From the Department of Physiology, University of
Texas Southwestern Medical Center, Dallas, Texas 75235 and the
¶ Department of Medicine and Physiology, University of Tennessee,
Memphis, Tennessee 38163
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ABSTRACT |
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A central function of cystic fibrosis
transmembrane conductance regulator (CFTR)-expressing tissues is the
secretion of fluid containing 100-140 mM
HCO3 Except for the sweat gland (1-3), all
CFTR1-expressing cells of
various ductal systems absorb Cl In most CFTR-expressing tissues, HCO3 Most models of HCO3 The observation that the luminal fluid is acidic with high
Cl Culture of NIH 3T3 Cells--
Mock-transfected NIH 3T3 cells or
NIH 3T3 cells stably transfected with WT or Site-directed Mutagenesis--
The pCMVNot6.2 plasmids
containing human WT or Expression of WT and Mutant CFTR in HEK 293 Cells--
HEK 293 cells were maintained in DMEM-HG supplemented with 10% fetal calf
serum, and plated on coverslips. On the following day, WT or mutant
CFTR plasmids and green fluorescent protein (GFP)-expressing plasmids
(Life Technologies, Inc.) were transfected into 293 cells using the
Fugene mammalian transfection kit (Boehringer Mannheim) according to
instructions provided by the manufacturer. Briefly, the mixture of
plasmids and Fugene solution (pCMVNot6.2, 1.5 µg; pCMVGFP, 1.5 µg;
Fugene, 12 µl) was incubated in 100 µl of DMEM for 30 min before
addition to the culture media. The cells were used for
immunocytochemistry or pHi measurements 48-72 h
after transfection.
Immunocytochemistry--
HEK 293 cells transfected with
expressing vectors were stained with a rat polyclonal anti-C-terminal
CFTR antibody (Ab) R3194 (19) and/or mouse monoclonal anti-rat Grp78
(BiP) antibody (StressGen Biotechnologies, Victoria, BC, Canada) to
determine their expression patterns using a published procedure (20).
For double-labeling, primary and secondary incubations were repeated
with antibodies against the second protein of interest. Images were
obtained using a Bio-Rad MRC 1024 confocal microscope.
Intracellular pH Measurements--
The coverslips with cells
attached to them were washed once with a Hepes-buffered solution and
assembled to form the bottom of perifusion chamber. The Hepes-buffered
solution contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, 10 Hepes (pH 7.4 with NaOH). In the case of 293 cells, the level of transfection was
estimated from GFP fluorescence. High GFP-expressing cells were
identified by viewing GFP fluorescence at excitation wavelength of 475 nm. GFP fluorescence was recorded and used to compare CFTR expression
in different experiments. Subsequently, cells were loaded with BCECF by
a 10-min incubation at room temperature in Hepes-buffered solution
containing 2.5 µM BCECF-AM. BCECF fluorescence was at
least 10-fold higher than the original GFP fluorescence. After BCECF
loading the cells were perfused with a
HCO3 Patch Clamp--
Cl AE in Cells Stably Transfected with CFTR--
The first set of
experiments to study regulation of AE activity by CFTR was performed in
NIH 3T3 cells stably transfected and expressing high levels of CFTR
protein. This particular model system has been used to extensively
characterize the properties of CFTR Cl
Stimulation of CFTR-expressing 3T3 cells with forskolin caused a
time-dependent intracellular acidification that was
complete after 3 min of incubation at 37 °C. This acidification was
observed only in cells expressing CFTR in all experiments tested
(n = 17) and was not inhibited by DIDS
(n = 9). Furthermore, removal of Cl
The finding that after forskolin stimulation, the
pHi changes due to changes in transcellular
Cl
The lack of effect imparted by changes in membrane potential and
inhibitors of CFTR Cl Expression and Localization of WT CFTR and CFTR Mutants in HEK 293 Cells--
All the experimental protocols used to identify the
HCO3
Many CFTR mutants, including some used in the present work, are known
folding mutants (26, 27) that are rapidly degraded by the
ubiquitin-dependent proteasome system (28) before
substantial amount of the protein reaches the plasma membrane.
Therefore, we first determined the expression and localization of the
CFTR mutants used in the present work. To distinguish plasma membrane and ER localized CFTR, CFTR localization was compared with that of the
ER-resident chaperone BiP (29). Fig. 6
(a and b) shows that WT CFTR is expressed in the
plasma membrane and CFTR expression correlated with expression of GFP.
In numerous cells examined, expression of WT CFTR and all mutants
correlated very well with expression of GFP. Fig. 6 (e and
f) shows the correlation between expression of GFP and
WT CFTR and Anion Exchange Activity in HEK 293 Cells--
The WT
CFTR and GFP constructs were used to determine whether expression of
CFTR in 293 cells affected AE activity as observed in stably
transfected NIH 3T3 cells (Figs. 1-5). Fig.
