, loss of pulmonary function, and pancreatic insufficiency (5). This genetic disease is among the most prevalent in the human population, occurring in ~1 of 2,500 Caucasian live births. CFTR is a member of the structurally related ATP-binding cassette family of transmembrane transport proteins whose other members include P-glycoprotein (Pgp, the MDR1 gene product), multidrug resistance-associated protein and other related multidrug resistance proteins, and the sulfonylurea receptor (21).
Of the more than 850 individual CFTR mutations leading to the CF
phenotype that have been described, the most important is F508
(phenylalanine deletion at amino acid position 508), which is seen in
~70% of all CF patients. The
F508-CFTR mutation results in
improper folding and trafficking of the CFTR protein, leading to its
degradation in the endoplasmic reticulum by the 26S proteosome machinery of the cell (23). However, importantly,
if this mutant protein is folded and expressed in the membrane by
experimental cell culture techniques such as glycerol (20)
or low temperature treatments (3), it functions normally
as a chloride channel. Moreover, it is estimated that restoration of
functional CFTR expression to ~10% of normal levels would be
sufficient to ameliorate the symptoms of the disease in vivo
(19). Thus there is great interest in development of
strategies that can enhance
F508-CFTR cell surface expression, which
may be clinically useful in treatment of CF patients.
There is evidence of coregulation of Pgp and CFTR in epithelial cells,
although the mechanism and level of this overlapping regulation is
currently unknown (2). Previous work in our laboratory demonstrated both transcriptional and posttranscriptional effects of
low-dose, noncytotoxic treatments with the cancer chemotherapy drugs
mitomycin C (MMC) and doxorubicin (Dox) on expression of Pgp in various
cell lines and in a mouse in vivo model (6, 7). We
therefore hypothesized that CFTR expression might also be modified by
these or related drugs under similar treatment conditions. We recently
demonstrated that MMC increased total protein and cell surface
functional expression of wild-type CFTR in HT-29 and T84 human colon
cells (15a). In this study, we investigated the effects of Dox
on CFTR and F508-CFTR expression. We report here that low-dose Dox
(0.25 µM) significantly increased functional cell surface expression
of CFTR and
F508-CFTR in T84 and Madin-Darby canine kidney (MDCK)
cells, respectively, and that this was primarily a result of
posttranscriptional effects.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture and treatment.
Human colon adenocarcinoma T84 cells (American Type Culture Collection,
Manassas, VA) were maintained in DMEM/F-12 media (Life Technology,
Rockville, MD) containing L-glutamine supplemented with
10% fetal bovine serum (FBS) and antibiotics (Life Technology). An
isogenic line of MDCK/C-7 cells stably expressing green fluorescent protein (GFP) tagged to the NH2 terminus of F508-CFTR
(MDCK/
F508) were created in a fashion similar to a cell line
expressing GFP tagged to the NH2 terminus of CFTR
(MDCK/CFTR) described elsewhere (17, 18). This cell line
and parental MDCK-C7 cells were maintained in minimum essential media
(MEM, Life Technology) supplemented as above. Additionally, 0.3 mg/ml
of G418 Sulfate (Sigma, St. Louis, MO) was added to the MDCK/
F508
media for maintaining the selection pressure. For immunoblot analysis
and RT-PCR following drug treatment, cells were grown in six-well
plates (Costar, Cambridge, MA) to confluence, washed with PBS,
and treated with or without 0.25 µM Dox (Sigma). After this,
completed media were replenished and cells were harvested at various
time points. For short-circuit current measurements and cell-surface
biotinylations, cells were cultured on Transwell-type filters (Corning,
Ithaca, NY). The medium was completely changed every 24 h, and
experiments were performed 14 days postseeding.
