From the Renal-Electrolyte Division, Department of
Medicine, University of Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania 15261, the Departments of § Medicine and
¶ Physiology, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104,
St Vincent's Institute of
Medical Research, Fitzroy, Victoria 3065, Australia, and the
** Departments of Medicine and Biochemistry, Dartmouth
Medical School, and the Department of Biological Sciences, Dartmouth
College, Hanover, New Hampshire 03755
Received for publication, October 17, 2002, and in revised form, November 7, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cystic fibrosis
transmembrane conductance regulator (CFTR) Cl The cystic fibrosis transmembrane conductance regulator
(CFTR)1 is a plasma membrane
Cl AMPK is a ubiquitous serine/threonine kinase with orthologues in all
eukaryotes that exists as a heterotrimer with a catalytic CFTR is unique among ion channels in its requirement for ATP binding
and hydrolysis to support channel activity. We previously discovered
that the COOH-terminal regulatory sequence of the AMPK The most serious CF-associated morbidity results from lung pathology.
Defective Cl Reagents and Chemicals--
All reagents and chemicals used were
purchased from Sigma unless otherwise noted.
Cell Culture, Cloning and Transfection--
Calu-3 cells (ATCC
HTT-55) were maintained as described (15). CHO cells stably transfected
with CFTR (CHO-BQ2 cells), kindly provided by Dr. J. Riordan, were
maintained as described (16).
AMPK- Short Circuit Current Measurements--
Calu-3 cells were seeded
at confluent density (5 × 105 cells/well) on 1-cm
permeable supports (Costar Snapwells, catalog no. 3407), and grown in
DMEM/F12 medium (Invitrogen) plus 10% (v/v) fetal bovine serum.
The apical bathing medium was removed 24-48 h after the cells were
seeded to establish an air interface, and the medium was then replaced
every 24-48 h. After ~7-12 days, the cells formed a confluent
monolayer that held back fluid, maintaining an apical air interface.
Transepithelial short circuit current (Isc)
measurements were performed after an additional 10-28 days in culture.
The experimental bath solutions contained 120 mM NaCl, 25 mM NaHCO3, 3.3 mM
KH2PO4, 0.8 mM
K2HPO4, 1.2 mM MgCl2,
1.2 mM CaCl2, and 10 mM glucose.
Mannitol was substituted for glucose in the apical bath to eliminate a
contribution of Na+-glucose cotransport to
Isc, as described (19). The pH of this solution
was 7.4 when gassed with a mixture of 95% O2 and 5%
CO2 at 37 °C. For each experiment, two paired Snapwell
inserts were mounted in Ussing chambers interfaced with a
voltage-current clamp amplifier (Physiological Instruments, San Diego,
CA) and electronic chart recorder (PowerLab, ADInstruments, Grand
Junction, CO). The monolayers were continuously voltage clamped to 0 mV
after fluid resistance and asymmetry voltage compensation. Changes in transepithelial resistance were calculated using Ohm's law from the
current excursions resulting from periodic 5-mV bipolar voltage pulses.
Under conditions of these experiments, Isc is
equivalent to net Cl Whole-cell Patch Clamp Experiments--
All experiments were
performed at room temperature. Fire-polished borosilicate pipettes
(catalog no. 1B150F-4, World Precision Instruments, Inc., Sarasota, FL)
had resistances of ~3-7 megaohms. Bath and pipette solutions used
were as described previously (20). Recordings were made using an
Axopatch 200A amplifier (Axon Instruments, Inc.) interfaced with
PULSE+PULSEFIT software (HEKA Electronics, Lambrecht/Pfalz,
Germany) on a Power Macintosh computer. Liquid junction and tip
potentials were corrected for each pipette after immersion into the
bath solution. Corrections for cellular capacitance and series
resistance were made immediately after sealing and breaking into
whole-cell mode. The plasma membrane was held at Cell-attached Patch Clamp Experiments--
Pipette resistances
were ~7-20 megaohms. The bath solution contained 140 NaCl
mM, 2 mM MgCl2, 0.1 mM
CaCl2, 1 mM EGTA, 1 mM MgATP, 10 mM HEPES (pH 7.5), and 1 µM forskolin. The
pipette solution was the same but without ATP and forskolin. Current
traces were obtained using an Axopatch 200B amplifier at ±60 mV
pipette potential, filtered at 100 Hz, digitized at 2 kHz, and recorded
to hard disk using PULSE+PULSEFIT software. Because changes in the
apparent open probability (Po) and number of
active channels (N) were observed in cell-attached patches
during the first 5 min of recording, only subsequent current traces of
at least 5 min duration were used for data analysis (TAC software,
Bruxton, Seattle, WA). The number of channels in a patch was assumed to
be the maximum number of open channel current levels observed during
the experiment. The data were fitted and modeled using IGOR PRO 4.0 software (WaveMetrics, Lake Oswego, OR), as described (15). Mean open
and closed times for each condition were derived from the means of all
of the mean open and closed times calculated for each individual
experiment. To calculate mean open and closed times of data from
patches containing >1 channel, we used the following formulas derived
from Horn and Lange (21) as shown in Equations 1 and 2,
AMPK Kinase Assays--
Polarized Calu-3 cells were grown on
Costar Transwells (catalog no. 3460) as described above for short
circuit current measurements. For the experiment, the medium was
replaced with DMEM/F12 plus 1 mM
5-amino-4-imidazolecarboxamide riboside (AICAR) (or DMEM/F12 alone for
controls) on both the apical and basolateral sides and then incubated
for 2 h at 37 °C with 5% CO2. Before lysis, cells were washed twice on both sides with ice-cold PBS. Lysis buffer (LB) contained 20 mM Tris.Cl, 50 mM NaCl, 50 mM NaF, 5 mM sodium
pyrophosphate, 250 mM sucrose, and 1% Triton X-100 (pH
7.4, with NaOH). Complete protease inhibitor cocktail (1×, Roche
Molecular Biochemicals), 1 mM phenylmethylsulfonyl fluoride, and 2 mM dithiothreitol were added to the LB just
