1 Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; Departments of 2 Medicine and 5 Physiology, University of Pennsylvania School of Medicine, and 3 Wistar Institute, Philadelphia, Pennsylvania 19104; and 4 Departments of Medicine and Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cystic fibrosis transmembrane
conductance regulator (CFTR) is a cAMP-activated, ATP-gated
Cl channel and cellular conductance regulator, but the
detailed mechanisms of CFTR regulation and its regulation of other
transport proteins remain obscure. We previously identified the
metabolic sensor AMP-activated protein kinase (AMPK) as a novel protein interacting with CFTR and found that AMPK phosphorylated CFTR and
inhibited CFTR-dependent whole cell conductances when coexpressed with
CFTR in Xenopus oocytes. To address the physiological
relevance of the CFTR-AMPK interaction, we have now studied polarized
epithelia and have evaluated the localization of endogenous AMPK and
CFTR and measured CFTR activity with modulation of AMPK activity. By immunofluorescent imaging, AMPK and CFTR share an overlapping apical
distribution in several rat epithelial tissues, including nasopharynx,
submandibular gland, pancreas, and ileum. CFTR-dependent short-circuit
currents (Isc) were measured in polarized T84
cells grown on permeable supports, and several independent methods were used to modulate endogenous AMPK activity. Activation of endogenous AMPK with the cell-permeant adenosine analog
5-amino-4-imidazolecarboxamide-1-
-D-ribofuranoside (AICAR) inhibited forskolin-stimulated CFTR-dependent
Isc in nonpermeabilized monolayers and
monolayers with nystatin permeabilization of the basolateral membrane.
Raising intracellular AMP concentration in monolayers with basolateral
membranes permeabilized with
-toxin also inhibited CFTR, an effect
that was unrelated to adenosine receptors. Finally, overexpression of a
kinase-dead mutant AMPK-
1 subunit (
1-K45R) enhanced
forskolin-stimulated Isc in polarized T84
monolayers, consistent with a dominant-negative reduction in the
inhibition of CFTR by endogenous AMPK. These results indicate that AMPK
plays a physiological role in modulating CFTR activity in polarized
epithelia and suggest a novel paradigm for the coupling of ion
transport to cellular metabolism.
ion transport; cell metabolism; epithelial transport; chloride channel; cystic fibrosis
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE CYSTIC FIBROSIS (CF) transmembrane conductance
regulator (CFTR) is localized to the apical membrane of epithelial
cells lining various tissues, including the lungs, gastrointestinal tract, exocrine pancreas, and sweat ducts (62). CF is
associated with abnormal epithelial solute and fluid transport due to
mutations in CFTR that reduce its plasma membrane expression or
activity (58, 61). CFTR is a cAMP-dependent protein kinase
(PKA)-activated, ATP-gated Cl channel that belongs to the
ATP-binding cassette (ABC) family of transporters. A unique feature of
CFTR is the requirement for ATP binding and hydrolysis to support
channel activity. It has been proposed that this ATP requirement may
enable CFTR activity to be coupled to cellular metabolism (21,
47). In addition to its role as an apical membrane
Cl
channel, it appears that CFTR also acts as a cellular
"conductance regulator," coordinating transepithelial solute and
fluid transport by modulating the activities of several other plasma
membrane transport proteins (35). The mechanisms involved
in this modulation by CFTR are not yet clear but in some cases could
involve CFTR-dependent ATP efflux from the cell (14, 50,
56). CFTR might also influence activities of other transport
pathways by direct or indirect protein-protein interactions
(35).
Several recently identified CFTR-interacting proteins may be important
in regulating CFTR channel activity and/or plasma membrane expression,
including syntaxin 1A, via its interaction with the NH2-terminal tail of CFTR (8, 45, 46), the PDZ
domain-containing proteins NHERF (43, 48, 52) and CAP70
(59), and the µ2-subunit of the AP-2 adaptor protein
complex (60), via interactions with the COOH-terminal
tail. We discovered (26) that the 1 (catalytic)-subunit of the AMP-activated protein kinase (AMPK) was a strong and consistent interactor with the COOH tail of CFTR in a yeast two-hybrid screen.
AMPK is a serine/threonine kinase that exists as a heterotrimer
composed of a catalytic -subunit and regulatory
- and
-subunits. Multiple isoforms (
1,
2,
1,
2,
1,
2,
and
3) exist, with differing tissue distributions and presumed
substrates (33) and orthologs in all eukaryotes
(28). The
-subunit contains an NH2-terminal
catalytic domain and a COOH-terminal regulatory domain involved in
interactions with the
- and
-subunits. The kinase activity
increases during conditions of metabolic stress, in response to
elevated intracellular AMP-to-ATP ratios (30). Activation
of the kinase involves both the binding of AMP to allosteric site(s),
probably involving both the
- and
-subunits (7, 63),
and phosphorylation by an upstream AMPK kinase
(29). Kinase activity is also regulated by
association of the
- and
-subunits, binding of which relieves an
autoinhibitory interaction between the catalytic and regulatory domains
of the
-subunit (12). The earliest discovered
substrates of AMPK were important rate-limiting biosynthetic enzymes
(e.g., HMG-CoA reductase and acetyl-CoA carboxylase; Ref.
28). Phosphorylation by AMPK inhibits their enzymatic
activities, thereby acting to preserve cellular ATP stores under
conditions of metabolic depletion. Consequently, AMPK is believed to
act as a metabolic sensor in cells, responding to changes in cellular
energy charge by regulating ATP-utilizing and -generating metabolic
pathways (28). In addition, there has been a recent surge
in reports linking AMPK to other cellular functions, e.g., the
regulation of glucose-dependent gene expression in pancreatic
-cells, cellular glucose uptake in contracting muscle, and the
activation of endothelial nitric oxide synthase during tissue hypoxia
(9, 36, 37). Furthermore, mutations in AMPK have been
linked to diseases such as familial hypertrophic cardiomyopathy
(5) and the Wolff-Parkinson-White syndrome
(23). Finally, modulation of AMPK may play an important
role in the pathogenesis and treatment of common disorders such as type
II diabetes mellitus (64) and obesity (40).