Relative RT-PCR. Total RNA from T84 cells was isolated using TRIAZOL reagent (Life Technology). One microgram of total RNA from T84 cells was reverse-transcribed using the RNA PCR Core Kit (Perkin Elmer, Foster City, CA) in 20 µl total volume. The cDNAs were divided into two equal aliquots and amplified using 0.5 µl of AmpliTaq DNA polymerase (5 U/µl), 20 pM upper and lower primers specific for human CFTR (upper: 5'-CCT GAA CCT GAT GAC ACA CT-3', lower: 5'-TAA AAC TGC GAC AAC TGC TA-3') or 1.25 µl of 5 µM 18S RNA specific primers and Competimers (Ambion, Austin, TX) in a ratio 3.5:6.5. Prior titration experiments ensured that amplification of both CFTR and 18S RNA products were within linear range. The cDNA products were separated by electrophoresis on 1% agarose gels (Life Technology) and stained with ethidium bromide to confirm proper product sizes.
Immunoblotting.
Cells were treated with solvent or Dox, washed once with cold PBS, and
scraped in a small volume of lysis buffer (50 mM Tris · HCl, pH
6.8, 150 mM NaCl, 1% Nonidet P-40) containing a protease inhibitor
cocktail (Roche Biochemicals, Indianapolis, IN). Protein was
extracted from the samples by a 30-min incubation on ice with intermediate vortexing and frozen at 70°C. Protein in each sample was quantified by the bichinchoninic acid assay (Pierce, Rockford, IL). SDS-PAGE was performed on 4-15% minigels using 50 µg of protein lysate per lane. Proteins were separated and
transferred to polyvinylidene difluoride membrane (Millipore, Bedford,
MA) at 120 mA for 1 h in Towbin's transfer buffer (25 mM Tris,
192 mM glycine, 12% methanol). After transfer, the blots were
blocked and probed sequentially, first with polyclonal A2 rabbit
anti-CFTR antisera (25) (generously provided by Dr.
W. Skach, Oregon Health Sciences Institute) overnight at 4°C and then
with a horseradish peroxidase (HRP)-labeled anti-rabbit secondary polyclonal antibody (Amersham, Piscataway, NJ). Membranes were washed six times in PBS + 0.3% Tween 20 (PBST) at room
temperature for 10 min each in between. The blots were
developed by enhanced chemiluminescence (ECL) Blaze substrate (Pierce)
and exposed to film.
Cell-surface biotinylation and immunoprecipitation.
T84 cells were biotinylated on the apical plasma membrane with a
commercially available biotinylation kit (Roche Biochemicals). After
treatment at 4°C with LC-(+)-biotin-hydrazide, cells were washed with cold PBS and harvested in lysis buffer on ice, and total
protein in each sample was quantified as described above. Two hundred
micrograms of protein from each sample was immunoprecipitated using
polyclonal A2 rabbit anti-CFTR antisera (1:1,000) and Protein A-agarose
(Roche Biochemicals). The beads were washed in PBST, and immunoreactive
proteins were separated by SDS-PAGE and detected by streptavidin-HRP
and ECL as described above. The F508-CFTR-expressing MDCK cells were
immunoprecipitated similarly but immunoblotted using a monoclonal
anti-GFP antibody (Clontech Laboratories, Palo Alto, CA). In other
experiments, CFTR was immunoprecipitated with A2 as described above and
detected with the M3A7 monoclonal anti-CFTR antibody (12),
a generous gift of Dr. J. Riordan (Mayo Institute, Scottsdale, AZ).
Measurement of short circuit current.