7 shows representative traces, and Figs.
8 and 9
summarize the results of multiple experiments. In these experiments GFP
fluorescence was measured prior to loading with BCECF. Based on
intensity of GFP fluorescence, the transfected cultures were divided
into two groups: those that express low to moderate levels and those
that express high levels of the transgene.
Expression of CFTR in 293 cells was sufficient to increase the
DIDS-inhibitable pHi increase upon removal of
external Cl
Of all known HCO3
The complete (Fig. 10a) or partial (Fig. 10b)
resistance of the Cl
In conclusion, we believe that the combined results in NIH 3T3 and HEK
293 cells provide strong evidence for regulation of AE activity by
CFTR. It is important to reiterate that such regulation required the
activation of CFTR by cAMP but did not require Cl CFTR Mutants and AE Activity--
To begin to elucidate the
mechanism by which CFTR domains regulate AE activity, the effect of
several mutations in CFTR that have been previously characterized in
terms of CFTR Cl
Another series of mutations in NBD2 that are known to affect channel
activity (Fig. 12) indicate that there
is no correlation between Cl
Taken together, the results presented here show that CFTR regulates
Cl
Kinetic, pharmacological, and molecular data indicate that it is
highly unlikely that the
Cl
Previously, CFTR has been shown to modulate the activity of
several ion channels and transporters. The best studied example is
regulation of epithelial Na+ channels (ENaC) (34).
Co-expression of CFTR and ENaC in Madin-Darby canine kidney cells
resulted in attenuation of ENaC activity upon stimulation of CFTR with
cAMP (34). This regulation appears to be by direct interaction between
the two proteins as it can be reproduced with purified proteins
reconstituted into planar lipid bilayers (35). CFTR was also proposed
to regulate the inward rectifying K+ channel ROMK2 (36) in
a mechanism similar to the regulation of the ATP-sensitive
K+ channel by the sulfonylurea receptor in pancreatic
The regulation of AE by CFTR demonstrated here may be of
particular physiological significance to understanding
the pathophysiology of cystic fibrosis. It is, therefore, of interest
to determine which of the known AE isoforms is regulated by CFTR and
whether such regulation exists in a native CFTR-expressing tissue.
Recently, we obtained evidence for regulation of
Cl. High levels of HCO3
maintain
secreted proteins such as mucins (all tissues) and digestive enzymes
(pancreas) in a soluble and/or inactive state. HCO3
secretion is impaired in CF in all CFTR-expressing,
HCO3
-secreting tissues examined. The mechanism
responsible for this critical problem in CF is unknown. Since a major
component of HCO3
secretion in CFTR-expressing cells
is mediated by the action of a Cl
/HCO3
exchanger (AE), in the present work we examined the regulation of AE
activity by CFTR. In NIH 3T3 cells stably transfected with wild type
CFTR and in HEK 293 cells expressing WT and several mutant CFTR,
activation of CFTR by cAMP stimulated AE activity. Pharmacological and
mutagenesis studies indicated that expression of CFTR in the plasma
membrane, but not the Cl
conductive function of CFTR was
required for activation of AE. Furthermore, mutations in NBD2 altered
regulation of AE activity by CFTR independent of their effect on
Cl
channel activity. At very high expression levels CFTR
modified the sensitivity of AE to
4,4'-diisothiocyanatostilbene-2,2'-disulfonate. The novel finding of
regulation of Cl
/HCO3
exchange by CFTR
reported here may have important physiological implications and
explain, at least in part, the impaired HCO3
secretion in CF.
INTRODUCTION
Top
Abstract
Introduction
References
and secrete
HCO3
(4-6). Since the discovery that
Cl
transport is defective in CF (7) and that CFTR
functions as a Cl
channel (8), Cl
transport
by CFTR-expressing tissues has been extensively studied (9). By
contrast, transcellular HCO3
secretion
is poorly understood (5), and little studied, even though
HCO3
secretion is impaired in CF
(10).
secretion has electrogenic and electroneutral components (4-6). The
electrogenic component is assumed to be mediated by an unknown
HCO3
channel or due to
HCO3
transport through CFTR itself
(11, 12). The electroneutral component is assumed to be mediated by a
Cl
/HCO3
exchange
activity. However, direct evidence for a
Cl
/HCO3
exchange
activity in the luminal membrane is limited to the perfused pancreatic
(13) and submandibular ducts (14).