CFTR is linked to cellular ATP in several important ways. CFTR consumes
ATP in its gating, is regulated by its phosphorylation state, and may
modulate ATP efflux from cells (14). Thus we considered
that the interaction between CFTR and the metabolic sensor AMPK could
have potential significant cell physiological implications. Although we
found (26) that the regulatory domain of the AMPK
-subunit binds the CFTR COOH-terminal tail, and that AMPK
phosphorylated CFTR in vitro and inhibited cAMP-activated CFTR
conductances in Xenopus oocytes, the physiological relevance of this protein-protein interaction has not yet been demonstrated in
epithelial tissues and cells that endogenously express both proteins
and in which CFTR plays an important transport role. In this study we
examined the localization of endogenous CFTR and AMPK in various
epithelial tissue sections. In addition, we used several approaches to
modulate endogenous AMPK activity and then measured the effects on
CFTR-dependent short-circuit currents (Isc) in
polarized T84 cell monolayers. The T84 secretory epithelial cell line,
derived from a human colon adenocarcinoma, has been extensively
characterized with respect to CFTR activity and function (3). Our results suggest that AMPK inhibits CFTR
Cl
channel activity in polarized T84 cells. This
AMPK-dependent inhibition of CFTR may be physiologically relevant
in general for polarized epithelia, providing a novel mechanism for
the modulation of CFTR activity as a function of cellular metabolic state.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents and chemicals. All reagents and chemicals used were purchased from Sigma (St. Louis, MO) unless otherwise noted.
Immunofluorescent staining.
The preparation of various rat tissues and immunofluorescent staining
of frozen sections were performed with rabbit polyclonal primary
anti-1-AMPK antibodies (54) or anti-CFTR-COOH-terminal tail antibodies (pAbC-term.B, a generous gift from Dr. John Marshall, Genzyme Pharmaceuticals, Cambridge, MA) and biotinylated secondary anti-rabbit IgG along with rhodamine-conjugated avidin
(Boehringer-Mannheim, Indianapolis, IN) exactly as described previously
(26). For each tissue shown, contiguous sections were used
for immunostaining AMPK-
1 and CFTR so that similar structures could
be compared. Hematoxylin and eosin staining of additional frozen
sections from the same block was also performed for reference to aid in
the identification of tissue structures. The tissues were obtained from
the Morphology Core of the Institute for Gene Therapy, University of Pennsylvania.
Ussing chamber Isc measurements.
T84 cells were seeded at confluent density on 1.0-cm permeable supports
(Costar Snapwells no. 3407) and grown in DMEM-F-12 medium. All
medium was removed and replaced with fresh medium every 24-48 h.
T84 cell monolayers were grown with medium on both the apical and
basolateral sides, and inserts were used for Ussing chamber experiments
5-12 days after seeding the cells. For experiments using intact
T84 cell monolayers (see Figs. 2 and 5), the experimental bath
solutions contained (in mM) 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose. Mannitol was substituted for glucose in the apical bath to eliminate the
contribution of Na+-glucose cotransport to
Isc, as previously described (17).
The pH of this solution was 7.4 when gassed with a mixture of 95% O2-5% CO2 at 37°C. For each experiment two
paired Snapwell inserts were mounted in Ussing chambers interfaced with
a voltage-current clamp amplifier (Physiologic Instruments, San Diego,
CA) and an electronic chart recorder (PowerLab; ADInstruments, Grand
Junction, CO), and the monolayers were continuously short-circuited by
voltage clamping to 0 mV, after fluid resistance and asymmetry voltage compensation. Changes in transepithelial resistance
(RT) were monitored and calculated with Ohm's
law from the current excursions resulting from periodic 2- or 5-mV
bipolar voltage pulses. T84 cell monolayers with
RT 1,000 ohm · cm2 under basal conditions were
used for experimentation. Net stimulated Isc
reported for each experiment was calculated by subtracting the baseline
Isc measured before stimulation from the peak
Isc measured after stimulation.
Overexpression of mutant AMPK subunits in polarized T84 cells by
lentiviral transduction.
Hemagglutinin (HA)-tagged wild-type or K45R mutant AMPK -subunits
were cloned into a transfer vector (pHR') as follows. Full-length AMPK-
1 or AMPK-
1-K45R gene fragments were amplified by
high-fidelity PCR (PFU-Turbo; Stratagene, La Jolla, CA) with
pEBG-AMPK-
1 or -
1-K45R plasmids (20) as templates
and specific primers designed to create a NotI site followed
in frame by an HA epitope tag on the 5' (sense) end and a
BamHI site on the 3' (antisense) end. These fragments were
then subcloned into the NotI/BamHI sites of the
vector pAAV-2, downstream of a cytomegalovirus (CMV) promoter. The CMV
promoter + AMPK-
1 gene fragments were then isolated from pAAV-2
by cutting with NheI and BamHI before cloning
into the SpeI and BamHI sites of pHR'. All primer
sequences and plasmid maps used are available on request. Plasmid
inserts were verified by DNA sequencing. This pHR'-AMPK-
1 (or
-
1-K45R) plasmid, the helper packaging construct pCMV
R8.2
(encoding the HIV helper function), and a plasmid encoding the
vesicular stomatitis virus G (VSV-G) envelope were then used for triple
transfection of HEK-293T cells to produce replication-incompetent
HIV-based VSV-G pseudotyped virus as described previously
(34).
AMPK kinase assay.