prior to addition of 120 µl of LB mixture to the apical side of each well. After rocking the samples for 15 min at 4 °C, lysates from each well were collected. For each condition (AICAR and control), three
sets of lysates pooled from two wells each were pelleted at 14,000 × g at 4 °C for 10 min. Protein concentrations of
supernatants were estimated by the Bio-Rad protein assay. AMPK activity
was measured against the SAMS peptide following immunoprecipitation of
the kinase from cell lysates with an anti- Surface Biotinylation Assays--
CHO-BQ2 cells transfected with
pTracer vector alone, pTracer-HA- AMPK Activation Inhibits CFTR Currents in Polarized Calu-3
Cells--
We have recently shown that AMPK modulates CFTR-mediated
Cl Whole-cell CFTR Conductance Is Inversely Modulated with Endogenous
AMPK Activity Modulation--
To confirm that the observed
AMPK-dependent inhibition of Isc
occurred via modulation of CFTR activity, whole-cell patch clamp measurements were made using different methods to modulate the activity
of endogenous AMPK (Fig. 2). Forskolin
increased the whole-cell conductance within 1-2 min and shifted the
reversal potential to ~
Consistent with the above results obtained from transepithelial
measurements, AICAR treatment inhibited the forskolin-stimulated capacitance-normalized whole-cell conductance by 37% compared with
untreated controls (Fig. 2B). To specifically modulate AMPK activity using distinct approaches, Calu-3 cells were transfected with
mutant AMPK cDNAs. Two constructs were used that alter endogenous AMPK activity. AMPK- Cell Surface Expression of CFTR Is Unaffected by Modulation of
Endogenous AMPK Activity--
The inhibition of CFTR by AMPK observed
in lung Calu-3 cells is in accord with observations made in the T84
colonic cell line,2 which also involved endogenous AMPK and
CFTR as well as in Xenopus oocytes engineered to express
both proteins (7). In all three cellular systems, activation of
AMPK activity resulted in diminished CFTR activity, but the mechanisms
that underlie this inhibition are unknown. Because total whole-cell or
tissue CFTR-mediated anion currents have been measured in all of these
cell systems, AMPK modulation of CFTR activity could conceivably occur
through changes in the amount of CFTR expressed at the plasma membrane and/or through changes in CFTR single channel properties
(Po and/or single-channel conductance). The
activation of AMPK in skeletal muscle has been shown to modulate the
amount of GLUT4 glucose transporters in the plasma membrane (29). To
determine whether changes in CFTR cell surface expression could account
for the observed modulation, biotinylation assays were performed to
determine the amount of CFTR in the plasma membrane. For these
experiments, CHO-BQ2 cells stably expressing CFTR were transiently
transfected with either vector alone or HA-tagged dominant negative or
constitutive-activating AMPK mutants. The fraction of plasma
membrane-localized CFTR was determined in forskolin-treated transfected
cells using a cell surface biotinylation protocol as described under
"Experimental Procedures." A typical experiment is shown in Fig.
3A that compares CFTR
(top) and exogenous AMPK (HA; bottom) expression
in the unbound (not present in plasma membrane) and bound (present in
plasma membrane) fractions from lysates of cells transfected with
pTracer vector alone or HA-tagged CFTR Channel Gating Is Inhibited by Activation of Endogenous AMPK
Activity--
The preceding results suggested that the predominant
mechanism by which AMPK modulates CFTR activity involves effects on the single channel properties of CFTR. To address this issue, we performed cell-attached patch clamp measurements of single CFTR channels in
Calu-3 cells (Fig. 4). As evidenced by
the equal amplitudes of gating-associated current amplitudes under the
two conditions, the single-channel conductance was not different in
control cells from those transfected with the activating mutant
AMPK- In the present study we have demonstrated that
endogenous AMPK can modulate the activity of CFTR in lung serous
epithelial cells (Figs. 1 and 2). This AMPK-dependent
modulation of CFTR activity could not be accounted for by changes in
the amount of CFTR expressed at the plasma membrane (Fig. 3). Rather,
CFTR inhibition following constitutive AMPK activation was mediated
through effects on CFTR single channel gating; i.e. AMPK
activation significantly inhibited the Po of
CFTR by stabilizing the closed conformation of the channel (Fig. 4).
Until now, the most well established kinase regulation of CFTR activity
was that associated with PKA and, to a lesser extent, protein kinase C,
both of which act to stimulate gating (30). Whereas specific
phosphorylation of CFTR is believed to underlie these effects, the
molecular mechanisms involved in AMPK regulation of CFTR remain to be
elucidated. Two general possibilities remain to be investigated. First,
AMPK may exert its effects directly on CFTR. The catalytic subunit of
AMPK directly binds to a region near the COOH terminus of CFTR, and CFTR can serve as a substrate for AMPK-dependent
phosphorylation in vitro (7). In Xenopus oocytes,
inhibition of CFTR-mediated Cl AMPK activity is exquisitely sensitive to changes in the cell metabolic
state, as relatively small changes in intracellular ATP levels produce
large changes in the intracellular AMP:ATP ratio due to the rapid
interconversion of ATP, ADP, and AMP by adenylate kinase (33). Because
AMPK responds to the cellular AMP:ATP ratio, it can serve as a primary
sensor of even minor changes in metabolic state. Therefore, AMPK may
become engaged not only following severe reductions in intracellular
ATP (e.g. that occur with ischemic or hypoxic injury) but
also during more subtle, "physiological" fluctuations of
intracellular ATP that may occur on a regular basis. Thus, the
interaction of CFTR with AMPK may provide a sensitive mechanism to
couple its activity to cell metabolic state, fine-tuning CFTR transport
activity on a short time scale in response to metabolic conditions.