secretion assume
that CFTR and the luminal
Cl
/HCO3
anion exchanger
(AE) are indirectly coupled. In these models Cl
absorbed
by the AE across the luminal membrane is secreted into the lumen by
CFTR to support further HCO3
secretion
(4, 15). However, if
Cl
/HCO3
exchange is
unaltered in CF, such a mechanism cannot adequately explain the
concomitant acidity of the secreted fluid and the impaired
Cl
absorption observed in CF (16-18). If
Cl
/HCO3
exchange is
responsible for the bulk of Cl
absorption and
HCO3
secretion and CFTR function is
required only for return of Cl
to the lumen, then in CF
Cl
absorption should be normal (normal
Cl
/HCO3
exchange) and
the secreted fluid should be acidic due to the limited supply of
luminal Cl
. This is not the case (16-18). Alternatively,
if Cl
reabsorption is singularly impaired in CF, the
model predicts that the high Cl
concentration in the
luminal fluid should increase HCO3
secretion by AE to produce an alkaline fluid with high Cl
concentration. Again, this is not observed.
concentration in CF (16-18) suggests that CFTR
regulates HCO3
secretion in
CFTR-expressing tissues. CFTR could regulate the electrogenic,
electroneutral, or both components of
HCO3
secretion. In the present work,
we explored the existence of these regulatory mechanisms in cells
stably or transiently expressing wild type (WT) or several mutated CFTR
constructs. We report that a cAMP-activated CFTR regulates
Cl
/HCO3
exchange
activity in several experimental systems. Expression of CFTR in the
plasma membrane was required for regulation of Cl
/HCO3
exchange, as
expression of several folding mutants, including
F508, had no effect
on Cl
/HCO3
exchange
activity. Surprisingly, the Cl
conductive function of
CFTR was not required for activation of Cl
/HCO3
exchange.
Furthermore, mutations in NBD2 altered regulation of AE activity by
CFTR independent of their effect on Cl
channel activity.
At very high expression levels, CFTR modified the sensitivity of
Cl
/HCO3
exchange to
DIDS. The novel finding of regulation of
Cl
/HCO3
exchange by CFTR
reported here may have important physiological implications and
explain, at least in part, the impaired
HCO3
secretion in CF.
EXPERIMENTAL PROCEDURES
F508 CFTR were kindly
provided by Dr. Michael J. Welch (University of Iowa, Iowa City, IA).
The cells were maintained in Dulbecco's modified Eagle's medium
containing 10 mM glucose (DMEM-HG) and 10% fetal calf
serum and plated on a sterile 22 × 40-mm coverslip at a density
of 2.5 × 105 cells/cm2 for intracellular
pH (pHi) measurements.
F508 CFTR cDNA were a generous gift from
Dr. Johanna Rommens (Hospital for Sick Children, Toronto, Canada).
Oligonucleotide-directed mutagenesis using the GeneEditor mutagenesis
kit (Promega, Madison, WI) was performed to generate the mutant CFTR in
the expression vector pCMVNot6.2. Briefly, mutants were selected based
upon the incorporation of a second-site mutation in
-lactamase,
which alters its substrate specificity allowing resistance of
transformed bacteria to cefotaxime and ceftriaxone in addition to
ampicillin. Incorporation of the mutation was verified by DNA
sequencing. The mutagenesis primers were as follows: P205S primer,
5'-CGT GTG GAT CGC TTC TTT GCA AGT GGC-3'; W846term, 5'-GAG CAT ACC AGC
AGT GAC TAC ATA GAA CAC ATA CCT TCG ATA TAT TAC-3'; G1247D/G1249E,
5'-GTG GGC CTC TTG GGA AGA ACT GAT TCA GAG AAG AGT ACT TTG TTA TCA
GC-3'; K1250M, 5'-CTT GGG AAG AAC TGG ATC AGG GAT GAG TAC TTT GTT ATC
AGC-3'; D1370N, 5'-GTA AGG CGA AGA TCT TGC TGC TTA ATG AAC CCA GTG CTC ATT TGG ATC-3'. Transfection-quality plasmid DNA was prepared using
reagents supplied by Qiagen (Valencia, CA).
-buffered solution and
pHi was measured by photon counting using the
recording setup (PTI Delta Ram, Brunswick, NJ) and the conditions
described previously (21). The
HCO3
-buffered solution contained (in
mM) 120 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, 5 Hepes, 25 NaHCO3 (pH 7.4 with NaOH) and was continuously gassed with 95% O2 and 5%
CO2. Cl
free solutions were prepared by
replacing Cl
with gluconate. When desired 5 µM forskolin was added to the perfusate from a stock
solution of 10 mM in Me2SO. BCECF fluorescence was recorded at excitation wavelengths of 490 and 440 nm at a resolution of 2/s. The 490/440 ratios were calibrated intracellularly by perfusing the cells with solutions containing 145 mM
KCl, 10 mM Hepes, 5 µM nigericin with pH
adjusted to 6.2-7.6, as described previously (13). The results of
multiple experiments were analyzed using paired or non-paired
Student's t-test as appropriate.
