Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755
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ABSTRACT |
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The F508 mutation
reduces the amount of cystic fibrosis transmembrane conductance
regulator (CFTR) expressed in the plasma membrane of epithelial cells.
However, a reduced temperature, butyrate compounds, and "chemical
chaperones" allow
F508-CFTR to traffic to the plasma membrane and
increase Cl
permeability in heterologous and nonpolarized
cells. Because trafficking is affected by the polarized state of
epithelial cells and is cell-type dependent, our goal was to determine
whether these maneuvers induce
F508-CFTR trafficking to the apical
plasma membrane in polarized epithelial cells. To this end, we
generated and characterized a line of polarized Madin-Darby canine
kidney (MDCK) cells stably expressing
F508-CFTR tagged with green
fluorescent protein (GFP). A reduced temperature, glycerol, butyrate,
or DMSO had no effect on 8-(4-chlorophenylthio)-cAMP
(CPT-cAMP)-stimulated transepithelial Cl
secretion across
polarized monolayers. However, when the basolateral membrane was
permeabilized, butyrate, but not the other experimental maneuvers,
increased the CPT-cAMP-stimulated Cl
current across the
apical plasma membrane. Thus butyrate increased the amount of
functional
F508-CFTR in the apical plasma membrane. Butyrate failed
to stimulate transepithelial Cl
secretion because of
inhibitory effects on Cl
uptake across the basolateral
membrane. These observations suggest that studies on heterologous and
nonpolarized cells should be interpreted cautiously. The GFP tag on
F508-CFTR will allow investigation of
F508-CFTR trafficking in
living, polarized MDCK epithelial cells in real time.
cystic fibrosis; Madin-Darby canine kidney; butyrate; glycerol; dimethyl sulfoxide; green fluorescent protein; cystic fibrosis transmembrane conductance regulator
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INTRODUCTION |
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THE CYSTIC FIBROSIS
TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR), a cAMP-activated
Cl channel, is expressed in a wide variety of epithelial
cells, including airway and kidney (13, 46). Mutations in
the CFTR gene lead to the genetic disease cystic fibrosis (CF), a
lethal autosomal recessive disorder (30, 40).
Approximately 70% of individuals with CF are homozygous for the
F508-CFTR mutation, which accounts for approximately 90% of all
mutant CFTR alleles (30). Trafficking of
F508-CFTR from
the endoplasmic reticulum (ER) to the apical plasma membrane of
epithelial cells is extremely inefficient. Moreover,
F508-CFTR has a
relatively short residence time in the plasma membrane compared with
wild-type CFTR (wt-CFTR) (20, 29). Thus little
F508-CFTR is expressed in the plasma membrane (8).
Because
F508-CFTR retains partial function as a cAMP-activated
Cl
channel (11, 28, 39), identification of
drugs that increase the amount and activity of
F508-CFTR in the
apical plasma membrane of airway epithelial cells would have important
implications for the treatment of CF.
Several experimental maneuvers increase F508-CFTR expression in the
plasma membrane. For example, growing cells in culture treated with
reduced temperature (9, 12, 14, 16, 17, 24, 41), butyrate
compounds (19, 24, 41), and so-called "chemical
chaperones" including DMSO and glycerol (41, 43) facilitate the expression of
F508-CFTR in the plasma membrane and
allow cAMP to enhance
F508-CFTR-mediated Cl
permeability (reviewed in Refs. 3 and 47). However,
because many of these studies were conducted in either nonpolarized or heterologous cells, the results should be interpreted cautiously, since
it is well known that protein trafficking is cell-type specific and
depends on the polarized state of the epithelium (reviewed in Refs.