CFTR appears to modulate the activities of various membrane
transport proteins and may thereby help to coordinate an ensemble of
proteins involved in transepithelial salt and water transport (34).
Thus, the AMPK-dependent regulation of CFTR may afford a
mechanism to couple epithelial transport in general to cellular metabolic status. In addition, AMPK might directly modulate the activity of other important membrane transport proteins independently of CFTR. Indeed, AICAR-induced activation of AMPK appears to induce increased transcription and insertion of glucose transporters into the
plasma membranes of muscle (29, 35, 36). Of note, the recent discovery
that certain mutations in the Finally, the results of this study illustrate the potential
utility of agents (e.g. AICAR or metformin (11)) that
enhance endogenous AMPK activity in the treatment of diseases where
inappropriately high CFTR activity plays a role, including certain
secretory diarrheas (3) and autosomal dominant polycystic kidney
disease (38). Conversely, if agents that could selectively inhibit AMPK
are discovered, they might be of benefit in diseases associated with inappropriately low CFTR activity (i.e. CF). A further
understanding of the detailed mechanisms involved in the AMPK-CFTR
regulatory interaction may also provide additional insights into the
pathogenesis of CF.
channel activity is important for fluid and electrolyte transport in
many epithelia including the lung, the site of most cystic fibrosis-associated morbidity. CFTR is unique among ion channels in
requiring ATP hydrolysis for its gating, suggesting that its activity
is coupled to cellular metabolic status. The metabolic sensor
AMP-activated kinase (AMPK) binds to and phosphorylates CFTR,
co-localizes with it in various tissues, and inhibits CFTR currents in
Xenopus oocytes (Hallows, K. R., Raghuram, V., Kemp, B. E., Witters, L. A. & Foskett, J. K. (2000)
J. Clin. Invest. 105, 1711-1721). Here we demonstrate
that this AMPK-CFTR interaction has functional implications in human
lung epithelial cells. Pharmacologic activation of AMPK inhibited
forskolin-stimulated CFTR short circuit currents in polarized Calu-3
cell monolayers. In whole-cell patch clamp experiments, the activation
of endogenous AMPK either pharmacologically or by the overexpression of
an AMPK-activating non-catalytic subunit mutant (AMPK-
1-R70Q)
dramatically inhibited forskolin-stimulated CFTR conductance in Calu-3
and CFTR-expressing Chinese hamster ovary cells. Plasma membrane
expression of CFTR, assessed by surface biotinylation, was not affected
by AMPK activation. In contrast, the single channel open probability of
CFTR was strongly reduced in cell-attached patch clamp measurements of
Calu-3 cells transfected with the AMPK-activating mutant, an effect due
primarily to a substantial prolongation of the mean closed time of the
channel. As a metabolic sensor in cells, AMPK may be important in
tuning CFTR activity to cellular energy charge, thereby linking
transepithelial transport and the maintenance of cellular ion gradients
to cellular metabolism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
channel expressed on the apical membranes in a wide
variety of epithelial tissues, including the lung, intestine, pancreas,
and reproductive tract (1). CFTR plays an important role in absorption and secretion of salt and water, with its channel activity often determining the rate of transepithelial transport in various tissues. The importance of native regulation of CFTR channel activity is underscored by pathological conditions, including cystic fibrosis (CF),
where mutations in CFTR cause reduced activity (2), and secretory
diarrhea, which results from excessive cellular stimulation of CFTR
(3). CFTR is activated in cells by hormone- and
neurotransmitter-induced cAMP signaling. The cAMP-dependent
protein kinase A (PKA) phosphorylates several residues in the
cytoplasmic R domain of CFTR, which activates gating by destabilizing
channel closed states (4). Activated CFTR channels can be inactivated
by phosphatases (5). PKA-mediated phosphorylation may also enhance
Cl
transport through the insertion of additional CFTR
channels into the plasma membrane (6). Whereas PKA-associated
phosphorylation/dephosphorylation is the most thoroughly understood
process regulating CFTR channel activity, other mechanisms may modulate
the activity of CFTR under various cellular conditions. In this regard,
AMP-activated protein kinase (AMPK) was discovered to interact with
CFTR and inhibit its activity (7).
subunit
and regulatory
and
subunits (8). In response to metabolic
stress and increasing intracellular AMP levels (9), AMPK phosphorylates
and inhibits several important rate-limiting biosynthetic enzymes,
thereby acting to preserve cellular ATP stores during metabolic
depletion (8). Recently, mutations in AMPK have been linked to human
diseases, including familial hypertrophic cardiomyopathy and
Wolff-Parkinson-White syndrome (10). Furthermore, modulation of AMPK
may also play an important role in the pathogenesis and treatment of
type II diabetes mellitus (11) and obesity (12).
subunit
directly interacts with the CFTR COOH-terminal tail. Our data indicate
that this interaction could be of physiological significance, because
AMPK and CFTR co-localize in epithelia, and AMPK phosphorylates CFTR
in vitro and inhibits cAMP-activated CFTR conductances in
Xenopus oocytes (7) and polarized colonic T84 cell
monolayers.2 However, the
mechanisms underlying inhibition of CFTR activity by AMPK have not been determined.
