Hyperexpression of recombinant CFTR in heterologous cells
alters its physiological properties
Raha
Mohammad-Panah,
Sophie
Demolombe,
David
Riochet,
Veronique
Leblais,
Gildas
Loussouarn,
Helene
Pollard,
Isabelle
Baró, and
Denis
Escande
Laboratoire de Physiopathologie et de Pharmacologie Cellulaires et
Moléculaires, Institut National de la Santé et de la
Recherche Médicale CJF96-01, Hôpital Hotel-Dieu, 44093 Nantes, France
 |
ABSTRACT |
We investigated whether high levels of expression of the cystic
fibrosis transmembrane conductance regulator (CFTR) would alter the
functional properties of newly synthesized recombinant proteins. COS-7,
CFPAC-1, and A549 cells were intranuclearly injected with a Simian
virus 40-driven pECE-CFTR plasmid and assayed for halide permeability
using the
6-methoxy-N-(3-sulfopropyl)quinolinium fluorescent probe. With increasing numbers of microinjected pECE-CFTR copies, the baseline permeability to halide dose dependently increased, and the response to adenosine 3',5'-cyclic monophosphate
(cAMP) stimulation decreased. In cells hyperexpressing CFTR, the high level of halide permeability was reduced when a cell metabolism poisoning cocktail was applied to decrease intracellular ATP and, inversely, was increased by orthovanadate. In CFPAC-1 cells
investigated with the patch-clamp technique, CFTR hyperexpression led
to a time-independent nonrectifying chloride current that was not
sensitive to cAMP stimulation. CFPAC-1 cells hyperexpressing CFTR
exhibited no outward rectifying chloride current nor inward rectifying
potassium current either spontaneously or under cAMP stimulation. We
conclude that hyperexpression of recombinant CFTR proteins modifies
their properties inasmuch as 1) CFTR
channels are permanently activated and not susceptible to cAMP
regulation and 2) they lose their capacity to regulate heterologous ionic channels.
cystic fibrosis transmembrane conductance regulator; cystic
fibrosis; expression; patch clamp; CFPAC-1 cells; A549 cells; epithelial cells
 |
INTRODUCTION |
CYSTIC FIBROSIS (CF), the most common lethal genetic
disease in Caucasians, is caused by mutations in a gene encoding a
1,480-amino acid membrane protein (25), the cystic fibrosis
transmembrane conductance regulator (CFTR). CFTR belongs to the ATP
binding cassette (ABC) superfamily of ATP-linked
transporters (16) and is directly involved in epithelial
Cl
secretion, since it is
itself an anionic channel (5) regulated by intracellular ATP and
protein kinase A-dependent phosphorylation. The CFTR protein is also a
regulator of other ionic channels implicated in water and salt
epithelial secretion such as the outward-rectifying Cl
channels (ORCC; see Ref.
11), the amiloride-sensitive Na+
channels (33), and the inward rectifying epithelial
K+ channels (21). The major
consequence of the most common deletion of phenylalanine 508 of the
CFTR gene is that the mutated protein is abnormally processed and
trafficked within affected epithelial cells so that the gene product is
virtually absent from the cell membrane (17). Pharmacological
treatments targeted to activate directly the CFTR protein would thus be
inefficient in the vast majority of CF patients, and the most promising
route to control the CF disease is presently gene therapy (27).
Complementation of respiratory epithelium with wild-type CFTR cDNA has
been achieved successfully both in vitro (10) and in vivo (2), and
several clinical trials using either viral or nonviral vectors have now been completed (27).
One preeminent problem for CF gene therapy is low in vivo efficiency
[<0.1% of transduced epithelial cells using an adenovirus vector, as assessed by in situ hybridization in the Knowles et al. (18)
protocol]. A partial solution to this problem could be to
hyperexpress wild-type CFTR transgene in the limited number of
successfully transduced cells with the hope to obtain amplification of
functional correction (so-called "bystander" effect) through Cl
movement via gap
junction from noncorrected cells into corrected adjacent cells for
secretion (6, 7). To this end, high-strength promoters are usually
chosen to drive wild-type CFTR cDNA in recombinant gene therapy
constructs. However, in the physiological setting, the number of CFTR
copies per respiratory epithelial cell is low (
20-100 channel
proteins/cell; see Ref. 13), and one may wonder whether hyperexpression
of wild-type CFTR impacts its physiological properties. The present
study was designed to gain further information on this important issue.
We have microinjected into the nucleus of CFTR-deficient cells various
concentrations of a Simian virus 40 (SV40)-driven plasmid encoding
wild-type CFTR, and we have constructed dose-effect relationships. The
present data show that CFTR hyperexpression profoundly affects its
regulation.