5 and 6). For example, the human low-density lipoprotein (LDL) receptor, when expressed in transgenic mice, is located in the apical membrane of renal tubules. By contrast, the LDL receptor
is expressed in the basolateral membrane of colonocytes and
enterocytes. In addition, in nonpolarized HT-29 intestinal cells,
wt-CFTR is localized in an intracellular compartment, whereas in
polarized HT-29 cells, wt-CFTR is expressed in the apical plasma membrane (32, 33). Moreover, cAMP induces the trafficking of wt-CFTR from an intracellular compartment to the plasma membrane in
polarized, but not nonpolarized, HT-29 cells (33). Several laboratories have studied
F508-CFTR trafficking in polarized epithelial cells. Many of these studies have failed to confirm observations made in nonpolarized and/or heterologous cells. For example, in polarized LLC-PK1 cells expressing
F508-CFTR, glycerol, sodium butyrate, or a reduced temperature (27°C) did not stimulate cAMP-stimulated Cl
secretion (2). In
addition, sodium butyrate had no effect on Cl
currents
across polarized human nasal airway epithelial cells expressing
F508-CFTR (7). Thus studies on polarized epithelial cells have not consistently confirmed results obtained in nonpolarized and/or heterologous cells.
Although LLC-PK1 cells expressing F508-CFTR form polarized
monolayers (2, 10, 20), additional polarized cell lines expressing
F508-CFTR would be valuable for secondary drug screening studies and to study
F508-CFTR trafficking. Moreover, addition of a
green fluorescent protein (GFP) tag on
F508-CFTR would allow investigation of
F508-CFTR trafficking in living, polarized
epithelial cells in real time. In preliminary studies, we were unable
to identify a robust human airway epithelial cell line that
consistently forms polarized monolayers. Thus we have stably expressed
F508-CFTR with a GFP tag in Madin-Darby canine kidney (MDCK) cells,
a well-established cell model in which to study protein trafficking in
polarized epithelial cells (35), and have conducted
studies to determine whether a reduced temperature, glycerol, butyrate,
or DMSO stimulate Cl
secretion in polarized cells. We
attached GFP to the NH2 terminus of
F508-CFTR to monitor
the subcellular localization of
F508-CFTR. GFP, a 27-kDa protein
from the jellyfish Aequorea victoria, generates a striking
green fluorescence, is resistant to photobleaching, and does not
require any exogenous cofactors or substrates to fluoresce
(37). We report that a reduced temperature, glycerol, sodium butyrate, or DMSO has no effect on
8-(4-chlorophenylthio)adenosine 3'5'-cyclic monophosphate
(CPT-cAMP)- stimulated transepithelial Cl
secretion.
However, when the basolateral membrane was permeabilized with nystatin,
sodium butyrate increased the CPT-cAMP-stimulated Cl
current across the apical plasma membrane. Sodium
butyrate failed to stimulate transepithelial Cl
secretion
because of inhibitory effects on Cl
uptake across the
basolateral membrane. Reduced temperature, DMSO, and glycerol had no
effect on the CPT-cAMP-stimulated Cl
current across the
apical plasma membrane. These observations demonstrate that data
obtained in heterologous, nonpolarized cell models should be
interpreted cautiously. The GFP tag on
F508-CFTR will allow
investigation of
F508-CFTR trafficking in living, polarized MDCK
epithelial cells in real time.
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METHODS |
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Expression vector.
The pGFP-F508-CFTR mammalian expression vector, driven by the
cytomegalovirus promoter, was constructed by methods described previously (34, 35). Proceeding from the NH2
to the COOH terminus, the resultant chimeric protein consists of
enhanced GFP, a flexible linker sequence of 23 amino acids, and
F508-CFTR. The cDNA coding for human
F508-CFTR was a generous
gift of Dr. William Guggino (Johns Hopkins University School of
Medicine, Baltimore, MD).
Cell culture and stable cell lines.