transport due to CFTR mutations results in
abnormal composition and/or volume of airway surface liquid, which
compromises airway clearance resulting in chronic infection and the
destruction of lung tissue (13, 14). Because detailed insights into the
mechanisms of CFTR regulation in the lung could provide new insights
into the pathogenesis of CF lung disease and suggest possible novel therapies, here we have investigated AMPK inhibition of CFTR activity in Calu-3 human lung serous cells. Using pharmacological, biochemical, and electrophysiological approaches, we demonstrate that AMPK is an
endogenous inhibitor of CFTR activity in lung submucosal gland serous
cells. Furthermore, we have determined that this inhibition is mediated
by effects on CFTR single channel gating.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-K45R and AMPK-
1-R70Q were cloned into the bicistronic
pTracer-CMV2 vector (Invitrogen), which constitutively expresses a
green fluorescent protein (GFP)-Zeocin fusion protein and the cloned
gene insert under the control of separate promoters. This plasmid
enabled transfected cells to be identified by their green fluorescence
for patch clamp experiments, which was mandatory because of low
transfection efficiency (
1%). Inserts were synthesized by high
fidelity PCR (PFU Turbo, Stratagene) using the pMT2-
1-K45R and
pMT2-
1-R70Q plasmids (17, 18) as templates and specific primers
(sequences available on request) that provided an
NH2-terminal hemagglutinin (HA) tag on each gene product.
All final clones were verified by DNA sequencing. Transfections were
performed using LipofectAMINE 2000 (Invitrogen) according to the
manufacturer's recommendations. For Calu-3 cells, 8-10 × 105 cells were seeded onto a 25-mm circular sterile glass
coverslip grown for 18-24 h prior to transfection and then used for
patch clamp experiments 2-3 days after transfection. For CHO-BQ2 cells transfected on cover slips, 1-2 × 105 cells were
initially seeded. For CHO-BQ2 cells transfected in 60-mm Petri dishes
to be used for biotinylation assays, 4 × 105 cells
were initially seeded. CHO-BQ2 cell transfection efficiency, as
assessed by GFP fluorescence, ranged from 25-50% in all experiments.
and/or
HCO
20 mV, and
current-voltage (I-V) plots were obtained from currents recorded in
response to a series of 850-ms voltage steps from
100 to +100 mV in
20-mV increments at 1.5-s intervals. Whole-cell conductance was
calculated as the slope at
20 mV of a fifth order polynomial fit to
the I-V data. Normalized conductance (pS/pF) was calculated as the mean
calculated whole-cell conductance under a given condition divided by
the whole-cell capacitance measured prior to the start of data
collection for that cell. Forskolin (1 µM) was added to
the bath solution to stimulate CFTR.
(Eq. 1)
where T is time and E is the total number of events (both
opening and closing).
(Eq. 2)
subunit antibody that
recognizes both catalytic subunit isoforms (18).
1-K45R, or pTracer-HA-
1-R70Q
(two dishes per condition) were used 2 days after transfection.
Biotinylation assays were performed based on a previously described
protocol (22). Cells were exposed to 1 µM forskolin in
growth medium at 37 °C for 10 min prior to washing twice in ice-cold
PBS (pH 8.0) and then adding ice-cold 1 mg/ml sulfo-NHS-SS-biotin
(Pierce) dissolved in PBS. Samples were rocked for 30 min at 4 °C,
then washed once in ice-cold PBS plus 1% BSA, and then washed in PBS.
Cells from two dishes were lysed in 250 µl of biotinylation lysis
buffer that contained 150 mM NaCl, 20 mM HEPES,
2 mM EDTA, and 1% Nonidet P-40 (pH 7.5, at room
temperature), with 1 mM phenylmethylsulfonyl fluoride and
1× complete protease inhibitor cocktail added just prior to lysis.
After incubating dishes on ice for 10 min, cells were scraped using a
cell lifter, and lysates were centrifuged at 14,000 × g at 4 °C. Supernatant protein concentrations were
measured using the Bio-Rad assay, and the samples were diluted in lysis
buffer to equalize all protein concentrations. 75 µl of a 50%
streptavidin-agarose slurry (Pierce) was then added to each sample
before rotating for 2 h at 4 °C. After gently pelleting the
beads, 10% of the supernatant (~30 µl) was aliquoted into 10 µl
of 4× Laemmli sample buffer (unbound fraction). The remainder of the
supernatant was removed, and the beads were washed twice in cold lysis
buffer and once in PBS. Then, 30 µl of 2× sample buffer containing
200 mM dithiothreitol was added to the beads and the
mixture was heated to 65 °C for 15 min to elute all proteins (bound
fraction). Samples were loaded onto a 4-12% gradient gel (NuPage,
Invitrogen), electrophoresed, and transferred to nitrocellulose
membranes. The membranes were cut into high molecular weight and low
molecular weight portions and then immunoblotted using primary
rabbit polyclonal anti-CFTR antibody (A2; kindly supplied by Dr. W. Skach) or mouse monoclonal anti-HA antibody (HA.11, Covance), followed
by the appropriate secondary antibody as described (7). After exposure
to film, band intensities were quantitated using a Molecular Dynamics
Personal Densitometer SI scanner and ImageQuant software (Amersham
Biosciences), with normalization of all bands to background levels on
the film. To calculate the percentage of biotinylated CFTR for each
condition, the intensity of the unbound fraction CFTR band was
multiplied by 10 before comparison with the bound fraction band.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
currents in both the Xenopus oocyte
expression system (7) and polarized colonic T84 cell
monolayers.2 However, the underlying mechanisms for
AMPK-dependent inhibition of CFTR have not yet been
determined. To examine whether AMPK regulates CFTR in polarized lung
epithelial cells, the Calu-3 human lung serous epithelial cell line,
which endogenously expresses both AMPK and CFTR (7, 23), was used.