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METHODS |
Cell cultures. COS-7 (an African green
monkey kidney-derived cell line) and A549 cells (an alveolar type II
epithelium-like cell line) were provided by the American Type Culture
Collection (Rockville, MD). COS-7 cells were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum and
antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin; all
from GIBCO, Paisley, Scotland) at 37°C in a humidified 5%
CO2-95% air incubator and were
regularly subcultured by enzymatic treatment with a solution of 0.25%
trypsin 1
EDTA in a Ca2+
and Mg2+-free phosphate buffer
solution (GIBCO). A549 cells were cultured in Ham's F-12 medium as
modified by M. E. Kaighn and supplemented with 10% fetal
calf serum. Pancreatic epithelioid CFPAC-1 cells issued from a
F508/
F508 patient with CF (29) were cultured as previously
reported (26). The CFPAC-PLJ6-CFTR clone, a kind gift from Dr. R. A. Frizzell (University of Pittsburgh, Pittsburgh, PA; see Ref. 9), was
obtained by stably transfecting CFPAC-1 cells with a retroviral vector
that contained the cDNA encoding wild-type CFTR gene. CFPAC-PLJ6-CFTR
cells were grown in the presence of geneticin (G418: 0.4 mg/ml; Calbiochem, La Jolla, CA).
Intranuclear injection of plasmids.
Cells were microinjected with plasmids at day
1 after plating on glass coverslips for 6-methoxy-N-(3-sulfopropyl)quinolinium
(SPQ) experiments or on coated plastic petri dishes (Nunclon; InterMed
Nunc, Roskilde, Denmark) for patch-clamp experiments. In this
procedure, the Eppendorf ECET microinjector 5246 system, the ECET
micromanipulator 5171 system, and a Nikon Diaphot inverted microscope
were used. Nuclear microinjection was performed with the Z (depth)
limit option using a 0.3-s injection duration and 40-60 hPa
injection pressure. Injection femtotips (internal diameter 0.5 ± 0.2 µm) were pulled from borosilicate glass capillaries. Plasmids
were diluted at a final concentration ranging from 5 to 350 µg/ml in
an injection buffer made of (in mM) 50 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), 50 NaOH, and 40 NaCl, pH 7.4. Fluorescein isothiocyanate
(FITC)-labeled dextran (0.5%) was also added to the injection medium
to stain successfully microinjected cells. Immediately before use, the injection medium containing the DNA sample was centrifuged at 13,000 revolutions/min for 20 min to pellet the particular material. Using
radiolabeled technetium-99 as a probe, we estimated that the nuclear
volume so injected was
10
12 liters. The
number of injected cDNA copies was thus estimated as 10,000 at a
plasmid concentration of 100 µg/ml. A plasmid construct, pECE-CFTR
[a kind gift from Dr. Pascale Fanen, Institut National de la
Santé et de la Recherche Médicale (INSERM), Hôpital
Henri Mondor, Créteil, France], in which the full-length
DNA encoding wild-type CFTR is placed under the control of a SV40
promoter and origin of replication was used for most experiments. SV40 large T antigen is a viral oncoprotein that transactivates viral promoters and induces synthesis in quiescent cells. Large T
antigen-expressing cells such as COS-7 cells support a high degree of
replication of transfected plasmids containing an SV40 origin of
replication. The same construct (pECE; see Ref. 12; a gift from Dr. E. Clauser, Collège de France, Paris, France) lacking the CFTR
insert was used for control experiments. Additional control experiments
were also performed with a pECE plasmid encoding the sulfonylurea
receptor (pECE-SUR; see Ref. 1; a kind gift from Dr. Lydia
Aguilar-Bryan, Baylor College of Medicine, Houston, TX) and with a
pSG5HN-EBCR plasmid encoding an epithelial basolateral
Cl
regulator (see Ref. 35;
a kind gift from Dr. Marcel A. Van Kuijck, University of
Nijmegen, The Netherlands). In preliminary experiments, we used a
reporter
-galactosidase expression vector (pRSV-LacZ; a gift
from Dr. Pierre Lehn, INSERM, Hôpital Robert Debré, Paris, France). To reveal exogenous
-galactosidase
activity, cells were fixed for 15 min with 0.5% formaldehyde
and then analyzed histochemically using
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-Gal).
SPQ fluorescence assay. Cells placed
on glass coverslips were loaded with the intracellular dye SPQ
(Molecular Probes) by incubation in
Ca2+-free hypotonic (50%
dilution) medium containing 10 mM SPQ at 37°C for either 6 min
(COS-7) or 10 min (CFPAC-1 and A549). The coverslip was mounted on the
stage of a Nikon Diaphot inverted microscope equipped for fluorescence
and illuminated at 360 nm. The emitted light was collected at 456 ± 33 nm by a high-resolution image intensifier coupled to a video camera
(Extended ISIS camera system; Photonic Science). The camera was
connected to a digital image processing board controlled by FLUO
software (Imstar, France). Single-cell fluorescence intensity was
measured from digital image processing and displayed against time.