current was recorded using the
whole cell configuration of the patch-clamp technique (22) as described
before (23). NIH 3T3 cells were released from culture dishes by a 30-s
treatment with trypsin-EDTA, washed twice with DMEM, and placed in a
perfusion chamber. Cl
current was isolated by using
Cl
as the only permeant ion in the pipette and bath
solutions. In all experiments the bath solution contained (in
mM) 140 N-methyl-D-glucamine chloride (NMDG-Cl), 1 MgCl2, 10 glucose and 10 Hepes (pH
7.4 with Tris), and the pipette solution contained 140 NMDG-Cl, 5 EGTA, 5 Tris-ATP, 5 MgCl2 and 10 Hepes (pH 7.2 with Tris). All
recordings were made at room temperature. Seals of 5-8 gigohms were
obtained on the cell surface prior to establishing the whole-cell
configuration. Macroscopic currents were recorded using the Axopatch-1B
patch clamp amplifier (Axon Instruments). Results were collected at 5 kHz after filtering at 2 kHz. The membrane potential was held at -40
mV to record the inward current.
RESULTS AND DISCUSSION
channel activity
(17). Mock-transfected cells of the same parental line were used as
controls. Significantly, results identical to mock-transfected cells
were obtained in cells stably transfected with
F508 CFTR (data not
shown). A standard protocol of removal and addition of Cl
to the incubation medium buffered with
HCO3
was used to follow
Cl
/HCO3
exchange
activity. All the changes in pHi reported here were dependent on the presence of HCO3
in the incubation media (data not shown). Fig.
1 illustrates the basic observation that
CFTR-expressing cells exhibited a forskolin-dependent activation of the AE. Fig. 2 summarizes
the results of 5-17 experiments under each condition. Removal of
Cl
from the incubation medium of mock-transfected cells
resulted in a slow and modest increase in pHi,
which was completely reversed on addition of Cl
to the
medium. Stimulation of control cells with 5 µM forskolin had no effect on basal level of pHi or the
pHi changes observed upon removal and readdition
of Cl
. Finally, treating the cells with 0.5 mM DIDS, a blocker of
Cl
/HCO3
exchange
activity (24), nearly abolished pHi changes resulting from changes in transcellular Cl
concentration.
These properties are commonly used to demonstrate Cl
/HCO3
exchange
activity in cells (13, 14, 24). CFTR-expressing cells showed marginal
statistical difference in
Cl
/HCO3
exchange
activity under resting conditions when compared with mock-transfected
cells (p = 0.11), or cells stably transfected with
F508 CFTR (data not shown). Interestingly,
Cl
/HCO3
exchange
activity observed in resting cells expressing CFTR was inhibited by
DIDS to the same extent as that measured in control cells (Fig. 2).
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Fig. 1.
Effect of forskolin on AE activity in NIH 3T3
cells. NIH 3T3 cells attached to glass coverslips were loaded with
BCECF and perfused with HCO3 -buffered
solutions. As indicated by the bars, the cells were perfused
with a Cl
-free solution before and after stimulation with
5 µM forskolin. In panel a, the cells were
transfected with empty vectors. Identical results were obtained in
cells stably transfected with
F508 CFTR (data not shown). In
panel b, the cells were stably transfected with
WT CFTR. In both experiments the cells were treated with 0.5 mM DIDS after the stimulation with forskolin. Upper
deflection in all traces indicates increase in
pHi.
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Fig. 2.
AE activity in CFTR-stimulated cells.
The protocols of Fig. 1 were used to measure the rate and extent of
pHi changes due to removal and addition of
Cl to mock transfected or WT CFTR-expressing NIH 3T3
cells before (Con, control) and after stimulation with 5 µM forskolin and before and after treatment with 0.5 mM DIDS. The results of 5-17 experiments were summarized
to calculate the mean ± S.E. After forskolin stimulation all
pHi changes in WT CFTR-expressing cells were
much higher than those in mock transfected cells. Before forskolin
stimulation the pHi changes in cells expressing
WT CFTR trended to be higher than those in control cells, although the
differences did not reach statistical significance (p = 0.11).
from
the incubation medium of forskolin-stimulated, CFTR-expressing cells
caused a rapid and a large increase in pHi that was reversed upon readdition of Cl
to the medium (Fig.
1b). Fig. 2 shows that after forskolin stimulation the rate
of pHi change due to changes in transcellular Cl
gradient in CFTR-expressing cells is 8-fold faster
than that before forskolin stimulation in the same cells, or before and after forskolin stimulation in control cells. Thus, the increased rate
of pHi changes required both expression of CFTR and activation of the protein by cAMP-dependent mechanisms.
It is well established that CFTR-mediated Cl
channel
activity is regulated by a cAMP-dependent phosphorylation (2, 7, 8).
gradient are resistant to inhibition by DIDS (Figs.
1b and 2) was unexpected. Since the same NIH 3T3 cell line
was used to suggest that CFTR may function as a Cl
and a
HCO3
-permeable channel (25), we
considered the possibility that the pHi changes
illustrated in Figs. 1 and 2 are due to CFTR functioning as a
HCO3
channel. Several lines of
evidence indicate that this is not the case. In contrast to Poulsen
et al. (25), in more than 10 experiments, we did not see any
effect of HCO3
addition on
pHi of forskolin-stimulated acidified NIH 3T3
cells (using the protocol of Fig. 1b in Ref. 25).