Short-circuit current (Isc) was measured across
monolayers of T84 cells grown on filters. Cells were treated with Dox
(0.25 µM, 24 h) beginning at 14 days after seeding, and the
media were changed to remove residual drug before
Isc measurement. Isc was measured in cells in the presence of amiloride (105 M) in
the apical solution to block the potential contribution of sodium
transport to Isc. In the presence of amiloride,
8-(4-chlorophenylthio)-cAMP (CPT-cAMP)-stimulated
Isc in T84 cells is equivalent to electrogenic Cl
secretion (1). The CFTR antagonist
diphenylamino carboxylic acid (DPC) and the
Cl
/HCO
Fluorescent chloride efflux assay. Cells were grown to confluence in six-well plates and loaded with 10 mM N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE, Molecular Probes, Eugene, OR) overnight as described in detail previously (24). Briefly, cells were incubated in a chloride-containing, nitrate-free buffer for chloride concentration equilibration inside and outside of the cells, and background fluorescence was recorded over 3 min. After this, the buffer was changed to a chloride-free, nitrate-containing buffer. Both buffers contained 100 µM CPT-cAMP (Roche Biochemicals) to stimulate CFTR. In the presence of chloride, MQAE is caged and the probe's fluorescence is quenched. Upon changing over to a chloride-free, nitrate-containing buffer, a chloride gradient is established, which results in the rapid exchange of chloride for nitrate in the cells. Over the first few minutes, the rate of increase in free MQAE fluorescence is then proportional to the number of chloride channels at the membrane. Increase in MQAE fluorescence was monitored over 20 min after buffer exchange. The rates of chloride efflux were calculated from the slopes of the fluorescence curves over the first 3 min. A Cytofluor II plate reader (PerSeptive Biosystems, Framingham, MA) equipped with a 360 nm excitation/460 nm emission filter set was used to measure MQAE fluorescence in the cells.
Software. Densitometric quantification and image processing were carried out using Adobe Photoshop (Adobe Software, San Jose, CA) and NIH Image (National Institutes of Health, Bethesda, MD) software packages. All statistical analyses were performed using Instat and Prism software (Graphpad Software, San Diego, CA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effects of Dox on CFTR expression in T84 cells. In all experiments
described below (except Fig. 2C), cells were treated with 0.25 µM Dox for up to 24 h, which caused no overt cytotoxicity or change in cell viability as measured by trypan blue exclusion assay.
This dose is 20-fold lower than the half-maximal lethal concentration
(LC50) for Dox in these cells (J. W. Hamilton et al.,
unpublished results). The effects of Dox on CFTR mRNA
expression were examined in T84 cells by a semiquantitative RT-PCR
assay. Treatment of cells for up to 24 h with 0.25 µM Dox had
little or no effect on steady-state CFTR mRNA expression (Fig.
1). In contrast, Dox treatment
significantly altered CFTR protein levels. A 24-h Dox treatment
increased CFTR protein by approximately twofold (Fig. 2,
A-C), particularly the "C" band, which
represents the mature, fully glycosylated form of CFTR (Fig.
2A). Dose-response and time
course studies indicated that the optimal treatment was 0.25 µM Dox
for 24 h (Fig. 2, B and C). An increase in
CFTR protein was also observed after immunoprecipitation with the A2
antibody and subsequent immunoblotting with a second CFTR-specific
monoclonal antibody, M3A7 (Fig. 2, D and E).
|
|
To assess whether Dox specifically increased membrane CFTR expression, we performed selective cell-surface biotinylation of apical membrane proteins followed by immunoprecipitation with a CFTR-specific polyclonal antibody and detection using HRP-conjugated streptavidin. As demonstrated in Fig. 2, F and G, Dox-treated cells, compared with control cells, had an approximately twofold increase in levels of plasma membrane-associated CFTR that was detected as a single 170-kDa band (mature "C" band) on the immunoblots (Fig. 2E). Taken together, these experiments indicate that Dox treatment of T84 cells leads to enhanced CFTR protein expression at the cell surface.
Experiments were then conducted to assess whether the increased plasma
membrane expression of CFTR resulted in enhanced chloride secretion in
T84 cells. When we used an MQAE fluorescence-based assay, a 24-h Dox
treatment increased chloride permeability approximately twofold
relative to control, as shown in Fig.
3B. Electrogenic chloride secretion was also measured across monolayers of T84 cells
(Fig. 3D) in an Ussing chamber. Dox treatment increased CFTR-associated chloride secretion approximately twofold compared with
control. Thus, when we used two different assays for CFTR function, Dox
significantly increased CFTR-mediated chloride permeability in
association with increased levels of membrane-associated CFTR in these
cells.
|
Effects of Dox on F508-CFTR expression in MDCK cells.