Fluorescence intensity was standardized according to F = (F
Fo)/Fo × 100, where F is fluorescence and
Fo is the fluorescence
intensity measured in the presence of
I
(36). The membrane
permeability to halides was determined as the rate of SPQ dequenching
upon perfusion with nitrates. At least three successive data points
were collected immediately after the
-containing medium was applied
and then fitted using a linear regression analysis. The slope of the
relation so measured reflected the membrane permeability to halide. The control Tyrode solution for SPQ experiments contained (in mM) 145 NaCl,
4 KCl, 1 MgCl2, 1 CaCl2, 5 HEPES, and 5 glucose, pH adjusted to 7.4 with NaOH.
I
and
media were identical to the
control Tyrode solution except that
I
or
replaced
Cl
as the dominant
extracellular anion. All extracellular medium used also contained 10 µM bumetamide to inhibit the
Cl
-cation cotransporter.
Control wash-out experiments showed that, under our experimental
conditions, intracellular SPQ fluorescence declined by only 10.5 ± 2.1% (n = 18) for a period of time as long as 2 h.
Whole cell patch-clamp recordings.
Whole cell currents with either the nystatin permeabilized-patch or the
ruptured-patch arrangements of the patch-clamp technique were recorded
as previously described (3). A petri dish containing cells was placed
on the stage of an inverted microscope and continuously superfused with
the standard extracellular solution. Patch pipettes with a tip
resistance 2.5-5 M
were electrically connected to a patch-clamp amplifier (axopatch 200A; Axon Instruments, Foster City, CA). Stimulation, data recording, and analysis were performed by a software
made by Gérard Sadoc (distributed by DIPSI Industrie, Asnière, France) through an analog-to-digital converter (Tecmar TM100 Labmaster; Scientific Solution, Solon, OH). Depolarizing voltage
ramps were applied at a frequency of 0.2 Hz from
80 to +60 mV
(depolarization rate: 46.7 mV/s; holding potential:
60 mV). In
experiments aimed to record the ORCC, the membrane voltage was stepped
in sequence from a holding potential of
60 to
100 mV for
150 ms, then back to
60 mV for another 150 ms, and then to +60
mV for 500 ms. Current-voltage relationships were constructed with
voltage steps applied for 500 ms every 2 s from
60 mV to various
potentials between
100 and +60 mV. A microperfusion system allowed local application and rapid change of the different
experimental solutions warmed at 35°C. The standard
Cl
-containing extracellular
solution was similar in composition to that used for SPQ experiments.
In experiments targeted to record pure
Cl
currents,
Na+ and
K+ were substituted for
Cs+ in the extracellular medium;
the pipette solution was made of (in mM) 74.5 CsCl, 70.5 aspartic acid,
5 HEPES, 1 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 0-5 MgATP, and 5 glucose, pH 7.2 with CsOH. In
experiments targeted to record K+
currents, the extracellular medium contained (in mM) 145 tris(hydroxymethyl)aminomethane (Tris), 2 K2SO4,
1 CaSO4, 1 MgSO4, and 5 glucose, pH 7.4 with H2SO4.
For ruptured-patch recordings, the pipette solution contained (in mM) 5 Tris, 72.5 K2SO4,
1 EGTA, 0-5 MgATP, and 5 glucose, pH 7.2 with
H2SO4,
whereas, for the nystatin permeabilized-patch arrangement, the pipette
solution contained (in mM) 5 Tris and 72.5 K2SO4,
pH 7.2 with
H2SO4.
For permeabilized-patch recordings, the extracellular solution was
supplemented with 60 mM mannitol to compensate osmotic imbalance caused
by the use of nystatin (3).
Drugs. Intracellular adenosine
3',5'-cyclic monophosphate (cAMP) was increased with a
mixture made of 10 µM forskolin plus 400 µM
8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (both from Sigma Chemical, St. Louis, MO). On occasion, 100 µM 3-isobutyl-1-methylxanthine (Sigma) was added to the cAMP stimulating cocktail. Diphenylamine-2-carboxylic acid (DPC; 500 µM; Fluka Chemical) was used as a CFTR channel blocker (31). Sodium orthovanadate (Na3VO4;
LC Laboratories, Wolum, MA) was used at a final concentration of 1 mM.
With the aim to decrease intracellular ATP, we used a cell-poisoning
cocktail made of sodium cyanide (2 mM; Aldrich Chemical),
2,4-dinitrophenol (0.1 mM; Sigma), carbonyl cyanide m-chlorophenyl-hydrazone (5 µM;
Sigma), iodoacetic acid (5 µM; Aldricht), and
-2-desoxy-D-glucose (5 mM; Sigma). Drugs were dissolved
in dimethyl sulfoxide so that the final concentration of the solvent
was 1
.
Statistics. Patch-clamp and SPQ
measurements are presented as means ± SE. Statistical significance
of the observed effects was assessed by means of the standard
t-test.
 |
RESULTS |
Intranuclear injection of reporter gene and kinetics
of CFTR expression. Using bacterial LacZ as a reporter
gene, we first evaluated the efficacy of our intranuclear injection
procedure. Injected cells were identified by FITC-dextran fluorescence
predominating either in the nucleus or in the cytoplasm. At a pRSV-LacZ
plasmid concentration of 100 µg/ml, 73.8 ± 8.1% (mean ± SE;
n = 151; 9 assays) of
intranuclearly injected COS-7 cells showed blue nuclear staining with
X-Gal revelation performed 24 h postinjection. In the CFPAC-1 cell
line, the percentage of intranuclearly injected cells demonstrating
-galactosidase expression was 78.7 ± 7.9% (n = 73; 7 assays) with a
pRSV-LacZ concentration of 100 µg/ml. As previously reported by
others (32), cells microinjected in the cytoplasm with
-galactosidase expression plasmid demonstrated no transgene
expression (n = 194 for COS-7 and
n = 130 for CFPAC-1).