Furthermore, depolarization of the plasma membrane with 5 mM Ba2+ (n = 5; data not
shown), 100 mM K+ (see Fig. 10) or 125 mM K+ (data not shown) had no effect on the
changes in pHi observed on removal and addition
of Cl
as would be expected is CFTR was functioning as a
HCO3
channel. Additional evidence that
CFTR conductance was not responsible for the pHi
changes in Figs. 1 and 2 is provided by testing the effect of
inhibitors of CFTR Cl
channel activity. Fig.
3 shows the effect of 100 µM DPC and 100 µM glibenclamide on
Cl
channel activity of CFTR-expressing cells. At 100 µM these blockers inhibited CFTR-dependent
Cl
current by at least 90% (Fig. 3c).
Notably, these blockers had no effect on the ability of CFTR to
stimulate pHi changes upon Cl
removal or addition in a forskolin-dependent manner (Fig.
4).
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Fig. 3.
Stimulation of Cl current in
NIH 3T3 cells expressing WT CFTR. The whole cell configuration of
the patch-clamp technique was used to measure Cl
current
in cells internally perfused through the patch pipette and bathed in
Na+- and K+-free, NMDG-Cl-containing medium.
Cl
current was stimulated by exposing cells to 5 µM forskolin. When the current reached maximal value, the
cells were perfused with a solution containing 100 µM DPC
(a) or 100 µM glibenclamide (b).
Panel c summarizes the results from the indicated
number of experiments to give the mean ± S.E. of the current as
percentage of the maximal current measured before addition of the
drugs.
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Fig. 4.
Effect of CFTR Cl channel
inhibition on AE activity. NIH 3T3 cells stably transfected with
WT CFTR and incubated in HCO3
-buffered
media were stimulated with 5 µM forskolin and transiently
exposed to Cl
-free medium before and after incubation
with 100 µM DPC (a) or 100 µM
glibenclamide (b). Similar results were obtained in at least
three experiments under each experimental condition.
channel activity strongly suggested
that the pHi changes observed on removal and
addition of Cl
are not mediated by an electrogenic
pathway. Rather, it appears that expression and stimulation of CFTR by
cAMP activated an electroneutral HCO3
transport mechanism. If this pathway transports Cl
in
exchange for HCO3
, then the
pHi changes should be a function of
intracellular Cl
content. Fig.
5 shows the protocol used to test this
prediction. The cells were first treated with DIDS to prevent the
initial changes in pHi due to Cl
removal. Then the cells were incubated in a
HCO3
-buffered, Cl
-free
medium for 1 min (Fig. 5a), 60 min (Fig. 5b), or
various times between 5 and 30 min (data not shown) to deplete
intracellular Cl
. The cells were then stimulated with
forskolin to activate CFTR and, thus,
Cl
/HCO3
exchange.
Progressive depletion of intracellular Cl
resulted in a
graded inhibition of forskolin-activated pHi increase (Fig. 5). Readdition of Cl
to the incubation
medium resulted in a pronounce acidification, as expected from
HCO3
i/Cl
o
exchange. Removal and readdition of Cl
in these cells
showed the expected changes in pHi (Fig. 5). In
additional experiments we incubated the cells in a HEPES-buffered, Cl
-free medium for 30-60 min to deplete intracellular
Cl
. Such incubations were as effective in inhibiting the
effect of forskolin on pHi in the presence of
HCO3
as the incubation in
HCO3
-buffered, Cl
-free
medium shown in Fig. 5b (data not shown).
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Fig. 5.
Dependence of
pHi changes on intracellular
Cl content. NIH 3T3 cells stably transfected with WT
CFTR and incubated in HCO3
-buffered
media were treated with 0.5 mM DIDS before incubation in
Cl
-free medium (a and b). After 1 min (a) or 1 h (b) of incubation in
Cl
-free medium, the cells were stimulated with 5 µM forskolin while still in Cl
-free medium.
Approximately 3 min after forskolin stimulation, the cells were
incubated in Cl
-containing medium, which caused a rapid
reduction in pHi. Subsequently the cells were
subjected to another round of incubation in Cl
-free and
Cl
-containing media while still incubated with 0.5 mM DIDS and stimulated with 5 µM forskolin.