The effects of Dox treatment on functional cell surface expression of
the CFTR folding mutant
F508-CFTR were then assessed with these same
techniques. CFTR-associated chloride secretion was measured in an
Ussing chamber across monolayers of parental MDCK-C7 cells and two
isogenic MDCK cell lines stably expressing either GFP-tagged human CFTR
or human GFP-
F508-CFTR. Dox treatment had no effect on chloride
secretion in MDCK-C7 parental cells (Fig.
4A). However, in MDCK cells
expressing the mutant GFP-
F508-CFTR, Dox treatment caused an
~2.3-fold increase in CFTR-mediated chloride secretion compared with
untreated control cells (Fig. 2A). The increase in
GFP-
F508-CFTR expression was confirmed by Western blotting of these
cells (Fig. 4C). It is important to note that the control
level of chloride secretion in these cells was similar to that of the
parental MDCK cells expressing the canine CFTR (Fig. 4A),
suggesting that there was essentially very little or no functional
F508-CFTR expression in the plasma membrane of these cells before
Dox treatment.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We report that a single, noncytotoxic dose of Dox increased the
amount of functional CFTR in the plasma membrane of T84 cells and the
amount of functional F508-CFTR in the plasma membrane of MDCK cells.
In the T84 cells, this appeared to be principally a posttranscriptional
effect because there was little effect on CFTR mRNA expression. In both
cell models, Dox caused a significant increase in total cellular CFTR
protein levels and a proportionate increase in CFTR-associated chloride
secretion. These results suggest that analogs may be identified that
will be clinically useful in ameliorating the CF disease phenotype in
human CF patients.
The purpose of these studies was to obtain proof of principle for this
class of drugs. Although the exact mechanistic basis behind this effect
is currently being investigated, one possibility is that Dox increases
the stability of the F508-CFTR nascent forms, allowing them to fold
properly. Dox may alter the kinetics of
F508-CFTR biogenesis
influencing the interaction of the nascent protein with cellular
chaperones. Previous reports indicate that heat shock cognate
protein (HSC70) facilitates the early steps of
F508-CFTR biogenesis
that appear to be important for maturation of the protein
(22), and compounds that are capable of altering this
association facilitate
F508-CFTR (9) expression and
chloride secretion. Another possibility is that Dox directly interacts with and alters the folding, stability, or trafficking of
F508-CFTR during its biogenesis. For example, mutations in Pgp that alter its
folding and plasma membrane trafficking, including a mutation that is
comparable to
F508-CFTR, can be corrected by treatment of cells with
Pgp substrate drugs (15). The authors proposed that such
drugs acted as "chemical chaperones" that interact with Pgp and
allow proper folding of the nascent molecule to the functional, mature
form. A similar phenomenon could occur for
F508-CFTR, although this
same study indicated no effect of these treatments on
F508-CFTR
expression indicating that the effect was Pgp specific. However,
anthracycline analogs were not tested in that study. Other drugs have
also been described that are capable of altering both CFTR and Pgp
protein levels. For example, butyrate compounds have been shown to be
capable of stimulating both Pgp (16) and CFTR
(18) protein expression. We found that another cancer
chemotherapy drug, mitomycin C, also increased functional cell surface
expression of CFTR in T84 and HT-29 cells, although the effects were
more modest than those of Dox (R. Maitra et al., unpublished observations).
Alternatively, Dox may inhibit degradation of misfolded
F508-CFTR, which might favor proper folding and maturation. However, previous studies by Kopito and colleagues (11, 23)
demonstrated that inhibition of the 26S proteosomal degradation pathway
per se does not enhance
F508-CFTR folding, but rather leads to
aggregation. Because other proteolytic pathways are involved
in CFTR degradation (8), this possibility cannot be
completely ruled out. These and other possibilities remain to be
explored in future studies.
Perhaps most interestingly, a recent clinical observation was made
(13) in a CF patient with F508-CFTR mutation in one allele and G673X stop mutation in the other allele who had
fibrosarcoma. This patient demonstrated significantly improved lung
function and inhibition of his Pseudomonas aeruginosa
infection after combination chemotherapy with cyclophosphamide and the
anthracycline epirubicin. This is an intriguing clinical
observation that should be further investigated systematically in CF
cancer patients receiving chemotherapy.