One of the well-identified advantages of injecting plasmids
intranuclearly is that expression of the transgene is fast (32). To
gain further insight into expression delay under our experimental conditions, CFPAC-1 cells were first loaded with SPQ, and a cell under
microscope observation was injected intranuclearly at
time 0 with the pECE-CFTR expression
plasmid at 200 µg/ml. The extracellular medium was then switched
sequentially between nitrate and iodide-containing solutions so that
the membrane permeability to halides, determined as the rate of SPQ
dequenching upon perfusion with nitrates, was monitored over time.
These experiments were conducted in the continuous presence of the cAMP
enhancing cocktail to activate neosynthesized CFTR channels. Cells
injected with pECE vector alone lacking the CFTR insert served as
control. As shown in Fig. 1, the membrane permeability to halide progressively increased with time in
pECE-CFTR-injected cells but not in cells injected with the pECE
plasmid alone. On average, the membrane permeability to halides
increased from 0.163 ± 0.039 min
1 (mean ± SE) up to
0.804 ± 0.136 min
1 over
a 1-h period of time in 13 cells injected with pECE-CFTR. From 24 experiments, we determined that the delay to observe a significant
increase in the membrane permeability to halides was 49 ± 7 min.
Additional experiments were also performed in COS-7 cells. In three
experiments, the membrane permeability to halides increased from 0.125 min
1 up to 0.839 min
1 over 2 h and peaked at
1.340 min
1 6 h
postinjection. Nevertheless, our results demonstrate that functional
recombinant CFTR channels were produced within <2 h postinjection of
the pECE-CFTR plasmid. Further experiments were usually performed 24 h
postinjection.

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Fig. 1.
Fast expression of recombinant cystic fibrosis transmembrane
conductance regulator (CFTR) channel protein after intranuclear
injection of 200 µg/ml pECE-CFTR. Relative
6-methoxy-N-(3-sulfopropyl)quinolinium
(SPQ) fluorescence from a single cell injected at time
0 (open circles) and from a noninjected neighboring
cell (continuous line) is plotted against time. Extracellular medium
was switched sequentially between nitrate (filled horizontal bars) and
iodide (open horizontal bars) media. cAMP enhancing cocktail was
present throughout. Inset: membrane
permeability to halide (p expressed in
min 1) determined as the
initial rate of dequenching in the nitrate solution (circles, cells
injected with pECE-CFTR; crosses, cells injected with pECE vector
alone) and plotted against time postinjection.
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CFTR hyperexpression in COS-7, CFPAC-1, and A549
cells. We then varied the concentration of the injected
pECE-CFTR plasmid and measured corresponding membrane permeability to
halide using the SPQ assay. When COS-7 cells were injected with a low
concentration of the pECE-CFTR plasmid (i.e., <5 µg/ml), the
membrane permeability under baseline was not significantly different
from cells injected with the pECE vector alone (Figs.
2A and
3A). As expected, the permeability to halide greatly increased upon application of the cAMP
enhancing cocktail (Figs. 2A and
3A). By contrast, COS-7 cells injected with a plasmid
concentration >5 µg/ml exhibited an increased membrane permeability
under baseline and were less responsive to cAMP stimulation (Figs. 2,
A and
D, and 3A). In Fig.
2D, the membrane permeability under
cAMP stimulation expressed relative to the membrane permeability under
baseline was determined in COS-7 cells injected with various
concentrations of pECE-CFTR and was plotted as a function of the
membrane permeability under baseline. This relation demonstrates that
the more COS-7 cells were permeable to halide under baseline conditions
the less responsive they were to cAMP stimulation. Typically, COS-7
cells injected with 200 µg/ml pECE-CFTR exhibited a 40-fold increased
baseline permeability that did not increase further with cAMP (Figs. 2, A and
D, and 3A). Interestingly,
the baseline permeability in COS-7 cells hyperexpressing CFTR exceeded
the cAMP-stimulated permeability determined in cells injected with a
lower plasmid concentration. Comparable findings were also obtained in
CFPAC-1 cells and in A549 cells, although in these cells, the membrane permeability under baseline increased only at the highest pECE-CFTR concentration tested (i.e., >200 µg/ml; Figs. 2,
B and
C, and 3). In CFPAC-1 and in A549
cells injected with 350 µg/ml, the membrane permeability under
baseline increased ~10- and 7-fold, respectively. We never observed
such behavior in cells injected with the pECE plasmid
alone (n = 77 for COS-7;
n = 12 in CFPAC-1; n = 15 in A549) or in cells injected
with cDNA encoding for other ABC proteins such as pECE-SUR (350 µg/ml; n = 27 for COS-7 and n = 33 for CFPAC-1) or pSG5HN-EBCR
(350 µg/ml; n = 38 for COS-7 and
n = 19 for CFPAC-1).