Similar results were observed in at least three experiments under
each experimental condition.
transporter activated by
forskolin in CFTR-expressing 3T3 cells except for the lack of
inhibition by DIDS point to a
Cl
/HCO3
exchanger. These
include (a) requirement for a
HCO3
gradient, (b)
requirement for a Cl
gradient, (c)
independence from Na+o, (see below),
(d) electroneutrality, and (e) insensitivity to
Cl
channel blockers. A possible explanation for these
observations is that high level expression of CFTR in 3T3 cells
activated the anion exchanger and modified its sensitivity to DIDS. To
test this hypothesis and provide additional evidence for regulation of
anion exchange by CFTR, we examined the effect of transient expression
of WT and mutant CFTR on anion exchange in HEK 293 cells. To identify
the transfected cells and evaluate the extent of protein expression,
the cells were co-transfected with GFP and the various CFTR plasmids.
F508 CFTR. In agreement with previous reports (30), it can be seen
that
F508 CFTR is retained in the ER and excluded from the plasma
membrane. Fig. 6, c and d shows the localization of WT CFTR relative to that of BiP, and g and h
show the localization of P205S CFTR. It is clear that WT CFTR was
present in the ER and plasma membrane whereas P205S CFTR localized in
the ER. Fig. 6 (i-k) shows the plasma membrane localization
of K1250M CFTR, D1370N CFTR, and the double mutant G1247D/G1249E CFTR,
respectively. Another mutant used in the present work is W846term CFTR,
which includes amino acids 1-845 of WT CFTR. This construct could not be localized with the C-terminal specific antibodies used to detect the
other constructs. However, a similar C-terminal truncation at Asp-836
has been shown to be expressed in plasma membrane of HeLa cells as a
functioning Cl
channel (31).
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Fig. 6.
Localization of WT CFTR and mutant CFTR in
HEK 293 cells. HEK 293 cells were transfected with plasmids
carrying the indicated CFTR-expressing genes and cotransfected with
plasmids carrying the gene for GFP. About 48 h after transfection,
the cells were fixed and stained with Ab specific for CFTR and the
ER-resident chaperone BiP. The transfected cells were identified in the
confocal microscope by measuring GFP fluorescence. Examples for such
fluorescence are given in panels b and
f. In all experiments examined, there was excellent
correlation between expression of GFP and WT CFTR or any of the mutant
CFTR. The cellular distribution of CFTR in GFP-expressing cells was
identified by a rhodamine staining. When the correlation between BiP
and CFTR mutants was studied, BiP was detected with Ab specific to BiP,
which were stained with fluorescein-coupled secondary Ab. The Ab used
are listed below each image. Panels a and
b, c and d, e and
f, and g and h are from the same
cells. Please note the expression of WT CFTR in the plasma membrane.
Panels e and g show the predominant ER
localization of F508 and P205S CFTR, respectively. Panels
i, j, and k show the expression of the
indicated CFTR mutants in the plasma membrane. Localization similar to
that shown in each panel was seen in virtually every cell expressing
the respective construct. (Original magnification in panels
c-f, ×400; original magnification in all other panels,
×600.)
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Fig. 7.
Effect of WT CFTR on AE activity of HEK 293 cells. HEK 293 cells were transiently transfected with WT CFTR and
the effect of forskolin stimulation, 0.5 mM DIDS, or 0.1 mM DPC on AE activity was measured using the protocol of
Fig. 1. Panel a shows the results obtained in
cells transfected with pCMVGFP and the empty vector for CFTR and
expressed high levels of GFP. Panel b shows the
results obtained with cells expressing modest levels of WT CFTR as
judged from the intensity of GFP fluorescence. Panels
c and d show results obtained with cells
expressing high levels of WT CFTR.
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Fig. 8.
Relationship between CFTR expression and AE
activity. HEK 293 cells were transfected with empty vector and GFP
(mock, open bars) or WT CFTR and GFP. Based on
intensity of GFP fluorescence excited at 475 nm and measured at
emission wavelengths of 530 nm, the cells were divided into two groups:
medium (four experiments, hatched bars) and high
(eight experiments, closed bars) expressors
before loading with BCECF and measurement of AE activity as in Fig. 7.
The figure shows the mean ± S.E. of the indicated number of
experiments before and after forskolin stimulation.
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Fig. 9.
Pharmacological characterization of
CFTR-regulated AE activity. HEK 293 cells transiently transfected
with high levels of WT CFTR were stimulated with 5 µM
forskolin, and the protocol of Fig. 7 was used to evaluate the effect
of 0.1 mM DPC, 0.1 mM glibenclamide
(Glib), or 0.5 mM DIDS on
pHi changes induced by removal and addition of
Cl . The figure shows the mean ± S.E. of the number
of experiments performed. DIDS inhibited the activity by approximately
60%.
and prior to stimulation with forskolin. The
increase in AE activity in unstimulated cells was statistically
significant only at high levels of WT CFTR expression (Fig. 8;
p = 0.018). Stimulation of cells expressing moderate or
high levels of WT CFTR with forskolin caused an initial acidification,
as was observed in NIH 3T3 cells (Fig. 1). Stimulation with forskolin
dramatically increased AE activity and the increased activity
(n = 16) correlated with the extent of WT CFTR
expression (Fig. 8). Figs. 7d and 9 show that inhibitors of
CFTR Cl
current, DPC and glibenclamide, had no
measurable effect on AE activity after stimulation with forskolin.