Polarized T84 cells were grown on Costar Transwells as described in
Ussing chamber Isc measurements. The
medium was replaced with DMEM-F-12 + 1 mM
5-amino-4-imidazolecarboxamide-1--D-ribofuranoside (AICAR) (or DMEM-F-12 alone for controls) on both the apical and basolateral sides and then incubated for 2 h at 37°C and 5%
CO2. Before lysis, cells were washed twice on both sides
with ice-cold PBS. Lysis buffer (LB) contained (in mM) 20 Tris-Cl, 50 NaCl, 50 NaF, 5 Na-pyrophosphate, 250 sucrose, and 1% Triton X-100 (pH 7.4 with NaOH). CPIC (1×; Roche), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 2 mM dithiothreitol (DTT) were added to the LB just before
addition of 120 µl of LB mixture to the apical side of each well.
After the samples were rocked for 15 min at 4°C, lysates from each
well were collected. For each condition two sets of lysates, each
pooled from two wells, were pelleted at 14,000 g at 4°C
for 10 min. Protein concentrations of supernatants were estimated by
the Bradford technique (Bio-Rad). AMPK activity was measured against
the SAMS peptide (HMRSAMSGLHLVKRR) after immunoprecipitation of the
kinase from cell lysates with an anti-
-subunit antibody that
recognizes both catalytic subunit isoforms (27).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We previously discovered (26) that the catalytic
-subunit of AMPK interacts with the CFTR COOH-terminal tail by using
independent yeast two-hybrid and biochemical ("pull-down")
techniques. It was demonstrated that CFTR can serve as a substrate for
AMPK-mediated phosphorylation in vitro. Importantly, exogenous
coexpression of AMPK with CFTR inhibited cAMP-stimulated CFTR whole
cell conductances in Xenopus oocytes, as measured with the
two-electrode voltage-clamp technique. Through expression of various
mutant AMPK-
1 subunits, it was determined that both binding of
AMPK-
1 to CFTR and an active kinase domain appeared to be required
to confer this inhibition in oocytes. These studies raised the
possibility that the interaction of AMPK with CFTR may have
physiological implications. To test the relevance of the AMPK-CFTR
interaction in physiologically relevant cell types, here we have
examined the cellular and subcellular distribution of both proteins by
immunofluorescent staining in various rat epithelial tissues and have
performed functional studies in polarized T84 epithelial cell
monolayers that endogenously express both proteins (3,
26).
Immunofluorescent staining to map localization of AMPK and CFTR in
epithelia.
We previously showed (26) that AMPK is expressed in
various CFTR-expressing cell lines. Overlapping cellular and
subcellular localizations of the two proteins in epithelial tissues
would be expected if the interaction between AMPK and CFTR is direct and physiologically relevant in vivo. To this end, immunofluorescent staining was performed in contiguous tissue sections of various rat epithelia, including nasopharynx, submandibular gland,
pancreas, and ileum (Fig. 1). To
facilitate direct comparisons between AMPK-stained structures (Fig. 1,
A, D, G, and J) and
CFTR-stained structures (Fig. 1, B, E,
H, and K), in Fig. 1 arrows indicate similar
structures in the corresponding contiguous tissue sections. Light
micrographs of similar hematoxylin and eosin-stained sections from the
same block are also shown to aid in the identification of relevant tissue morphology (Fig. 1, C, F, I,
and L). In typical fluorescence micrographs of rat
nasopharynx revealing submucosal gland ducts and acini, CFTR staining
was very distinct at the apical membranes of surface epithelial cells
lining the ducts and acini (Fig. 1B). AMPK staining was also
observed in these same submucosal gland structures, but it was overall
more diffuse than that of CFTR yet was also prominent near the apical
membranes (Fig. 1A). A control for nonspecific staining (no
primary antibody added) is shown in Fig. 1C. Similar control
immunostains in which no primary antibodies were used also revealed
very minimal background staining in the other tissues examined (not
shown). In micrographs of rat submandibular gland, CFTR was distinctly
localized to the apical membranes in certain small and large ducts and
in acini (Fig. 1E). AMPK expression in submandibular gland
was greatest in surface epithelial cells lining ducts, with a slight
apical predominance (Fig. 1D). In rat pancreas both proteins
stained strongly in the surface epithelial cells that line the large
ducts shown (Fig. 1, G and H). CFTR staining in
the pancreas was also present at the apical membranes of certain small
ducts (Fig. 1H), whereas AMPK stained in a more diffuse
reticular pattern in areas surrounding the large duct (Fig.
1G), which probably represent secretory acini (cf. Fig.
1I). Finally, in rat ileum AMPK was found predominantly at
the apical membrane of epithelial cells in the crypts of the villi and
to a lesser extent on the apical surfaces of cells lining the villi
(Fig. 1J). CFTR was also localized at the apical membrane of
crypts, but not in the surface epithelial cells situated more distally
along the villi (Fig. 1K), consistent with previous studies (55).
|
Pharmacological activation of AMPK inhibits endogenous
CFTR-dependent currents in polarized T84 cell monolayers.
Previous published studies characterized the various transport proteins
that are expressed on the apical and basolateral membranes of polarized
T84 cells. In T84 cell polarized monolayers studied under the
conditions of these experiments, net Cl secretion via
CFTR is responsible for almost all of the ionic conductance of the
apical membrane (3). Net basolateral transport of
Cl
likely occurs via
Na+-K+-2Cl
cotransporters, acting
in concert with the Na+-K+-ATPase and
K+ channels to recycle Na+ and K+
across the basolateral membrane (3, 16, 18).
|
AMPK-dependent inhibition of CFTR in T84 cells with basolateral
membrane permeabilization.
Because currents measured across intact monolayers under short-circuit
conditions traverse both membranes in series, it was possible that the
observed inhibition of Isc by AICAR represented, at least in part, inhibition of basolateral membrane transport pathways. To examine the effects of AMPK on the apical membrane CFTR
Cl conductance specifically, the basolateral membrane was
permeabilized with either nystatin (Fig.