In this study, the Dox treatment regimen at which significant
F508-CFTR effects were observed (0.25 µM) is more promising from a
clinical pharmacology perspective. This Dox concentration is ~20-fold
lower that its LC50 in these cell systems, suggesting that
one might also be able to achieve these effects in vivo at a comparably
low dose that is well below those used in cancer chemotherapy and that
can elicit significant "nontarget" toxicity. Dox itself is unlikely
to be useful in a CF clinical setting due to its cumulative systemic
toxicity. However, it is likely, given that there are thousands of
anthracyclines and their derivatives that have been developed and
characterized over the past 50 years, that analogs can be discovered or
developed that share the beneficial properties of Dox in increasing
F508-CFTR expression while avoiding its cumulative toxicities. The
long-term goal of this research is to develop clinically useful drugs
for the systemic treatment of CF patients with the aim of ameliorating
their disease phenotype.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors thank Dr. A. Givan for help with flow cytometry.
![]() |
FOOTNOTES |
---|
This work was supported by grants from the National Institutes of Health to B. A. Stanton, and from the Cystic Fibrosis Foundation to B. A. Stanton and J. W. Hamilton. J. W. Hamilton was also partially supported by the Norris Cotton Cancer Center, and R. Maitra was partially supported by a research grant to J. W. Hamilton from Bristol-Myers Squibb.
Address for reprint requests and other correspondence: J. W. Hamilton, Dept. of Pharmacology & Toxicology, Dartmouth Medical School, 7650 Remsen, Hanover, NH 03755-3835 (E-mail josh.hamilton{at}dartmouth.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.
Received 1 February 2000; accepted in final form 17 November 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barrett, KE.
Positive and negative regulation of chloride secretion in T84 cells.
Am J Physiol Cell Physiol
265:
C859-C868,
1993
2.
Breuer, W,
Slotki IN,
Ausiello DA,
and
Cabantchik IZ.
Induction of multidrug resistance downregulates the expression of CFTR in colon epithelial cells.
Am J Physiol Cell Physiol
265:
C1711-C1715,
1993
3.
Denning, GM,
Anderson MP,
Amara JF,
Marshall J,
Smith AE,
and
Welsh MJ.
Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive.
Nature
358:
761-764,
1992[ISI][Medline].
4.
Drumm, ML,
Wilkinson DJ,
Smit LS,
Worrell RT,
Strong TV,
Frizzell RA,
Dawson DC,
and
Collins FS.
Chloride conductance expressed by F508 and other mutant CFTRs in Xenopus oocytes.
Science
254:
1797-1799,
1991[ISI][Medline].
5.
Ferrari, M,
and
Cremonesi L.
Genotype-phenotype correlation in cystic fibrosis patients.
Ann Biol Clin (Paris)
54:
235-241,
1996[ISI][Medline].
6.
Ihnat, MA,
Lariviere JP,
Warren AJ,
La Ronde N,
Blaxall JRN,
Pierre KM,
Turpie BW,
and
Hamilton JW.
Suppression of P-glycoprotein expression and multidrug resistance by DNA cross-linking agents.
Clin Cancer Res
3:
1339-1346,
1997[Abstract].
7.
Ihnat, MA,
Nervi AM,
Anthony SP,
Kaltreider RC,
Warren AJ,
Pesce CA,
Davis SA,
Lariviere JP,
and
Hamilton JW.
Effects of mitomycin C and carboplatin pretreatment on multidrug resistance-associated P-glycoprotein expression and on subsequent suppression of tumor growth by doxorubicin and paclitaxel in human metastatic breast cancer xenografted nude mice.
Oncol Res
11:
303-310,
1999[ISI][Medline].
8.
Jensen, TJ,
Loo MA,
Pind S,
Williams DB,
Goldberg AL,
and
Riordon JR.
Multiple proteolytic systems, including the proteosome, contribute to CFTR processing.
Cell
83:
129-135,
1995[ISI][Medline].
9.