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Fig. 2.
Overexpression and hyperexpression of CFTR channels in COS-7, CFPAC-1,
and A549 cells. Relative SPQ fluorescence is plotted against time.
A-C:
open horizontal bars indicate iodide-containing solution, whereas
filled horizontal bars indicate nitrate-containing solution. Filled
inverted triangles indicate the time at which the cAMP- enhancing
cocktail was applied. A: COS-7 cells
injected with the pECE plasmid alone (continuous line) or with 5 µg/ml (filled circles), 100 µg/ml (open triangles), or 200 µg/ml
(open circles) pECE-CFTR. B: CFPAC-1
cells injected with the pECE plasmid alone (continuous line) or with
100 µg/ml (filled circles), 200 µg/ml (open triangles), or 350 µg/ml (open circles) pECE-CFTR. C:
A549 cells injected with the pECE plasmid alone (continuous line) or
with 100 µg/ml (filled circles) or 350 µg/ml (open circles)
pECE-CFTR. D: response to cAMP
stimulation determined as the ratio of the membrane permeability under
cAMP stimulation (pcAMP) in the
presence of the cAMP enhancing cocktail to the membrane permeability
under baseline (pbaseline) and plotted
as a function of pbaseline (in
min 1) . Each data point
was obtained from different individual COS-7 cells injected with
various plasmid concentrations ranging from 5 to 350 µg/ml.
Continuous line is a hyperbolic fit.
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Fig. 3.
Plasmid concentration dependence of baseline (open bars) and
cAMP-stimulated (filled bars) permeability to halide
(p in
min 1) in COS-7
(A), CFPAC-1 (B), and A549
(C) cell lines. C, noninjected control cells; pECE,
cells injected with the pECE plasmid alone (100 µg/ml);
[pECE-CFTR], cells injected with various plasmid
concentrations ranging from 5 to 350 µg/ml. Data are means ± SE
with the number of experiments (n)
between 17 and 113. Y-axis for COS-7
and CFPAC-1 cells is discontinuous. Statistical significance
vs. control cells injected with pECE alone for baseline permeability
(open bars) or vs. cells before cAMP stimulation for cAMP-stimulated
permeability: * P < 0.05, ** P < 0.01, and
*** P < 0.001. Absence of
asterisks denotes nonsignificant differences.
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This set of experiments suggested that cells injected with a low
pECE-CFTR plasmid concentration expressed recombinant
Cl
CFTR channels that were
closed under basal conditions and opened in response to cAMP-mediated
phosphorylation, whereas cells injected with a high pECE-CFTR plasmid
concentration possessed permanently opened recombinant
Cl
channels that were not
susceptible to cAMP-dependent protein kinase (PKA)
phosphorylation. The former were defined as overexpressing cells,
whereas the latter were defined as hyperexpressing cells.
Pharmacological modulation of hyperexpressed CFTR
conductance in COS-7 cells. We next explored the
pharmacology of hyperexpressed recombinant CFTR channels using the SPQ
assay. The sensitivity of hyperexpressed CFTR channels to DPC (500 µM) was similar to that of overexpressed CFTR protein (Fig.
4). The effects of vanadate stimulation on
hyperexpressed CFTR channels were also explored. Vanadate substituted
for inorganic phosphate at the ATP-binding fold to stabilize CFTR
channels in an open state (4). As shown in Fig.
5, vanadate increased baseline membrane
permeability in COS-7 cells hyperexpressing CFTR. This effect was not
reversible upon washout of the drug. Finally, we altered the cell
metabolism with the objective to explore the sensitivity of
hyperexpressed channels to decreased intracellular ATP. Cells were
submitted to a metabolism poisoning cocktail containing cyanide (Fig.
6). Upon perfusion with the poisoning
cocktail, the membrane permeability under baseline decreased,
suggesting that hyperexpressed CFTR channels retained their sensitivity
to intracellular ATP.

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Fig. 4.
Effect of diphenylamine-2-carboxylic acid (DPC) on SPQ fluorescence in
CFTR expressing COS-7 cells. A:
typical experiment performed in COS-7 cell overexpressing CFTR and
injected with 5 µg/ml pECE-CFTR. B:
typical experiment performed in a COS-7 cells hyperexpressing CFTR and
injected with 200 µg/ml pECE-CFTR (open circles are from an injected
cell, whereas open triangles are from a noninjected neighboring cell).
cAMP [10 µM forskolin + 400 µM
8-(4-chlorophenylthio)adenosine 3',5'-cyclic
monophosphate] and DPC (500 µM) were applied as indicated. Open
horizontal bars indicate iodide-containing solution, whereas filled
horizontal bars indicate nitrate-containing solution.