MDCK type I cells (C7 clone, a generous gift of Dr. Hans Oberleithner)
stably expressing GFP-F508-CFTR were established by methods
described previously (35). The C7 clonal line expresses very low levels of cAMP-stimulated Cl
currents
attributable to wt-CFTR (18, 31, 35). Three independent stable cell lines expressing GFP-
F508-CFTR were studied to exclude the possibility that results were attributable to clonal variation. MDCK I cells stably expressing GFP-wt-CFTR and parental MDCK I cells
(C7) have been characterized and described in detail (34, 35). Previously, we demonstrated that addition of GFP to the NH2 terminus of wt-CFTR had no effect on CFTR localization,
trafficking, or biophysical properties (35). Moreover,
others have shown that GFP has no effect on the synthesis and
degradation of wt-CFTR (25). In preliminary pulse-chase
studies, we observed that GFP does not affect the rate of biosynthesis
or degradation of
F508-CFTR. For simplicity, we refer to the
chimeric GFP proteins as
F508-CFTR and wt-CFTR.
Immunocytochemistry. Cells grown on glass slides or Transwell filters were fixed in paraformaldehyde and prepared for laser scanning confocal microscopy as described previously (35). The ER was identified by indirect immunofluoresence with the use of an anti-BiP polyclonal antibody (StressGen Biotechnology, Victoria, BC, Canada) to detect the ER-resident protein BiP, followed by an anti-rabbit Texas Red-labeled secondary antibody (Molecular Probes, Eugene, OR), as described previously (35).
SDS-PAGE and Western blotting.
F508-CFTR and wt-CFTR were detected on membranes blocked with 5%
nonfat dry milk in Tris-buffered saline (TBS)-0.02% Tween 20 using a
monoclonal CFTR antibody (M3A7; Chemicon International, Temecula, CA)
(26, 27), followed by anti-mouse horseradish peroxidase
(HRP)-conjugated secondary antibody (Amersham, Arlington Heights, IL)
as described previously (35). Blots were developed by
enhanced chemiluminescence (ECL) using Hyperfilm ECL (Amersham).
Cell surface biotinylation.
Selective apical membrane biotinylation and immunoprecipitation of
GFP-CFTR and F508-CFTR were performed as described in detail
previously (34, 35). All steps were performed at 4°C. Proteins were separated by SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes as described previously (35). Biotinylated GFP-CFTR fusion proteins were
immunoprecipitated with a GFP antibody (1:1,000; Clontech, Palo Alto,
CA) and were detected on membranes blocked with 5% nonfat dry milk in
TBS-0.02% Tween 20 using a monoclonal CFTR antibody (M3A7) followed by
anti-mouse HRP-conjugated secondary antibody (1:5,000; Amersham).
Short-circuit current.
Short-circuit current (Isc) was measured across
MDCK cell monolayers stably expressing F508-CFTR or wt-CFTR or
across MDCK cells not expressing the CFTR transgene, as described
previously (35). In all experiments, amiloride
(10
5 M) was present in the apical bath solution to
inhibit electrogenic Na+ absorption. Under these
conditions, cAMP-stimulated Isc is referable to
CFTR-mediated Cl
secretion from the basolateral to the
apical solution (35, 36). To measure the Cl
currents across the apical membrane, we permeabilized the basolateral membrane with nystatin (200 µg/ml). When the basolateral membrane was
permeabilized, Isc measured in the presence of a
transepithelial Cl
ion gradient directed from the
basolateral to the apical solution (140 vs. 14 mM: Cl
was
replaced on an equimolar basis with gluconate; Ca2+ was
increased from 0.5 to 2 mM in the gluconate-containing solution to
maintain Ca2+ activity similar in both solutions)
represented the Cl
current across the apical membrane, as
described previously (35). Cl
currents are
reported as the difference between the peak value of
Isc following CPT-cAMP (100 µM) minus the
steady-state Isc after inhibition of the
CPT-cAMP-stimulated CFTR Cl
current by
diphenylamine-2-carboxylic acid (DPC; 3 mM).
Statistical analyses. Differences between means were compared by using the unpaired, two-tailed Student's t-test or by ANOVA followed by a post hoc test (Dunnett's or Tukey's test) as appropriate using Instat v2.01 statistical software (GraphPad, San Diego, CA). Data are expressed as means ± SE. P < 0.05 is considered significant.
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RESULTS |
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Characterization of F508-CFTR stable cell lines.