Jiang, C,
Fang SL,
Xiao YF,
O'Connor SP,
Nadler SG,
Lee DW,
Jefferson DM,
Kaplan JM,
Smith AE,
and
Cheng SH.
Partial restoration of cAMP-stimulated CFTR chloride channel activity in F508 cells by deoxyspergualin.
Am J Physiol Cell Physiol
275:
C171-C178,
1998
10.
Jilling, T,
and
Kirk KL.
The biogenesis, traffic, and function of the cystic fibrosis transmembrane conductance regulator.
Int Rev Cytol
172:
193-241,
1997[ISI][Medline].
11.
Johnston, JA,
Ward CL,
and
Kopito RR.
Aggresomes: a cellular response to misfolded proteins.
J Cell Biol
143:
1883-1898,
1998
12.
Kartner, N,
and
Riordon JR.
Characterization of polyclonal and monoclonal antibodies to cystic fibrosis transmembrane conductance regulator.
Methods Enzymol
292:
629-652,
1998[ISI][Medline].
13.
Lallemand, JY,
Stoven V,
Annereau JP,
Boucher J,
Blanquet S,
Barthe J,
and
Lenoir G.
Induction by antitumoral drugs of proteins that functionally complement CFTR: a novel therapy for cystic fibrosis?
Lancet
350:
711-712,
1997[ISI][Medline].
14.
Li, C,
Ramjeesingh M,
Reyes E,
Jensen T,
Chang X,
Rommens JM,
and
Bear CE.
The cystic fibrosis mutation (F508) does not influence to chloride channel activity of CFTR.
Nat Genet
3:
311-316,
1993[ISI][Medline].
15.
Loo, TW,
and
Clarke DM.
Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.
J Biol Chem
272:
709-712,
1997
15a.
Maitra R, Shaw CM, Stanton BA, and Hamilton JW. Functional
enhancement of CFTR expression by mitomycin C. Cell Physiol
Biochem. In press.
16.
Morrow, CS,
Nakagawa M,
Goldsmith ME,
Madden MJ,
and
Cowan KH.
Reversible transcriptional activation of mdr1 by sodium butyrate treatment of human colon cancer cells.
J Biol Chem
269:
10739-10746,
1994
17.
Moyer, BD,
Loffing J,
Schwiebert EM,
Loffing-Cueni D,
Halpin PA,
Karlson KH,
Ismailov II,
Guggino WB,
Langford GM,
and
Stanton BA.
Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in Madin-Darby canine kidney cells.
J Biol Chem
273:
21759-21768,
1998
18.
Moyer, BD,
Loffing-Cueni D,
Loffing J,
Reynolds D,
and
Stanton BA.
Butyrate increases apical membrane CFTR but reduces chloride secretion in MDCK cells.
Am J Physiol Renal Physiol
277:
F271-F276,
1999
19.
Pilewski, JM,
and
Frizzell RA.
Role of CFTR in airway disease.
Physiol Rev
79:
S215-S255,
1999[Medline].
20.
Sato, S,
Ward CL,
Krouse ME,
Wine JJ,
and
Kopito RR.
Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation.
J Biol Chem
271:
635-638,
1996
21.
Schneider, E,
and
Hunke S.
ATP-binding cassette (ABC) transport systems: functional and structural aspects of the ATP-hydrolyzing subunits/domains.
FEMS Microbiol Rev
22:
1-20,
1998[ISI][Medline].
22.
Strickland, E,
Qu BH,
Millen L,
and
Thomas PJ.
The molecular chaperone Hsc70 assists the in vitro folding of the N-terminal nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem
272:
25421-25424,
1997
23.
Ward, CL,
Omura S,
and
Kopito RR.
Degradation of CFTR by the ubiquitin-proteosome pathway.
Cell
83:
121-127,
1995[ISI][Medline].
24.
West, MR,
and
Malloy CR.
A microplate assay measuring chloride ion channel activity.
Anal Biochem
241:
51-58,
1996[ISI][Medline].
25.
Xiong, X,
Bragin A,
Widdicombe JH,
Cohn J,
and
Skach WR.
Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator.
J Clin Invest
100:
1079-1088,
1997