3) or S. aureus
-toxin
(Fig. 4) to render the apical membrane
conductance rate limiting. The basolateral bathing solution was
substituted with a solution containing a low Cl
concentration to establish a mucosa-to-serosa Cl
concentration gradient as described previously (15). For
the results shown in Fig. 3, T84 cell monolayers were preincubated in
serum-free medium with or without the addition of 1 mM AICAR for 2 h before being mounted in Ussing chambers (time 0). Nystatin (180 µg/ml) was added to the basolateral bath at the indicated time
to permeabilize the basolateral membrane to small monovalent ions.
Under these conditions any changes in observed
Isc in response to cell activation should be
directly attributable to changes in the apical membrane conductance,
which is dependent on the activity of CFTR. After ~20-30 min, to
allow Isc to reach a steady state, 4 µM
forskolin was added to the basolateral bath to stimulate apical
membrane CFTR channel conductance. Under these asymmetrical conditions
the Cl
gradient was reversed, so activation of apical
membrane CFTR conductance caused a rapid downward current deflection
(Fig. 3A). This forskolin-dependent stimulation was
partially reversed (~50%) by subsequent 250 µM glibenclamide
treatment (and more so with further glibenclamide treatments; not
shown), suggesting that the current was mediated predominantly by
apical CFTR. The forskolin-activated CFTR-dependent Cl
current was inhibited by 24.1 ± 3.0% (P < 0.05, paired t-test, n = 4) in the
AICAR-pretreated monolayers relative to paired controls (Fig.
3B). These results are therefore in agreement with those obtained with nonpermeabilized monolayers.
|
|
Modulation of AMPK-dependent inhibition of endogenous CFTR by
mutant kinase overexpression.
To manipulate AMPK activity by a nonpharmacological approach, we
specifically altered its kinase activity with a dominant-negative approach. A Lys to Arg (K45R) point mutation at the active site in the
kinase domain of the -subunit of AMPK renders the kinase catalytically dead (12). Chronic overexpression of this
kinase-dead
-K45R mutant in cultured cells (63) and in
transgenic mice (44) appears to cause a downregulation of
the endogenous wild-type
-subunit through competition for binding to
the endogenous regulatory
- and
-subunits. Therefore,
overexpression of the
-K45R subunit has a dominant-negative effect
on AMPK activity in vivo (44). We exploited this
observation by using lentiviral transduction to overexpress either the
wild-type AMPK-
1 subunit or the dominant-negative AMPK-
1-K45R
subunit in polarized T84 cell monolayers as a specific method to
modulate the activity of AMPK (Fig.
5). The lentiviral transduction system was selected because it was shown recently to
transduce intact airway epithelium in vivo with very high efficiency and stability (34). Expression of the exogenous HA-tagged
AMPK-
1 subunits in the monolayers was confirmed by immunoblotting
for the HA epitope tag in T84 cellular lysates obtained from the
permeable supports after each Ussing chamber experiment (Fig.
5C). We hypothesized that overexpression of the
dominant-negative AMPK-
1-K45R mutant might result in enhanced
CFTR-mediated Cl
currents compared with wild-type
AMPK-
1, if CFTR activity was tonically inhibited by the wild-type
kinase. The magnitudes of the Isc activated by
the combined treatment of forskolin and thapsigargin were then compared
between the two groups of monolayers (Fig. 5, A and
B). Compared with the wild-type AMPK-
1-transduced
monolayers, the mutant AMPK-
1-K45R-transduced T84 cell monolayers
exhibited a 22.4 ± 4.6% greater stimulated
Isc (P < 0.01, paired
t-test, n = 6). This result therefore
suggests that overexpression of the dominant-negative AMPK-
1-K45R
mutant in polarized T84 cells reduced the inhibition of CFTR by
endogenous AMPK, i.e., inhibition of endogenous AMPK activity resulted
in a disinhibition (or stimulation) of CFTR. Because control
experiments suggested that only ~50% of the T84 cells in the
monolayer under these conditions expressed the exogenous protein (Fig.
5D), it is likely that this result underestimates the true
magnitude of the dominant-negative effect.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The maintenance of ionic gradients across the plasma membrane by various cellular pump and leak pathways is vital in general for normal cellular functioning and in particular for the coordinated transport of solutes and fluids across polarized epithelia. Cellular transport processes consume a substantial proportion of total cellular metabolic energy and thus would be expected to respond closely to changes in cellular metabolic status. Our recent discovery of a functional interaction between AMPK and CFTR suggests a novel mechanism for such coupling between ion transport and cellular metabolism. The results obtained in the present study now suggest that the AMPK-CFTR interaction is physiologically relevant in polarized epithelia.
AMPK and CFTR share an overlapping apical distribution in various rat
epithelial tissues (Fig. 1). Previous yeast two-hybrid and biochemical
techniques demonstrated that the COOH-terminal regulatory domain of the
AMPK-1 catalytic subunit binds to the COOH-terminal tail of CFTR
(26). The overlapping localization of the two proteins in
epithelial cells may suggest first that the proteins do indeed interact
at the apical plasma membrane and, more speculatively, that the apical
localization of AMPK is a consequence of its interaction with CFTR.
However, because AMPK exhibits a more diffuse subcellular distribution
compared with CFTR, it is clear that binding to CFTR cannot be the sole determinant of AMPK localization. Indeed, the AMPK
-subunit has been
shown to be myristoylated (41), and this modification may generally promote localization of the AMPK holoenzyme to membranes. Localization of the kinase to the apical membrane might enable AMPK to
effectively and directly modulate the function of CFTR as well as other
nearby membrane proteins. Future studies of AMPK localization, using
epithelial tissues from CFTR knockout mice or CF patients, may provide
insights into the possible role of CFTR as an AMPK-anchoring protein.
In our examination of AMPK immunolocalization, it appeared that AMPK cellular expression levels, as assessed by the intensity of staining, were high in the very metabolically active surface epithelial cells and generally lower in the deeper interstitial and connective tissue regions of the tissue sections (Fig. 1). Epithelial cells generally expend a large percentage of total cellular metabolic energy on membrane transport processes, and their metabolic consumption rate is highly correlated with their rate of active ion transport (39). The fact that cellular expression levels of this metabolic-sensing kinase appear to correlate with cellular metabolic activity in epithelial tissues is perhaps not surprising because previous studies demonstrated increased AMPK expression in highly metabolically active cells in other tissues, such as neurons in the brain (13) and ventricular myocytes in rat hearts with pressure-overload hypertrophy (57).