Again, these results are similar to those found in NIH 3T3 cells stably
expressing WT CFTR (Fig. 4).
transport pathways
(including HCO3
conductance and the
Na+-HCO3
cotransporters),
only the AE is electroneutral and its activity is independent of
Na+ (24). Hence, as a further test for the
HCO3
transport activity stimulated by
CFTR we determined the effect of membrane potential and external
Na+ on this activity using two experimental conditions. In
the first set of experiments, cells incubated in
HCO3
-buffered solutions in which all
NaCl was replaced with KCl and all NaHCO3 was replaced with
choline-HCO3
. In these solutions, as
needed, Cl
was replaced with gluconate using
K+-gluconate. To prevent intracellular acidification due to
incubation of the cells in these Na+-free solutions, all
solutions also contained 5 µM
Na+/H+ exchange inhibitor,
ethyl-isopropyl-amiloride. Under these conditions removal of external
Na+ still caused substantial intracellular acidification,
probably due to the activity of a
Na+-HCO3
cotransporter
(data not shown). However, the effect of removal and addition of
Cl
was identical to those illustrated in Fig.
10 using the second experimental
protocol. In these experiments external Na+ was reduced
from 140 to 40 mM, which removed the need to include ethyl-isopropyl-amiloride in the incubation medium and almost eliminated the initial acidification on reduction in external Na+. The membrane potential was strongly depolarized by
increasing external K+ from 5 to 100 mM. Fig.
10 shows that membrane depolarization had no effect on the
pHi changes observed on removal and addition of
Cl
in NIH 3T3 (Fig. 10a) or HEK 293 cells
(Fig. 10b) expressing high levels of CFTR.
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Fig. 10.
Effect of membrane depolarization on
CFTR-stimulated AE activity. NIH 3T3 cells (a) stably
transfected or HEK cells (b) transiently transfected with
high levels of WT CFTR were perfused with
HCO3 -buffered solutions. After strong
membrane depolarization by increasing external K+ to 100 mM, the cells were incubated in Cl
-free and
then Cl
-containing high K+ medium before and
after stimulation with 5 µM forskolin. The cells were
then incubated with 0.5 mM DIDS. As was found in normal
K+ (5 mM) medium, DIDS had no effect in NIH 3T3
cells and only partially inhibited AE activity in 293 cells expressing
high WT CFTR levels. Identical results were obtained when cells were
incubated in Na+-free media containing 125 mM
K+, 25 mM
choline-HCO3
, and 5 µM
ethyl-isopropyl-amiloride to inhibit Na+/H+
exchange activity (data not shown). Similar results were obtained in at
least three experiments under each condition.
-dependent
pHi changes to 0.5 mM DIDS was
preserved under high K+ conditions. However, a detailed
examination of the result in 293 cells show that the sensitivity of AE
activity to inhibition by DIDS was a function of the level of WT CFTR
expression. At moderate expression levels of WT CFTR, DIDS nearly
abolished AE activity. However, at high expression levels of WT CFTR
DIDS inhibited only about 60% of AE activity (Figs. 7, b
and c, and 9). This may account, at least in part, for the
resistance of CFTR-stimulated AE activity in NIH 3T3 to DIDS (Figs. 1
and 2).
transport by CFTR. The increased activity observed in non-stimulated cells expressing high levels of CFTR probably reflects tonic activation of CFTR in resting cells as a result of routine cell handling during an experiment.
channel activity were assessed. Fig.
11 shows the results obtained with CFTR
mutants that did not affect AE activity.
F508 and P205S CFTR are
known maturation mutants (26, 27) that do not reach the plasma membrane
of 293 cells (Fig. 6). Hence, it was not surprising that they had no
effect on AE activity. CFTR truncated at Asp-836 (between the R domain
and NBD2) was reported to maintain Cl
channel activity
when expressed in HeLa cells (31). However, expression of a similar
construct truncated at Trp-846 in 293 cells was insufficient to
activate AE (Fig. 11c).
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Fig. 11.
Folding CFTR mutants and N-terminal half
CFTR had no effect on AE activity. HEK 293 cells were
cotransfected with plasmids carrying GFP and P205S CFTR (a),
F508 CFTR (b), or Trp-846 termination codon CFTR
(c). Immunocytochemical assays verified that cells
expressing high levels of GFP also expressed high levels of the
mutants. Cells expressing high levels of GFP were used to test the
effect of forskolin stimulation on AE activity by the standard protocol
of Cl
removal and addition. Expression of the above
mutants did not activate AE before or after forskolin stimulation.