C: halide permeability is expressed as
a percent of predrug values in pECE (CFTR )-injected cells, in
cells overexpressing CFTR (CFTR+), and in cells hyperexpressing CFTR
(CFTR+++). ** Statistical significance with
P < 0.01 vs. cells injected with
pECE alone. Numbers within bars are n
values.
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Fig. 5.
Effect of vanadate on SPQ fluorescence in CFTR hyperexpressing COS-7
cells. A: superimposed SPQ
fluorescence obtained in control (Ctrl; open squares), after 15 min
vanadate (VO4; open circles), and
after washout of the drug (open triangles). Same cell throughout. Open
horizontal bars indicate iodide-containing solution, whereas filled
horizontal bars indicate nitrate-containing solution.
B: average data from 7 different cells
injected with 200 µg/ml pECE-CFTR (filled circles) and from 9 cells
injected with 100 µg/ml pECE (open squares). Halide permeability in
the presence of vanadate is expressed relative to its predrug values.
* P < 0.05 and
** P < 0.01 vs. cells injected
with pECE alone.
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Fig. 6.
Effect of cyanide on SPQ fluorescence in CFTR hyperexpressing COS-7
cells. A: superimposed SPQ
fluorescence in the same cell obtained in control (open squares) and
after 5 min (CN5'; open circles), 15 min (CN 15'; open
triangles), and 25 min (CN 25'; open diamonds) superfusion with a
cell-poisoning cocktail containing cyanide (CN; 2 mM). Open horizontal
bars indicate iodide-containing solution, whereas filled horizontal
bars indicate nitrate-containing solution.
B: average data from 7 cells injected
with 200 µg/ml pECE-CFTR (filled circles) and from 9 cells injected
with 100 µg/ml pECE (open squares). Halide permeability in the
presence of cyanide is expressed relative to its predrug values.
*** P < 0.001 vs. cells
injected with pECE alone.
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Patch-clamp recordings of hyperexpressed CFTR
current. Patch-clamp experiments performed in 350 µg/ml pECE-CFTR-injected CFPAC-1 or COS-7 cells confirmed that a
high-amplitude time-independent Cl
current was yet
available in the absence of cAMP stimulation (Fig.
7A) and
did not increase further upon stimulation with forskolin. On average,
the current amplitude at +60 mV was 28.6 ± 10.2 pA/pF (n = 6) in CFPAC-1 cells injected with
350 µg/ml pECE-CFTR but only 3.5 ± 0.6 pA/pF
(n = 15;
P < 0.001) in CFPAC-1 cells injected with the pECE vector alone.

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Fig. 7.
Whole cell recordings of Cl
currents under K+-free conditions
in CFPAC-1 cells. A: from
left to
right, superimposed current traces of
the background current in a CFPAC-1 cell injected with pECE vector
alone (CFTR ), in a CFPAC-PLJ6-CFTR cell [CFTR+
(PLJ6)], in a cell injected with 100 µg/ml pECE-CFTR (CFTR+),
and in a cell injected with 350 µg/ml pECE-CFTR (CFTR+++). Protocol
consisted of voltage steps, 20 mV in increment, applied from 60
mV to various voltages between 100 and +60 mV. Vertical bar: 200 pA; horizontal bar: 100 ms.
B-E:
current-voltage curves of
Cl currents induced by cAMP
and obtained by digital subtraction.
Insets show corresponding current
traces (vertical bar: 200 pA; horizontal bar: 100 ms). Same voltage
protocol as in A.
B: parental CFPAC-1 cell
(CFTR ). C: CFPAC-PLJ6-CFTR cell
[CFTR+ (PLJ6)]. D: CFPAC-1
cell overexpressing CFTR and injected with 100 µg/ml pECE-CFTR.
E: CFPAC-1 cell hyperexpressing CFTR
and injected with 350 µg/ml pECE-CFTR. In
D and
E, external and intrapipette medium
contained only 30% Cl
(70% gluconate) to improve voltage control.
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Our next goal was to investigate whether hyperexpressed CFTR proteins
would retain their property to regulate other ionic channels. These
experiments were performed in CFPAC-1 cells. In a first series of
experiments, we used the CFPAC-PLJ6-CFTR clone, which stably expresses
a low level of wild-type CFTR protein. Under experimental conditions
that suppressed contaminating K+
currents, the average background current was 3.6 ± 0.5 pA/pF at +60
mV (n = 20) in CFPAC-PLJ6-CFTR cells,
not different from parental CFPAC-1 cells (2.9 ± 0.4 pA/pF at +60
mV; n = 11). cAMP stimulation
activated a Cl
conductance
in 24 out of 58 CFPAC-PLJ6-CFTR cells (mean current amplitude: 14.5 ± 2.8 pA/pF at +60 mV; n = 24) but
not in parental CFPAC-1 cells (Fig.
7B; n = 43). The current activated by cAMP in CFPAC-PLJ6-CFTR cells was
further investigated with a voltage step protocol as shown in Fig. 7,
B-E.