The immunolocalization studies indicate that the cellular localizations
of AMPK and CFTR overlap, but they provide no direct evidence that the
proteins interact or that the interaction has physiological relevance.
In the present study, we used several independent functional approaches
to modulate endogenous AMPK activity and then determined the effects on
endogenous CFTR-mediated Cl currents in polarized T84
cell monolayers. The T84 cell line is a widely used model of epithelial
Cl
secretion because it forms polarized, high-resistance
monolayers and expresses relevant transport proteins in its apical and
basolateral membranes (18). Of relevance for the present
studies, the Isc through T84 monolayers is
conducted entirely by CFTR at the apical membrane. Pretreatment with
the pharmacological AMPK activator AICAR inhibited the magnitude of
stimulated CFTR-dependent Isc in both intact T84
cell monolayers (Fig. 2) and nystatin-permeabilized monolayers, in
which the effects could be specifically localized to CFTR at the apical
membrane (Fig. 3). Furthermore, elevating intracellular [AMP]
directly through
-toxin permeabilization of the basolateral membrane
and dialysis into the cells of high concentrations of AMP from the
basolateral bath also inhibited CFTR-mediated Cl
currents
(Fig. 4). The latter effect could not be explained by activation of
adenosine receptors (Fig. 4, C and D), and it is unlikely to be due to a direct effect of AMP on CFTR, because no
AMP-dependent effects on CFTR single-channel gating have been observed
at concentrations up to 3 mM (49). The AMP-dependent inhibition of CFTR activity was also unlikely to be caused by intracellular conversion of AMP and ATP to ADP through adenylate kinase, because the adenylate kinase inhibitor AP5A was
present throughout the experiments. Thus the [ATP]i and
[ADP]i should have been relatively unaffected by
intracellular dialysis of AMP, so the inhibition observed with AMP
treatment under these conditions cannot be attributed to a reduction of
[ATP]i or to an elevation of [ADP]i, both
of which could be expected to directly inhibit CFTR gating. We
therefore conclude that the elevation of [AMP]i (and
therefore intracellular [AMP]-to-[ATP] ratio) inhibited CFTR by
directly activating endogenous intracellular AMPK. Finally, overexpression of a kinase-dead, dominant-negative AMPK mutant in the
polarized T84 cell monolayers enhanced CFTR-mediated transepithelial Cl
currents (Fig. 5). This result provides specific
evidence that AMPK can modulate CFTR activity in a relevant epithelial
system. The enhancement of CFTR-dependent Isc
seen after suppression of endogenous AMPK activity with the
dominant-negative mutant suggests that there may be a tonic level of
AMPK activity in these T84 cells under the conditions of these
experiments that inhibited CFTR activity. Indeed, cultured cells,
including epithelial cell lines, are chronically hypoxic when cultured
submerged in medium (2), as the T84 cells were for these
studies, which is a condition that would promote basal AMPK activity.
By the dominant-negative downregulation of this AMPK activity, a
disinhibition (or enhancement) of CFTR Isc could
be observed. It is also possible that overexpression of the wild-type
AMPK
1-subunit promoted endogenous AMPK activity and thereby
inhibited CFTR in the control group. For this to have occurred,
however, sufficient amounts of endogenous regulatory
- and
-subunits would need to be available to associate with the expressed
-subunit, because they are required to confer full AMPK activity.
The data presented here provide the first compelling evidence for a
physiologically relevant regulation of an ion channel (CFTR) by AMPK.
AMPK activity may also be important in the regulation of other membrane
transport proteins in vivo. It has been long observed that a loss in
membrane transport protein activity, expression, and polarity is
associated with ischemic injury to epithelial tissues [e.g.,
in the kidney (25)], although the mechanisms underlying
this effect are unclear. For example, hypoxia and cellular energy
depletion in epithelial tissues inhibit ATP-dependent membrane transporters such as the Na+-K+-ATPase and
P-glycoprotein, also known as multidrug resistance protein (MDR)
(6, 39). Pertinent to this study, Bell and Quinton
(4) previously measured Cl conductance in
-toxin-permeabilized T84 cells before and after metabolic depletion.
It was concluded that a nonhydrolytic, presumably direct, interaction
of ATP with CFTR was involved in the coupling between cellular
metabolic status and CFTR activity (4). A direct ATP-CFTR
interaction certainly occurs through ATP binding to the NBDs of CFTR.
However, it is unlikely that small changes in [ATP] would be
sufficient to tightly couple CFTR activity to cellular metabolic state
by this mechanism under normal physiological conditions because ambient
cellular [ATP] are in the millimolar range, which is generally much
higher than the saturating concentrations for ATP binding to the NBDs
of CFTR (1). Similarly, it has been noted that only modest
decreases in cellular [ATP], remaining within the millimolar range,
dramatically inhibit the activities of the
Na+-K+-ATPase and MDR, despite their having
affinities for ATP of <100 µM. This discrepancy has been
rationalized by proposals that large gradients of [ATP]i
exist in cells (6). We suggest that AMPK may provide a
more sensitive means of coupling changes in the cellular metabolic
state to transporter activity. Relatively small changes in
intracellular ATP levels result in large changes in the intracellular
AMP-to-ATP ratio because of the rapid interconversion of ATP, ADP, and
AMP by adenylate kinase (see Ref. 30 for review). Because
AMPK activity is sensitive to the AMP-to-ATP ratio, AMPK can serve as a
primary sensor of (even minor) changes in metabolic state and may
therefore play an important role in the initiation of cellular events
that occur both during physiological fluctuations of
[ATP]i and during pathological reductions in
[ATP]i (i.e., in response to ischemic or hypoxic
injury). Recently, mutations in the gene encoding the AMPK-
regulatory subunit (PRKAG2) in cardiac muscle were shown to be
responsible for familial cases of ventricular preexcitation arrhythmias
(Wolff-Parkinson-White syndrome), which led to the suggestion that AMPK
may be an important regulator of ion channels in the heart (23,
24). Of note, CFTR is expressed in heart muscle
(31) and has been postulated to play a role in the genesis
of cardiac arrhythmias (32). Also, ATP-sensitive
K+ (KATP) channels, which are composed of
inward rectifying K+ channel (Kir) subunits
coupled stoichiometrically with the sulfonylurea receptor SUR
(51), another ABC transporter closely related to CFTR, are
regulated by changes in cellular metabolic state and are important ion
channels found in the heart (22). Further studies are thus
warranted to investigate the potential role of AMPK in the modulation
of membrane transport proteins, which could lead to new insights into
the pathogenesis and treatment of many disorders, including
ischemic tissue injury and cardiac arrhythmias.