CFTR-dependent basal and forskolin-activated short circuit
currents were measured in polarized cells grown on permeable supports
and mounted in Ussing chambers. To test whether AMPK could modulate
endogenous CFTR activity, cells were exposed for 2 h prior to and
during the experiment to vehicle alone (control) or to 1 mM
AICAR, a cell-permeant purine synthesis intermediate that activates
endogenous AMPK in vivo (24) (Fig.
1). After steady-state basal currents
were measured, 4 µM forskolin was added to further
stimulate the CFTR-mediated Cl
conductance at the apical
membrane. To help ensure that the stimulated apical membrane
conductance was not rate-limited by the basolateral membrane
conductance, 2 µM thapsigargin, a Ca2+-ATPase
inhibitor, was then added to increase cytosolic Ca2+ and
thereby activate basolateral K+ conductances (25). Typical
experimental traces are shown in Fig. 1A. Activation of
endogenous AMPK with AICAR inhibited the baseline
Isc (by 47 ± 11%) as well as the
forskolin-stimulated Isc, measured in either the
presence or absence of thapsigargin (by 41 ± 1% with forskolin
alone and by 49 ± 3% with forskolin plus thapsigargin) as
compared with untreated paired controls (Fig. 1B). To verify
that AICAR treatment inhibited CFTR-mediated currents as a consequence
of the activation of endogenous AMPK kinase activity, we employed an
in vitro kinase assay using lysates prepared from
AICAR-treated or control Calu-3 cells. AICAR pretreatment stimulated
endogenous AMPK kinase activity to ~270% of control levels, as
assessed by this assay (Fig. 1C). Therefore, AICAR treatment
of Calu-3 monolayers stimulated AMPK activity, and this stimulation was
associated with an inhibition of CFTR-mediated transepithelial
currents. An independent experiment, described below, also indicated
that AICAR mediates its effects on CFTR activity specifically by
enhancing AMPK activity.
View larger version (39K):
[in a new window]
Fig. 1.
Effects of AICAR on polarized Calu-3 cell
monolayers. A, Isc traces from a
typical paired experiment under basal conditions and after the addition
of 4 µM forskolin and then 2 µM
thapsigargin to the basolateral bath. Cells were treated with 1 mM AICAR (or vehicle alone, Control) for 2 h before and then during recordings. Voltage pulses (± 5 mV) were
applied every 30 s to monitor transepithelial conductance.
B, summary of control versus AICAR-treated paired
experiments comparing mean (± S.E.) baseline
Isc values just prior to stimulation
(left) and peak values after forskolin stimulation
(middle) and after forskolin plus thapsigargin treatment
(right). p values comparing the measured
Isc from AICAR-treated and control monolayers
are shown for each condition (paired t test;
n = 4). C, kinase activities determined
using the SAMS peptide assay on endogenous AMPK immunoprecipitated from
Calu-3 monolayer lysates after a 2-h incubation with 1 mM
AICAR versus vehicle control (*, p < 0.001, unpaired t test, n = 3).
20 mV, close to the Cl
reversal potential (ECl), consistent with activation of
plasma membrane Cl
conductance (Fig. 2A,
center panel). Glibenclamide (500 µM) substantially inhibited the stimulated conductance,
especially at hyperpolarized voltages, suggesting that the
forskolin-induced increase in whole-cell conductance was due to
activation of CFTR (Fig. 2A, right
panel).
View larger version (38K):
[in a new window]
Fig. 2.
Effects of modulating AMPK activity on
whole-cell conductances in Calu-3 cells. A,
whole-cell current sweeps (y axis and lower x
axis) and superimposed I-V curves (y axis and upper
x axis) from representative control experiment before
(left) and after both the addition of 1 µM
forskolin (middle) and then 500 µM
glibenclamide (right). B, normalized conductances
of forskolin-stimulated cells treated for 2 h with 1 mM AICAR versus untreated controls (*,
p = 0.01, unpaired t test). C,
normalized conductances of mock (no DNA)-transfected cells and cells
transfected with the pTracer vector alone or the dominant negative
1-K45R or constitutive-activating
1-R70Q AMPKmutants (*,
p < 0.001 for
1-R70Q- versus both mock-
and
1-K45R-transfected cells, unpaired t tests).
D, normalized conductances of cells transfected with pTracer
alone or with the dominant negative
1-K45R AMPK mutant, with or
without a 2-h AICAR pretreatment (*, p < 0.005 for
pTracer + AICAR versus
1-K45R + AICAR, unpaired
t test). Data shown are the mean (± S.E.) normalized
conductances from 4 to 11 replicate experiments.
1-K45R contains a Lys to Arg (K45R) point mutation at the active site of the AMPK
subunit kinase domain that
renders the kinase catalytically inactive (17, 26). Overexpression of
this mutant in cultured cells (27) and in transgenic mice (28) causes a
down-regulation of the endogenous
subunit, presumably through
competition for binding to the endogenous
and
subunits. Therefore, overexpression of the
-K45R mutant has a dominant negative effect on AMPK activity in cultured cells and in
vivo (17, 28). Conversely, a
subunit mutant (
1-R70Q)
renders the AMPK heterotrimer constitutively active by causing
hyperphosphorylation of the
subunit in the activation loop (at
Thr-172) and rendering the enzyme relatively AMP-independent (18).