Close examination of the current traces revealed that the
Cl
current activated by
cAMP in CFPAC-PLJ6-CFTR was the sum of
1) a time-independent
Cl
current lacking
rectification that supposedly corresponded to CFTR current and
2) a time-dependent current that
slowly activated upon depolarization and exhibited outward
rectification (Fig. 7C). We
identified the latter as a
Cl
current flowing through
ORCC. CFTR and ORCC currents were also activated by
1) extracellular ATP in CFPAC-1
cells transduced or not with the wild-type CFTR gene
(n = 10; data not shown) and 2) cAMP stimulation in CFPAC-1 cells
injected with a low pECE-CFTR concentration (Fig.
7D). These latter experiments were
conducted in 30%
Cl
-containing solutions
(70% gluconate) to improve the quality of voltage control in injected
cells exhibiting large Cl
currents. Twenty-seven cells were recorded under 30%
Cl
: 10 out of 27 exhibited
cAMP-activated Cl
currents
compatible with CFTR plus ORCC coactivation as shown in Fig.
7D, 7 out of 27 showed
Cl
currents compatible with
CFTR activation alone, and 2 out of 27 with ORCC alone and 8 out of 27 did not respond to cAMP stimulation. Thus CFPAC-1 cells expressing
wild-type CFTR possess ORCC channels that activate in response to cAMP
stimulation, as previously demonstrated by Schwiebert et al. (31) in
9HTEo
epithelial cells.
Under K+-free conditions, CFPAC-1
cells injected with 350 µg/ml pECE-CFTR never exhibited ORCC current
neither at baseline nor under cAMP stimulation
(n = 25 cells; Fig.
7E). These investigations were conducted either in the ruptured-patch configuration with 5 mM MgATP in
the pipette solution or, alternatively, in the permeabilized-patch configuration. Additional experiments were also conducted in low 10 or
30% Cl
solution (28 cells)
or in iodide-containing solution (5 cells). These experiments were all
unsuccessful at identifying ORCC current.
In a study also conducted in CFPAC-1 cells (21), we have previously
identified an inwardly rectifying
K+ current that responds to cAMP
stimulation in the exclusive presence of functional CFTR channels.
Figure 8 shows that the cAMP-activatable inwardly rectifying K+ current was
recorded in CFPAC-1-PLJ6-CFTR cells and also in parental CFPAC-1 cells
injected with a low CFTR plasmid concentration
(n = 7). Under
Cl
-free conditions, no
inwardly rectifying K+ current was
identified either at baseline or under cAMP stimulation (16 cells;
ruptured-patch or permeabilized-patch configurations; Fig.
8C). On average, in injected CFPAC-1
cells bathed in Cl
-depleted
solutions, the background K+
current was 0.9 ± 0.1 pA/pF at +10 mV
(n = 16), not significantly different
from noninjected or pECE-injected cells (0.8 ± 0.1 pA/pF at +10 mV;
n = 13). From these data, we concluded
that hyperexpressed CFTR channels loose their property to regulate
heterologous Cl
or
K+ channels.

View larger version (8K):
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|
Fig. 8.
Inwardly rectifying K+ currents in
CFPAC-1 cells. cAMP-induced K+
currents obtained by digital subtraction and recorded with a ramp
protocol under Cl -free
conditions. Recordings are from a CFPAC-1 cell injected with pECE alone
(A), a CFPAC-PLJ6-CFTR cell
(B), a CFPAC-1 cell overexpressing
CFTR and injected with 100 µg/ml pECE-CFTR
(C), and a CFPAC-1 cell
hyperexpressing CFTR and injected with 350 µg/ml pECE-CFTR
(D).
|
|
 |
DISCUSSION |
In the present study, the use of direct intranuclear cDNA injection
provided us with the opportunity to vary the amount of recombinant CFTR
protein produced in a given cell and to evaluate the potentially
deleterious consequences of hyperexpression on CFTR physiology. Our
data suggest that CFTR hyperexpression alters the basic properties of
the recombinant protein in such a way that CFTR channels
1) are permanently opened in the
absence of cAMP stimulation and exhibit reduced sensitivity to
phosphorylation by PKA; 2) have a
conserved sensitivity to intracellular ATP regulation; and
3) lose their property to regulate
heterologous ionic channels. Using radiolabeled technetium, we
estimated that the intranuclearly injected volume was in the order of
10
12 liters, in accordance with previous calculations
made by others (22). Therefore, the number of copies injected in the
nucleus was in the range of 500-35,000 for plasmid concentrations
between 5 and 350 µg/ml. In comparison, common lipotransfection
methods bring about 100,000 copies in the cytoplasm (19).
Hyperexpression as reported here has previously been observed by Stutts
et al. (34) in NIH/3T3 fibroblasts stably transfected with a retroviral construct carrying wild-type CFTR: five fibroblastic clones were identified for expressing the CFTR protein using Western blot analysis.
Among these, three clones (nos. 3, 5, and 10) demonstrated a higher
level of CFTR expression by densitometric estimates of CFTR-specific
immunostaining. These clones exhibited depolarized membrane potential
at baseline, a consistent background
Cl
conductance, and an
altered sensitivity to cAMP stimulation. Typically, in
clone 5, which maintained the most
elevated basal current mediated by CFTR, there was no response of the
whole cell current to forskolin stimulation.