In summary, we have demonstrated that endogenous AMPK and CFTR share an apical membrane localization in epithelial tissues and that modulating endogenous AMPK activity in epithelial cells regulates the activity of CFTR. The AMPK-CFTR interaction may constitute a novel mechanism to link CFTR ion channel activity to cell metabolic status, thereby coupling cellular metabolism with transepithelial solute transport and the maintenance of cellular ion gradients.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank John Tazelaar, Evan Ray, Jill McCane, and Christine Richardson for excellent technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (F32-DK-09994 and K08-DK-59477 to K. R. Hallows, R01-DK-47757 to J. M. Wilson, and R01-DK-35712 to L. A. Witters) and the Cystic Fibrosis Foundation (to G. P. Kobinger, J. M. Wilson, and J. K. Foskett). G. P. Kobinger was the recipient of a fellowship from the Medical Research Council of Canada. J. M. Wilson owns equity in Targeted Genetics.
Address for reprint requests and other correspondence: J. K. Foskett, Dept. of Physiology, Univ. of Pennsylvania School of Medicine, B39 Anatomy-Chemistry Bldg., Philadelphia, PA 19104 (E-mail: foskett{at}mail.med.upenn.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 2, 2003;10.1152/ajpcell.00227.2002
Received 20 May 2002; accepted in final form 30 December 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aleksandrov, L,
Mengos A,
Chang X,
Aleksandrov A,
and
Riordan JR.
Differential interactions of nucleotides at the two nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem
276:
12918-12923,
2001
2.
Bebok, Z,
Tousson A,
Schwiebert LM,
and
Venglarik CJ.
Improved oxygenation promotes CFTR maturation and trafficking in MDCK monolayers.
Am J Physiol Cell Physiol
280:
C135-C145,
2001
3.
Bell, CL,
and
Quinton PM.
T84 cells: anion selectivity demonstrates expression of Cl conductance affected in cystic fibrosis.
Am J Physiol Cell Physiol
262:
C555-C562,
1992
4.
Bell, CL,
and
Quinton PM.
Regulation of CFTR Cl conductance in secretion by cellular energy levels.
Am J Physiol Cell Physiol
264:
C925-C931,
1993
5.
Blair, E,
Redwood C,
Ashrafian H,
Oliveira M,
Broxholme J,
Kerr B,
Salmon A,
Ostman-Smith I,
and
Watkins H.
Mutations in the gamma(2) subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis.
Hum Mol Genet
10:
1215-1220,
2001
6.
Broxterman, HJ,
and
Pinedo HM.
Energy metabolism in multidrug resistant tumor cells: a review.
J Cell Pharmacol
2:
239-247,
1991.
7.
Carling, D,
Clarke PR,
Zammit VA,
and
Hardie DG.
Purification and characterization of the AMP-activated protein kinase. Copurification of acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA reductase kinase activities.
Eur J Biochem
186:
129-136,
1989[Abstract].
8.
Chang, SY,
Di A,
Naren AP,
Palfrey HC,
Kirk KL,
and
Nelson DJ.
Mechanisms of CFTR regulation by syntaxin 1A and PKA.
J Cell Sci
115:
783-791,
2002
9.
Chen, ZP,
Mitchelhill KI,
Michell BJ,
Stapleton D,
Rodriguez-Crespo I,
Witters LA,
Power DA,
Ortiz de Montellano PR,
and
Kemp BE.
AMP-activated protein kinase phosphorylation of endothelial NO synthase.
FEBS Lett
443:
285-289,
1999[ISI][Medline].
10.
Corton, JM,
Gillespie JG,
Hawley SA,
and
Hardie DG.
5-Aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells?
Eur J Biochem
229:
558-565,
1995[Abstract].
11.
Coyne, CB,
Kelly MM,
Boucher RC,
and
Johnson LG.
Enhanced epithelial gene transfer by modulation of tight junctions with sodium caprate.
Am J Respir Cell Mol Biol
23:
602-609,
2000
12.
Crute, BE,
Seefeld K,
Gamble J,
Kemp BE,
and
Witters LA.
Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase.
J Biol Chem
273:
35347-35354,
1998
13.
Culmsee, C,
Monnig J,
Kemp BE,
and
Mattson MP.
AMP-activated protein kinase is highly expressed in neurons in the developing rat brain and promotes neuronal survival following glucose deprivation.
J Mol Neurosci
17:
45-58,
2001[ISI][Medline].
14.
Devidas, S,
and
Guggino WB.
The cystic fibrosis transmembrane conductance regulator and ATP.
Curr Opin Cell Biol
9:
547-552,
1997[ISI][Medline].
15.
Devor, DC,
Singh AK,
Frizzell RA,
and
Bridges RJ.
Modulation of Cl secretion by benzimidazolones. I. Direct activation of a Ca2+-dependent K+ channel.
Am J Physiol Lung Cell Mol Physiol
271:
L775-L784,
1996
16.