Cells were transfected with either of these constructs in a vector that
also contained the cDNA for the GFP, enabling transfected cells to
be identified for patch clamp electrophysiology. In cells expressing
the dominant negative
1-K45R mutant, the forskolin-activated
conductance was not significantly different from that observed in
control cells, although there was a trend toward activation (by 26%
compared with mock-transfected cells and 8% compared with vector
alone-transfected cells) (Fig. 2C). To ensure that the
transfected cells expressed the
1-K45R mutant and that it was indeed
exerting a dominant negative inhibitory effect on the endogenous AMPK
activity, we examined the effects of AICAR in the transfected cells.
Whereas AICAR treatment inhibited the forskolin-activated whole-cell
conductance in vector alone-transfected cells, as described above, it
had no inhibitory effect in cells that had been transfected with the dominant negative AMPK mutant (Fig. 2D). These results
therefore suggest, first, that the AICAR inhibition of CFTR conductance observed (Figs. 1 and 2B) occurred as a specific consequence
of the activation of endogenous AMPK and, second, that basal CFTR activity in Calu-3 cells is not significantly inhibited by AMPK under
the conditions of our experiments so that the dominant negative inhibition of the kinase had either no effect or a slight stimulatory one (i.e. relief from AMPK inhibition). More dramatic
results were obtained from cells expressing AMPK-
1-R70Q. The
forskolin-activated whole-cell conductance was profoundly inhibited (by
69% compared with mock-transfected cells and 73% compared with vector
alone-transfected cells) (Fig. 2C). Thus, specific
activation of endogenous AMPK in lung serous cells strongly inhibited
the activity of CFTR. In summary, these results show that modulation of
endogenous AMPK activity in Calu-3 cells using both pharmacological and
molecular expression approaches modulated endogenous CFTR activity in
this cell type as assessed by two distinct electrophysiological approaches.
1-K45R or
1-R70Q AMPK mutants.
Of interest, a significant proportion of exogenously expressed AMPK appeared in the bound fraction (Fig. 3A, bottom),
suggesting that the exogenously expressed AMPK subunits were either
directly biotinylated in the membrane or that they were tightly bound
to biotinylated integral membrane proteins (e.g. CFTR). This
result is consistent with earlier immunolocalization data, which
demonstrated a propensity for AMPK to localize near the (especially
apical) plasma membrane (7). There were no differences in the fraction
of plasma membrane-associated CFTR (~0.5) under control conditions
versus conditions where endogenous AMPK was inhibited
(HA-
1-K45R) or activated (HA-
1-R70Q) (Fig. 3B).3 To verify
that CHO-BQ2 cells were an appropriate cell type for these experiments,
we examined the effects of AMPK-
1-R70Q expression on CFTR-mediated
whole-cell conductance. As observed in Calu-3 cells, the expression of
this constitutive-activating mutant caused a dramatic, almost complete
inhibition of the forskolin-activated conductance as compared with
control and
1-K45R-AMPK-transfected cells (Fig. 3C).
CHO-BQ2 cells therefore behave similarly to Calu-3 cells. These results
therefore indicate that changes in plasma membrane expression of CFTR
cannot account for the AMPK-dependent modulation of CFTR
activity.
View larger version (17K):
[in a new window]
Fig. 3.
Lack of effects of modulating AMPK activity
on CFTR surface expression. CHO-BQ2 cells were transfected with
pTracer vector alone or the HA-tagged dominant negative 1-K45R or
constitutive-activating
1-R70Q-AMPK mutants followed by surface
labeling of plasma membrane proteins by biotinylation as described
under "Experimental Procedures." Cellular proteins were then
adsorbed to streptavidin-agarose and bound (plasma membrane) and
unbound (non-plasma membrane) fractions separated. A,
representative immunoblots probed with antibodies to CFTR
(top) or HA (bottom). For each transfection
condition shown, 10% of the unbound fraction (U) and 100%
of the bound fraction (B) was loaded onto the gel.
B, scanning densitometric determinations of the mean (± S.E.) fraction of biotinylated CFTR from five replicate experiments.
C, mean (± S.E.) normalized forskolin-stimulated
conductance measurements from 3-5 replicate experiments for each
transfection condition (*, p < 0.05 for HA-
1-R70Q
versus both pTracer vector alone-transfected and
HA-
1-K45R-transfected cells, unpaired t-tests).
1-R70Q (Fig. 4A). In contrast, the
Po was reduced by 64% (p < 0.001, unpaired t test) in the AMPK-
1-R70Q-overexpressing
cells (0.13 ± 0.03, n = 10) compared with vector
alone-transfected cells (0.36 ± 0.03, n = 11)
(Fig. 4B). Of note, this magnitude of
Po inhibition is comparable with that of the
forskolin-activated whole cell conductance (Fig. 2C),
suggesting that inhibition of single-channel gating can account for the
AMPK-dependent inhibition of CFTR. The decreased
Po in the AMPK-
1-R70Q-transfected cells was
due primarily to a substantial prolongation of the mean channel closed time (Fig. 4C), suggesting that AMPK activation stabilizes
the closed conformation of the CFTR channel.
View larger version (32K):
[in a new window]
Fig. 4.
Effects of AMPK activation on single CFTR
channels. Cell-attached patch clamp experiments were performed on
Calu-3 cells transfected with either pTracer vector alone (control) or
the constitutive-activating AMPK- 1-R70Q mutant. A,
typical traces of single-channel patches under each condition.