The reason why hyperexpression produces permanently opened CFTR
channels remains obscure. Honoré et al. (15) previously reported
that different amounts of cRNAs encoding Kv1.3
K+ channels produced biophysically
and pharmacologically distinguishable expressions of
K+ channel activity in
Xenopus oocytes; with a low amount of
cRNA, Kv1.3 channels exhibited time- and voltage-dependent inactivation and were fully blocked by 10 nM charybdotoxin, whereas, with a high
amount of cRNA, recombinant Kv1.3 channels did not inactivate and were
hardly blocked by charybdotoxin. Injection of cRNA at intermediate
concentrations induced K+ channels
with properties corresponding to a mixture of biophysical and
pharmacological properties observed for the inactivating and noninactivating K+ channels
obtained at low and high cRNA concentrations, respectively. Inactivating and noninactivating
K+ channels were also observed in
IM-9 human B lymphocytes stably transfected with Kv1.3 cDNA (15). Our
study suggests that a comparable behavior also exists with CFTR
channels. Most recently, Larsen et al. (20) reported that CFTR
Cl
channel clusters
spontaneously entered a mode of high open probability as a result of
cooperative interaction between neighboring channel proteins.
Previously, it was suggested that CFTR channels are tonically blocked
by the R domain plugging the channel pore and that phosphorylation on
serines by protein kinase A electrostatically repels the R domain,
allowing the passage of Cl
(8). Expression of a variant protein in which the R domain was deleted
resulted in the appearance of
Cl
channels that were
active in the absence of added cAMP and showed only a small additional
response to cAMP (24). One interpretation of our results is that
hyperexpression produces huge clusters in which the high density of
CFTR proteins creates charge-charge interactions between the different
R domains. Interaction may push these domains away from the channel
pore and maintain the channels in the open state in the absence of cAMP
stimulation. However, our results do not provide any experimental
support to this interpretation, and alternative explanations should
also be considered, including cytoskeleton interactions (14) or
synthesis of an immature form of the CFTR protein. Of interest is the
possibility that a portion of CFTR channels are constitutively active
at every level of CFTR expression. In cells overexpressing CFTR, the
proportion of constitutively active CFTR channels is too small to be
detectable with the assay we use. In cells hyperexpressing CFTR, the
number of constitutively active CFTR channels increases and is yet
detectable with the SPQ assay. Obviously, further experiments are
needed to get some insight into the mechanism leading to permanently opened CFTR channels during hyperexpression.
Potentially the farthest reaching implication of our results is that
hyperexpression of CFTR in targeted epithelial cells may affect CFTR
functions that are physiologically important in the organ targeted. For
example, this may concern the capacity of CFTR to regulate other
epithelial ionic channels and thereby to coordinate water and salt
secretion. The mechanism that governs regulation by CFTR of other ionic
channels is still unknown. One proposed hypothesis has been that the
CFTR channel pore drives ATP outside the cell membrane to activate
neighboring channels through an autocrine mechanism (30), although the
capacity of CFTR channels to conduct ATP has most recently been
disputed (23). Our own results also oppose the ATP hypothesis, since
one may expect that permanently opened CFTR channels as induced by
hyperexpression should also drive ATP to activate the ORCC channels
even in the absence of cAMP stimulation. Schiavi et al. (28) previously showed that high levels of wild-type CFTR expression lead to cell growth perturbation and can be deleterious to the normal function of
the cells. Our study extends this concept to the molecular physiology
of the CFTR protein itself. We anticipate that the level of CFTR
expression obtained with direct intranuclear plasmid injection may be
greater than that achieved in vitro with more conventional transfection
techniques (maybe with the exception of retroviral infection; see Ref.
34). Therefore, the deleterious consequences of CFTR hyperexpression as
observed with direct intranuclear cDNA injection in vitro may require
stronger promoters and/or highly more efficient transfection
vectors than currently used in in vivo gene therapy. However, with the
aim to obtain a bystander effect of CFTR gene transfer, higher-strength
promoters are currently developed for clinical use. Our study suggests
that hyperexpression and consequent alterations in CFTR molecular
physiology may potentially result from this search.
 |
ACKNOWLEDGEMENTS |
We thank Béatrice Leray for expert technical assistance with
cell cultures and Marie-Jo Louerat for plasmid amplification.
 |
FOOTNOTES |
This work was supported by grants from the Association Française
de Lutte contre la Mucoviscidose, the Institut National de la
Santé et de la Recherche Médicale, and Air Liquide. G. Loussouarn is recipient of a special grant from the Crédit
Mutuel.
Address for reprint requests: D. Escande, Laboratoire de
Physiopathologie et de Pharmacologie Cellulaires et Moléculaires,
Hôpital Hotel-Dieu, 44093 Nantes, France.
Received 27 January 1997; accepted in final form 9 October 1997.
 |
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