Devor, DC,
Singh AK,
Gerlach AC,
Frizzell RA,
and
Bridges RJ.
Inhibition of intestinal Cl secretion by clotrimazole: direct effect on basolateral membrane K+ channels.
Am J Physiol Cell Physiol
273:
C531-C540,
1997
17.
Devor, DC,
Singh AK,
Lambert LC,
DeLuca A,
Frizzell RA,
and
Bridges RJ.
Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells.
J Gen Physiol
113:
743-760,
1999
18.
Dharmsathaphorn, K,
and
Madara JL.
Established intestinal cell lines as model systems for electrolyte transport studies.
Methods Enzymol
192:
354-389,
1990[Medline].
19.
Dho, S,
Stewart K,
and
Foskett JK.
Purinergic receptor activation of Cl secretion in T84 cells.
Am J Physiol Cell Physiol
262:
C67-C74,
1992
20.
Dyck, JR,
Gao G,
Widmer J,
Stapleton D,
Fernandez CS,
Kemp BE,
and
Witters LA.
Regulation of 5'-AMP-activated protein kinase activity by the noncatalytic beta and gamma subunits.
J Biol Chem
271:
17798-17803,
1996
21.
Foskett, JK.
ClC and CFTR chloride channel gating.
Annu Rev Physiol
60:
689-717,
1998[ISI][Medline].
22.
Fujita, A,
and
Kurachi Y.
Molecular aspects of ATP-sensitive K+ channels in the cardiovascular system and K+ channel openers.
Pharmacol Ther
85:
39-53,
2000[ISI][Medline].
23.
Gollob, MH,
Green MS,
Tang AS,
Gollob T,
Karibe A,
Ali Hassan AS,
Ahmad F,
Lozado R,
Shah G,
Fananapazir L,
Bachinski LL,
Roberts R,
and
Hassan AS.
Identification of a gene responsible for familial Wolff-Parkinson-White syndrome.
N Engl J Med
344:
1823-1831,
2001
24.
Gollob, MH,
Seger JJ,
Gollob TN,
Tapscott T,
Gonzales O,
Bachinski L,
and
Roberts R.
Novel PRKAG2 mutation responsible for the genetic syndrome of ventricular preexcitation and conduction system disease with childhood onset and absence of cardiac hypertrophy.
Circulation
104:
3030-3033,
2001
25.
Green, J,
Abassi Z,
Winaver J,
and
Skorecki KL.
Acute renal failure: clinical and pathophysiologic aspects.
In: The Kidney, edited by Seldin DW,
and Giebisch G.. Philadelphia, PA: Lippincott Williams & Wilkins, 2000, p. 2329-2373.
26.
Hallows, KR,
Raghuram V,
Kemp BE,
Witters LA,
and
Foskett JK.
Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase.
J Clin Invest
105:
1711-1721,
2000
27.
Hamilton, SR,
Stapleton D,
O'Donnell JB,
Kung JT,
Dalal SR,
Kemp BE,
and
Witters LA.
An activating mutation in the gamma1 subunit of the AMP-activated protein kinase.
FEBS Lett
500:
163-168,
2001[ISI][Medline].
28.
Hardie, DG,
and
Carling D.
The AMP-activated protein kinasefuel gauge of the mammalian cell?
Eur J Biochem
246:
259-273,
1997[Abstract].
29.
Hardie, DG,
Carling D,
and
Carlson M.
The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell?
Annu Rev Biochem
67:
821-855,
1998[ISI][Medline].
30.
Hardie, DG,
and
Hawley SA.
AMP-activated protein kinase: the energy charge hypothesis revisited.
Bioessays
23:
1112-1119,
2001[ISI][Medline].
31.
Hart, P,
Warth JD,
Levesque PC,
Collier ML,
Geary Y,
Horowitz B,
and
Hume JR.
Cystic fibrosis gene encodes a cAMP-dependent chloride channel in heart.
Proc Natl Acad Sci USA
93:
6343-6348,
1996
32.
Horie, M,
Obayashi K,
Xie LH,
James AF,
and
Sasayama S.
Hormonal regulation of cardiac cystic fibrosis transmembrane conductance regulator chloride channels.
Jpn Heart J
37:
661-671,
1996[ISI][Medline].
33.
Kemp, BE,
Mitchelhill KI,
Stapleton D,
Michell BJ,
Chen ZP,
and
Witters LA.
Dealing with energy demand: the AMP-activated protein kinase.
Trends Biochem Sci
24:
22-25,
1999[ISI][Medline].
34.
Kobinger, GP,
Weiner DJ,
Yu QC,
and
Wilson JM.
Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo.
Nat Biotechnol
19:
225-230,
2001[ISI][Medline].
35.
Kunzelmann, K.
CFTR: interacting with everything?
News Physiol Sci
16:
167-170,
2001
36.
Kurth-Kraczek, EJ,
Hirshman MF,
Goodyear LJ,
and
Winder WW.
5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle.
Diabetes
48:
1667-1671,
1999[Abstract].
37.
Leclerc, I,
Kahn A,
and
Doiron B.
The 5'-AMP-activated protein kinase inhibits the transcriptional stimulation by glucose in liver cells, acting through the glucose response complex.
FEBS Lett
431:
180-184,
1998[ISI][Medline].
38.
Lienhard, GE,
and
Secemski, II
P1,P5-Di(adenosine-5')pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase.
J Biol Chem
248:
1121-1123,
1973
39.
Mandel, LJ,
and
Balaban RS.
Stoichiometry and coupling of active transport to oxidative metabolism in epithelial tissues.
Am J Physiol Renal Fluid Electrolyte Physiol
240:
F357-F371,
1981
40.
Minokoshi, Y,
Kim YB,
Peroni OD,
Fryer LG,
Muller C,
Carling D,
and
Kahn BB.
Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase.
Nature
415:
339-343,
2002[ISI][Medline].
41.