Arrows indicate the channel closed level. Data were recorded
at
60-mV pipette potential. B, mean
Po from at least 10 experiments performed for
each transfection condition with patches containing 1-6 active
channels (*, p < 0.001 compared with control, unpaired
t test). C, mean (± S.E.) open and closed times
for each transfection condition (see "Experimental Procedures" for
details; p values are shown). Because the
Po measured in the
1-R70Q transfected cells
was low, it is more likely that N was underestimated in
them, which would tend to cause an overestimation of
Po under that condition.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
currents by AMPK appeared
to require both binding as well as kinase activity (7). Second, AMPK
may exert its effects through other molecules. CFTR likely exists
within a complex of closely associated proteins, including those
involved in its PKA-mediated phosphorylation (20). PKA activates CFTR
gating by destabilizing the channel closed conformation (4), whereas
AMPK appears to inhibit CFTR gating by stabilizing the channel closed
state, the opposite effect. Thus, it is possible that AMPK antagonizes
the effects of PKA on CFTR channel gating by exerting its effects on
proteins involved in the phosphorylation or dephosphorylation of CFTR.
Alternatively, because the interaction of other proteins with CFTR may
influence CFTR gating (15, 31, 32), those interactions could be sites
of modulation by AMPK. It is interesting to note that the interaction
of CFTR with the PDZ domain-containing protein NHERF, which has
profound effects on CFTR gating (15), is mediated through sites located
immediately downstream from the region of CFTR involved in binding to
AMPK (7).
subunit of AMPK cause the
Wolff-Parkinson-White syndrome (10, 37), a disease that predisposes to
fatal cardiac arrhythmias, may imply a possible functional link between
AMPK and cardiac ion channels. Thus, further investigations into the
effects of AMPK on various membrane transport proteins may provide
important new insights into the pathophysiology of several disorders,
including ischemic epithelial injury and cardiac arrhythmias.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Christine Richardson for technical assistance and Drs. Daniel Mak and Jack Stutts for technical advice.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants F32-DK09994 and K08-DK59477 (to K. R. H.) and R01-DK35712 (to L. A. W.) and grants from the National Heart Foundation and National Health and Medical Research Council (to B. E. K.) and the Cystic Fibrosis Foundation (to J. K. F.).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.
To whom correspondence should be addressed: Dept. of
Physiology, University of Pennsylvania School of Medicine, B39
Anatomy/Chemistry Bldg., Philadelphia, PA 19104. Tel.: 215-898-1354;
Fax: 215-573-6808; E-mail: foskett@mail.med.upenn.edu.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M210621200
2 K. R. Hallows and J. K. Foskett, unpublished results.
3 This fraction of CFTR in CHO-BQ2 cells that is biotinylated is substantially higher than that determined in epithelial cells that endogenously express CFTR, such as T84 cells (K. R. Hallows and J. K. Foskett, unpublished results). It is, however, consistent with the observation that a very high proportion of CFTR in immunoblots of CHO-BQ2 cell lysates exists in the mature glycosylated (band C) form (cf. Ref. 7).
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; CF, cystic fibrosis; PKA, cyclic AMP-dependent protein kinase; AMPK, AMP-activated protein kinase; CHO, Chinese hamster ovary; GFP, green fluorescent protein; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; Isc, short circuit current; I-V, current-voltage; AICAR, 5-amino-4-imidazolecarboxamide riboside; PBS, phosphate-buffered saline.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Welsh, M. J., and Smith, A. E. (1995) Sci. Am. 273, 52-59[Medline] [Order article via Infotrieve] |
2. | Tsui, L.-C. (1992) Trends Genet. 8, 392-398[Medline] [Order article via Infotrieve] |
3. |
Kunzelmann, K.,
and Mall, M.
(2002)
Physiol. Rev.
82,
245-289 |
4. | Winter, M. C., and Welsh, M. J. (1997) Nature 389, 294-296[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Luo, J. X.,
Pato, M. D.,
Riordan, J. R.,
and Hanrahan, J. W.
(1998)
Am. J. Physiol.
274,
C1397-C1410 |
6. | Howard, M., Jiang, X., Stolz, D. B., Hill, W. G., Johnson, J. A., Watkins, S. C., Frizzell, R. A., Bruton, C. M., Robbins, P. D., and Weisz, O. A. (2000) Am. J. Physiol. 279, C375-C382 |
7. |
Hallows, K. R.,
Raghuram, V.,
Kemp, B. E.,
Witters, L. A.,
and Foskett, J. K.
(2000)
J. Clin. Invest.
105,
1711-1721 |
8. | Hardie, D. G., and Carling, D. (1997) Eur. J. Biochem. 246, 259-273[Abstract] |
9. | Hardie, D. G., Carling, D., and Carlson, M. (1998) Annu. Rev. Biochem. 67, 821-855[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Gollob, M. H.,
Seger, J. J.,
Gollob, T. N.,
Tapscott, T.,
Gonzales, O.,
Bachinski, L.,
and Roberts, R.
(2001)
Circulation
104,
3030-3033 |
11. |
Zhou, G.,
Myers, R., Li, Y.,
Chen, Y.,
Shen, X.,
Fenyk-Melody, J., Wu, M.,
Ventre, J.,
Doebber, T.,
Fujii, N.,
Musi, N.,
Hirshman, M. F.,
Goodyear, L. J.,
and Moller, D. E.