We characterized three independent clonal lines stably expressing
F508-CFTR. First, we conducted Western blots on cell lysates to
confirm CFTR protein expression. As demonstrated previously (35), in cells expressing wt-CFTR, but not in parental
nontransfected cells (i.e., C7), we detected two protein bands on
Western blots using a monoclonal CFTR antibody (Fig.
1A). Core glycosylated wt-CFTR
had an apparent molecular mass of 210 kDa (the so-called "band B"),
and maturely glycosylated wt-CFTR had an apparent molecular mass of 240 kDa (the so-called "band C"). These results confirm our previous
study demonstrating that GFP-wt-CFTR runs ~30 kDa larger than
anticipated by SDS-PAGE analysis (35). By contrast, in
cells expressing
F508-CFTR, we only detected core glycosylated band
B
F508-CFTR in cells treated with sodium butyrate (Fig. 1B). We could not detect
F508-CFTR expression in cells
not treated with butyrate (Fig. 1B). Moreover,
F508-CFTR
expression was low, even in butyrate-treated cells, relative to the
robust expression of wt-CFTR. Lower expression of
F508-CFTR relative
to that of wt-CFTR is due in part to the shorter half-life of
F508-CFTR (<4 h) relative to wt-CFTR (>24 h) (20,
29). As expected,
F508-CFTR was expressed in an intracellular
compartment, primarily in the ER (Fig.
2).
F508-CFTR could not be detected in
the apical plasma membrane of cells treated with sodium butyrate by
selective cell-surface biotinylation. By contrast, the maturely
glycosylated band C of wt-CFTR was detected in the apical plasma
membrane as described previously (35, 36).
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Low temperature, butyrate, glycerol, and DMSO do not increase
CPT-cAMP-stimulated transepithelial Cl secretion.
We examined the effect of reduced temperature, sodium butyrate,
glycerol, and DMSO on the ability of CPT-cAMP to stimulate Cl
secretion in polarized MDCK cells stably expressing
F508-CFTR. The effect of sodium butyrate on the CPT-cAMP-stimulated
Isc is summarized in Fig.
3. Cells were exposed to sodium butyrate
(1 or 5 mM) for 1, 3, or 5 days. Similar experimental maneuvers
stimulate Cl
permeability in several other cell types
expressing
F508-CFTR (1, 7, 19, 41). Stock solutions of
sodium butyrate were made immediately before addition to the cell
culture medium, and the butyrate containing cell culture medium was
changed daily. Although sodium butyrate dramatically increased
F508-CFTR protein expression (see Fig. 1B), butyrate had
no effect on CPT-cAMP-stimulated Isc (Fig. 3).
|
F508-CFTR channel activity and Cl
electrochemical
driving force.
The inability to detect an increase in CPT-cAMP-stimulated
Cl
secretion in cells treated with sodium butyrate in the
present study may be due to a low open probability of
F508-CFTR
channels in the plasma membrane (21, 22). To test this
possibility, we added genistein, a flavinoid that increases the open
probability of
F508-CFTR channels (1, 14), to the
apical bathing solution after addition of CPT-cAMP. Genistein (50 µM)
had no effect on Isc across control or sodium
butyrate-treated monolayers (5 mM for 2 days). The change in
Isc with genistein was 0.1 ± 1.1 µA/cm2 in control monolayers and 2.1 ± 0.8 µA/cm2 in monolayers treated with sodium butyrate
[n = 6 monolayers/group, P = NS (not
significant)]. Thus the lack of effect of sodium butyrate on
CPT-cAMP-stimulated Cl
secretion did not appear to be due
to a reduced activity of
F508-CFTR channels in the apical plasma membrane.
Apical membrane F508-CFTR currents.
To examine the effect of sodium butyrate, reduced temperature,
glycerol, and DMSO on Cl
currents across the apical
membrane of cells expressing
F508-CFTR, we permeabilized the
basolateral membrane with nystatin and measured CPT-cAMP-stimulated
Isc across the apical membrane as described in
METHODS. The data are summarized in Fig.