Mitchelhill, KI,
Michell BJ,
House CM,
Stapleton D,
Dyck J,
Gamble J,
Ullrich C,
Witters LA,
and
Kemp BE.
Posttranslational modifications of the 5'-AMP-activated protein kinase beta1 subunit.
J Biol Chem
272:
24475-24479,
1997
42.
Moore, F,
Weekes J,
and
Hardie DG.
Evidence that AMP triggers phosphorylation as well as direct allosteric activation of rat liver AMP-activated protein kinase. A sensitive mechanism to protect the cell against ATP depletion.
Eur J Biochem
199:
691-697,
1991[Abstract].
43.
Moyer, BD,
Denton J,
Karlson KH,
Reynolds D,
Wang S,
Mickle JE,
Milewski M,
Cutting GR,
Guggino WB,
Li M,
and
Stanton BA.
A PDZ-interacting domain in CFTR is an apical membrane polarization signal.
J Clin Invest
104:
1353-1361,
1999
44.
Mu, J,
Brozinick JT, Jr,
Valladares O,
Bucan M,
and
Birnbaum MJ.
A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle.
Mol Cell
7:
1085-1094,
2001[ISI][Medline].
45.
Naren, AP,
Nelson DJ,
Xie W,
Jovov B,
Pevsner J,
Bennett MK,
Benos DJ,
Quick MW,
and
Kirk KL.
Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms.
Nature
390:
302-305,
1997[ISI][Medline].
46.
Naren, AP,
Quick MW,
Collawn JF,
Nelson DJ,
and
Kirk KL.
Syntaxin 1A inhibits CFTR chloride channels by means of domain-specific protein-protein interactions.
Proc Natl Acad Sci USA
95:
10972-10977,
1998
47.
Quinton, PM,
and
Reddy MM.
Control of CFTR chloride conductance by ATP levels through non-hydrolytic binding.
Nature
360:
79-81,
1992[ISI][Medline].
48.
Raghuram, V,
Mak DD,
and
Foskett JK.
Regulation of cystic fibrosis transmembrane conductance regulator single-channel gating by bivalent PDZ-domain-mediated interaction.
Proc Natl Acad Sci USA
98:
1300-1305,
2001
49.
Schultz, BD,
Venglarik CJ,
Bridges RJ,
and
Frizzell RA.
Regulation of CFTR Cl channel gating by ADP and ATP analogues.
J Gen Physiol
105:
329-361,
1995[Abstract].
50.
Schwiebert, EM,
Egan ME,
Hwang TH,
Fulmer SB,
Allen SS,
Cutting GR,
and
Guggino WB.
CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.
Cell
81:
1063-1073,
1995[ISI][Medline].
51.
Seino, S.
ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies.
Annu Rev Physiol
61:
337-362,
1999[ISI][Medline].
52.
Short, DB,
Trotter KW,
Reczek D,
Kreda SM,
Bretscher A,
Boucher RC,
Stutts MJ,
and
Milgram SL.
An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton.
J Biol Chem
273:
19797-19801,
1998
53.
Song, L,
Hobaugh MR,
Shustak C,
Cheley S,
Bayley H,
and
Gouaux JE.
Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore.
Science
274:
1859-1866,
1996
54.
Stapleton, D,
Mitchelhill KI,
Gao G,
Widmer J,
Michell BJ,
Teh T,
House CM,
Fernandez CS,
Cox T,
Witters LA,
and
Kemp BE.
Mammalian AMP-activated protein kinase subfamily.
J Biol Chem
271:
611-614,
1996
55.
Strong, TV,
Boehm K,
and
Collins FS.
Localization of cystic fibrosis transmembrane conductance regulator mRNA in the human gastrointestinal tract by in situ hybridization.
J Clin Invest
93:
347-354,
1994[ISI][Medline].
56.
Sugita, M,
Yue Y,
and
Foskett JK.
CFTR Cl channel and CFTR-associated ATP channel: distinct pores regulated by common gates.
EMBO J
17:
898-908,
1998
57.
Tian, R,
Musi N,
D'Agostino J,
Hirshman MF,
and
Goodyear LJ.
Increased adenosine monophosphate-activated protein kinase activity in rat hearts with pressure-overload hypertrophy.
Circulation
104:
1664-1669,
2001
58.
Tsui, LC,
and
Buchwald M.
Biochemical and molecular genetics of cystic fibrosis.
Adv Hum Genet
20:
153-266,
1991[Medline].
59.
Wang, S,
Yue H,
Derin RB,
Guggino WB,
and
Li M.
Accessory protein facilitated CFTR-CFTR interaction, a molecular mechanism to potentiate the chloride channel activity.
Cell
103:
169-179,
2000[ISI][Medline].
60.
Weixel, KM,
and
Bradbury NA.
Mu 2 binding directs the cystic fibrosis transmembrane conductance regulator to the clathrin-mediated endocytic pathway.
J Biol Chem
276:
46251-46259,
2001
61.
Welsh, MJ,
and
Smith AE.
Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis.
Cell
73:
1251-1254,
1993[ISI][Medline].
62.
Welsh, MJ,
and
Smith AE.
Cystic fibrosis.
Sci Am
273:
52-59,
1995[ISI][Medline].
63.
Woods, A,
Azzout-Marniche D,
Foretz M,
Stein SC,
Lemarchand P,
Ferre P,
Foufelle F,
and
Carling D.
Characterization of the role of AMP-activated protein kinase in the regulation of glucose-activated gene expression using constitutively active and dominant negative forms of the kinase.
Mol Cell Biol
20:
6704-6711,
2000
64.
Zhou, G,
Myers R,
Li Y,
Chen Y,
Shen X,
Fenyk-Melody J,
Wu M,
Ventre J,
Doebber T,
Fujii N,
Musi N,
Hirshman MF,
Goodyear LJ,
and
Moller DE.
Role of AMP-activated protein kinase in mechanism of metformin action.
J Clin Invest
108:
1167-1174,
2001