(2001)
J. Clin. Invest.
108,
1167-1174 |
12. | Minokoshi, Y., Kim, Y. B., Peroni, O. D., Fryer, L. G., Muller, C., Carling, D., and Kahn, B. B. (2002) Nature 415, 339-343[CrossRef][Medline] [Order article via Infotrieve] |
13. | Smith, J. J., Travis, S. M., Greenberg, E. P., and Welsh, M. J. (1996) Cell 85, 229-236[Medline] [Order article via Infotrieve] |
14. |
Caldwell, R. A.,
Grubb, B. R.,
Tarran, R.,
Boucher, R. C.,
Knowles, M. R.,
and Barker, P. M.
(2002)
J. Gen. Physiol
119,
3-14 |
15. |
Raghuram, V.,
Mak, D.-O. D.,
and Foskett, J. K.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
1300-1305 |
16. |
Pasyk, E. A.,
and Foskett, J. K.
(1995)
J. Biol. Chem.
270,
12347-12350 |
17. |
Dyck, J. R. B.,
Gao, G.,
Widmer, J.,
Stapleton, D.,
Fernandez, C. S.,
Kemp, B. E.,
and Witters, L. A.
(1996)
J. Biol. Chem.
271,
17798-17803 |
18. | Hamilton, S. R., Stapleton, D., O'Donnell, J. B., Kung, J. T., Dalal, S. R., Kemp, B. E., and Witters, L. A. (2001) FEBS Lett. 500, 163-168[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Devor, D. C.,
Singh, A. K.,
Lambert, L. C.,
DeLuca, A.,
Frizzell, R. A.,
and Bridges, R. J.
(1999)
J. Gen. Physiol.
113,
743-760 |
20. |
Huang, P.,
Trotter, K.,
Boucher, R. C.,
Milgram, S. L.,
and Stutts, M. J.
(2000)
Am. J. Physiol.
278,
C417-C422 |
21. | Horn, R., and Lange, K. (1983) Biophys. J. 43, 207-223[Abstract] |
22. | Chang, X. B., Cui, L. Y., Hou, Y. X., Jensen, T. J., Aleksandrov, A. A., Mengos, A., and Riordan, J. R. (1999) Mol. Cell 4, 137-142[Medline] [Order article via Infotrieve] |
23. |
Shen, B.-Q.,
Finkbeiner, W. E.,
Wine, J. J.,
Mrsny, R. J.,
and Widdicombe, J. H.
(1994)
Am. J. Physiol.
266,
L493-L501 |
24. | Corton, J. M., Gillespie, J. G., Hawley, S. A., and Hardie, D. G. (1995) Eur. J. Biochem. 229, 558-565[Abstract] |
25. |
Devor, D. C.,
Singh, A. K.,
Bridges, R. J.,
and Frizzell, R. A.
(1997)
Am. J. Physiol.
272,
C976-C988 |
26. |
Crute, B. E.,
Seefeld, K.,
Gamble, J.,
Kemp, B. E.,
and Witters, L. A.
(1998)
J. Biol. Chem.
273,
35347-35354 |
27. |
Woods, A.,
Azzout-Marniche, D.,
Foretz, M.,
Stein, S. C.,
Lemarchand, P.,
Ferre, P.,
Foufelle, F.,
and Carling, D.
(2000)
Mol. Cell. Biol.
20,
6704-6711 |
28. | Mu, J., Brozinick, J. T., Valladares, O., Bucan, M., and Birnbaum, M. J. (2001) Mol. Cell 7, 1085-1094[CrossRef][Medline] [Order article via Infotrieve] |
29. | Kurth-Kraczek, E. J., Hirshman, M. F., Goodyear, L. J., and Winder, W. W. (1999) Diabetes 48, 1667-1671[Abstract] |
30. | Gadsby, D. C., and Nairn, A. C. (1999) Physiol. Rev. 79, S77-S107[Medline] [Order article via Infotrieve] |
31. | Wang, S. S., Yue, H. W., Derin, R. B., Guggino, W. B., and Lit, M. (2000) Cell 103, 169-179[Medline] [Order article via Infotrieve] |
32. |
Chang, S. Y., Di, A.,
Naren, A. P.,
Palfrey, H. C.,
Kirk, K. L.,
and Nelson, D. J.
(2002)
J. Cell Sci.
115,
783-791 |
33. | Hardie, D. G., and Hawley, S. A. (2001) Bioessays 23, 1112-1119[CrossRef][Medline] [Order article via Infotrieve] |
34. | Schwiebert, E. M., Benos, D. J., Egan, M. E., Stutts, M. J., and Guggino, W. B. (1999) Physiol. Rev. 79, S145-S166[Medline] [Order article via Infotrieve] |
35. | Abbud, W., Habinowski, S., Zhang, J. Z., Kendrew, J., Elkairi, F. S., Kemp, B. E., Witters, L. A., and Ismail-Beigi, F. (2000) Arch. Biochem. Biophys. 380, 347-352[CrossRef][Medline] [Order article via Infotrieve] |
36. | Zheng, D., MacLean, P. S., Pohnert, S. C., Knight, J. B., Olson, A. L., Winder, W. W., and Dohm, G. L. (2001) J. Appl. Physiol. 90, 1073-1083 |
37. |
Blair, E.,
Redwood, C.,
Ashrafian, H.,
Oliviera, M.,
Broxholme, J.,
Kerr, B.,
Salmon, A.,
Ostman-Smith, I.,
and Watkins, H.
(2001)
Hum. Mol. Genet.
10,
1215-1220 |
38. | O'Sullivan, D. A., Torres, V. E., Gabow, P. A., Thibodeau, S. N., King, B. F., and Bergstralh, E. J. (1998) Am. J. Kidney Dis. 32, 976-983[Medline] [Order article via Infotrieve] |