4, and representative experiments are
illustrated in Fig. 5. Only sodium
butyrate significantly increased the Cl
currents across
the apical membrane. In untreated monolayers expressing
F508-CFTR,
the CPT-cAMP-stimulated Isc was 11.8 ± 2.7 µA/cm2. Sodium butyrate increased the CPT-cAMP-stimulated
Isc in monolayers expressing
F508-CFTR to
33.6 ± 5.4 µA/cm2 (P < 0.05 compared with control). However, a reduced temperature, DMSO, or
glycerol did not increase the Cl
current across the
apical membrane (Fig. 4). Moreover, a combination of sodium butyrate
and reduced temperature did not increase the Cl
current
compared with sodium butyrate treatment alone (Fig. 4). In addition, a
combination of sodium butyrate and glycerol or sodium butyrate and DMSO
failed to increased the Cl
current compared with sodium
butyrate treatment alone (Fig. 4). These data, in combination with
studies described below, demonstrate that sodium butyrate increased the
amount of functional
F508-CFTR in the apical plasma membrane of
polarized MDCK cells.
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Sodium butyrate had no effect on endogenous wt-CFTR
Cl currents in parental MDCK I cells.
Because the parental MDCK I (C7) cell line expresses low levels
of endogenous wt-CFTR (31), it is possible that sodium
butyrate stimulated Cl
currents across the apical
membrane of cells stably expressing
F508-CFTR by increasing
endogenous wt-CFTR expression. To examine this possibility, we treated
MDCK I parental cells with sodium butyrate (5 mM for 2 days).
CPT-cAMP-stimulated transepithelial Cl
secretion was
2.5 ± 0.5 µA/cm2 in control untreated monolayers
and 0.5 ± 0.3 µA/cm2 in sodium butyrate-treated
monolayers (n = 6, P < 0.05). Next, the Cl
current across the apical plasma membrane was
measured in the presence of nystatin to permeabilize the basolateral
membrane. Sodium butyrate had no effect on the CPT-cAMP-stimulated
Cl
current across the apical membrane, which was 9.0 ± 1.5 µA/cm2 in untreated monolayers and 11.6 ± 0.9 µA/cm2 in sodium butyrate-treated monolayers (5 mM
for 2 days: n = 7 monolayers/group, P = NS). These data demonstrate that sodium butyrate does not increase the
functional expression of endogenous wt-CFTR in the apical membrane of
parental MDCK cells. Thus the effects of sodium butyrate in MDCK cells
expressing
F508-CFTR, described above, are due to increased
functional expression of
F508-CFTR in the apical plasma membrane and
not to the enhanced expression of endogenous wt-CFTR.
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DISCUSSION |
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A new polarized epithelial cell line in which to study F508-CFTR
trafficking.
We have established and characterized a new polarized cell line stably
expressing GFP-tagged
F508-CFTR. MDCK I cells form polarized
monolayers, are robust and easy to maintain in culture, express low
levels of endogenous CFTR, have Rt values ~400
· cm2, and are a well-established model in
which to study trafficking of ion channels. In addition, Ussing chamber
studies can be performed to monitor transepithelial Cl
secretion as well as Cl
secretion across the apical
plasma membrane. As expected,
F508-CFTR was expressed primarily in
the ER. However, sodium butyrate increased the functional expression of
F508-CFTR in the apical plasma membrane. The GFP tag on
F508-CFTR
will allow, in future studies, investigation of
F508-CFTR
trafficking in living, polarized MDCK epithelial cells in real time.
Butyrate increases functional F508-CFTR expression in the apical
plasma membrane.
Sodium butyrate increased the amount of functional
F508-CFTR in the
apical plasma membrane as determined by measuring the CPT-cAMP-stimulated, DPC-sensitive Cl
current across the
membrane. Thus, as in many cell types, sodium butyrate increases the
plasma membrane expression of
F508-CFTR. However, we did not detect
F508-CFTR in the apical membrane by cell surface biotinylation, and
we did not detect maturely glycosylated (band C)
F508-CFTR by
Western blot analysis. Similar results were observed in LLC-PK1 cells
stably expressing
F508-CFTR (20). Thus it is not
surprising that
F508-CFTR in the apical membrane was below the
detection limit of Western blot analysis and selective cell surface
biotinylation. Plasma membrane expression of
F508-CFTR in vivo is
also very low compared with the expression of wt-CFTR.