Similar observations were seen in at least three experiments with each
construct.
channel activity and
activation of AE, as predicted from the lack of effect of
Cl
channel blockers. For example, the G1247D/G1249E CFTR
double mutant was reported to have no Cl
channel activity
(32), was expressed in the plasma membrane (Fig. 6k), and
had no effect on AE activity (Fig. 12a). The K1250M CFTR
mutant had increased channel activity (32), was expressed in the plasma
membrane (Fig. 6i) and activated AE similar to WT CFTR (Fig.
12b). However, D1370N CFTR had nearly normal
Cl
channel activity (32) and was expressed in the plasma
membrane (Fig. 6j), but was unable to activate AE (Fig.
12c).
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Fig. 12.
Effect of CFTR mutations in NBD2 on AE
activity. HEK 293 cells were transfected with the indicated
constructs, all of which carrying mutations in NBD2. Cells expressing
high levels of GFP were used for experimentation. The G1247D/G1249E
double mutant was expressed in the plasma membrane but had no effect on
AE activity (a). The K1250M mutant was at least as effective
as WT CFTR in stimulating AE activity (b). The D1370N mutant
had minimal effect on AE activity (c). Similar results were
obtained in at least three experiments with each mutant.
/HCO3
exchange
activity in stably transfected NIH 3T3 cells and transiently transfected HEK 293 cells. The anion exchange activity stimulated by
CFTR has all the kinetic properties associated with anion exchange reported in many cell types (24). The only deviation was the relative
insensitivity of the exchange activity to DIDS. However, expression of
CFTR at high level was apparently responsible for this behavior. This
finding highlights the need for caution when using cell lines and
overexpression of CFTR to reach conclusions as to its function in
native tissues.
/HCO3
exchange
activated by CFTR was mediated by CFTR itself. This is concluded from
the findings that inhibitors and mutants of CFTR Cl
channel activity had no effect on
Cl
/HCO3
exchange
activity stimulated by CFTR. More importantly, this lack of correlation
demonstrates that transport of Cl
by CFTR was not needed
to observe increased exchange activity, although CFTR had to be
activated by a cAMP-dependent mechanism to exert its effect
on the AE. Thus, an activated conformation of the protein was needed
for activation of AE. This is further supported by the findings with
the
F508 and P205S CFTR maturation mutants, which showed that
expression of CFTR in the plasma membrane, rather than mere expression
of CFTR in the cells, was required for activation of AE. In this
respect the results obtained with D1370N CFTR are of particular
interest since this mutation in NBD2 did not ablate channel activity
(32) but eliminated regulation of AE activity by CFTR. Recently, it was
reported that the D1506A mutation of the sulfonylurea receptor 1 protein, which corresponds to D1370N of human CFTR, similarly failed to
stimulate the KATP channel (33). This points to the
importance of NBD2 in regulating other ion channel or transport
proteins. It will be important to determine whether NBD2 of CFTR by
itself can regulate AE activity. These experiments are in progress.
cells (37). Such a regulation may account for the
cAMP-dependent membrane repolarization in colonic crypt
base cells (6, 38). Finally, expression of CFTR in Xenopus
oocyte and stimulation with cAMP increased water permeability (39), as
if CFTR functions as or regulates a water channel. Similar to our
findings with AE, in all these cases, activation of CFTR by cAMP, but
not Cl
channel activity, was required for regulation of
the transporters. This conclusion is reinforced by our findings that
mutations in or truncation of NBD2 resulted in loss of regulation of AE
by CFTR, even when Cl
channel activity was retained.
/HCO3
exchange
activity by CFTR in the intestinal cell line T84 and, more importantly,
in duct cells of the mouse submandibular gland and
pancreas.2 In these studies, we also discuss the
physiological significance of regulation of AE by CFTR.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Michael J. Welch for kindly
providing us with NIH 3T3 cells stably transfected with CFTR. We
also thank Dr. Johanna Rommens for the generous gift of pCMVNot6.2
containing human WT or F508 CFTR cDNA.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants DE12309 and DK38938 (to S. M.) and DK49835 (to P. J. T.).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.
§ Current address: Dept. of Pharmacology, Yonsei University College of Medicine, Seoul 120-752, Korea.
Established Investigator of the American Heart Association.
** To whom correspondence should be addressed: Dept. of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9040. Tel.: 214-648-2593; Fax: 214-648-8685; E-mail: smuall{at}mednet.swmed.edu.
The abbreviations used are:
CFTR, cystic
fibrosis transmembrane conductance regulator; CF, cystic fibrosis; AE, Cl/HCO3
exchanger; WT, wild type; NBD, nucleotide binding domain; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate; GFP, green
fluorescent protein; BCECF-AM, 2'7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl
ester; DPC, N-phenylanthranilic acid; NMDG, N-methyl-D-glucamine; Ab, antibody; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; ENaC, epithelial Na+ channel.
2 M.G. Lee, J. Y. Choi, L. Xiang, E. Strickland, P. J. Thomas, and S. Muallem, unpublished results.
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REFERENCES |
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