Untoward effects of sodium butyrate on transepithelial
Cl secretion.
Why did sodium butyrate have no effect on CPT-cAMP-stimulated
transepithelial Cl
secretion but increase
CPT-cAMP-stimulated Cl
secretion across the apical plasma
membrane in MDCK cells expressing
F508-CFTR? Previously, we reported
that sodium butyrate increased the expression of wt-CFTR in the apical
membrane of MDCK I cells 25-fold and stimulated Cl
currents across the apical membrane 30-fold (36). However, transepithelial CPT-cAMP-stimulated Cl
secretion was
reduced because butyrate reduced the activity of the
Na+-K+-ATPase. In epithelial cells, the
Na+-K+-ATPase maintains a low intracellular
concentration of Na+, which is important for providing the
driving force for Cl
entry into the cell across the
basolateral membrane via the
Na+-K+-2Cl
cotransporter.
Cl
then exits the cell across the apical membrane through
CFTR Cl
channels. By reducing the activity of the
Na+-K+-ATPase, we speculated that sodium
butyrate increases intracellular Na+ concentration and
thereby reduces Cl
entry into the cell across the
basolateral membrane via the
Na+-K+-2Cl
cotransporter. The
decrease in Cl
entry would reduce intracellular
Cl
concentration and thereby inhibit Cl
secretion across the apical membrane. The present study also supports
the view that despite an increase in the amount of
F508-CFTR in the
apical membrane, sodium butyrate does not increase transepithelial CPT-cAMP-stimulated Cl
secretion, most likely because
sodium butyrate inhibits the Na+-K+-ATPase and
indirectly reduces Cl
uptake across the basolateral
membrane. It is possible, but not proven, that the inability of sodium
butyrate to stimulate Cl
secretion across polarized
epithelial MDCK cells expressing
F508-CFTR could be due to a similar
effect of butyrate on the Na+-K+-ATPase.
Additional experiments, beyond the scope of this report, are required
to elucidate the cellular mechanism(s) whereby butyrate inhibits
Na+-K+-ATPase activity in MDCK cells expressing
F508-CFTR.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Katherine Karlson and Bonita Coutermarsh for assistance with cell culture and Western blots, Dr. Alice Givan and Ken Orndorff for assistance with confocal microscopy, and Dr. Sandra Guggino for the suggestion to conduct the nystatin experiments.
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FOOTNOTES |
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These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-45881 and Cystic Fibrosis Foundation Grant STANTO97RO. J. Loffing was supported by a fellowship from the Swiss National Science Foundation. Confocal microscopy was performed at Dartmouth Medical School, in the Herbert C. Englert Cell Analysis Laboratory, which was established by a grant from the Fannie E. Rippel Foundation and is supported in part by the Core Grant of the Norris Cotton Cancer Center (CA 23108). The Dartmouth Cystic Fibrosis Cell Biology/Cell Culture Core (STANTO97RO) provided scientific and technical support.
Present address of D. Loffing-Cueni and J. Loffing: Institute of Anatomy, University of Zurich, 8006 Zurich, Switzerland.
Address for reprint requests and other correspondence: B. A. Stanton, Dept. of Physiology, 615 Remsen Bldg., Dartmouth Medical School, Hanover, NH 03755 (E-mail: bruce.a.stanton{at}dartmouth.edu).
1
We cannot formally exclude the possibility that
the GFP tag on F508-CFTR prevented glycerol, DMSO, or low
temperature from increasing
F508-CFTR expression in the plasma
membrane of MDCK cells. However, as noted above, our data are
consistent with reports by others that these maneuvers fail to increase
plasma membrane expression of untagged
F508-CFTR in polarized
epithelial cells.
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 15 March 2001; accepted in final form 1 August 2001.
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