Electrodiffusional ATP movement through the cystic fibrosis
transmembrane conductance regulator
Horacio F.
Cantiello1,2,
George
R.
Jackson Jr.2,
Claudio F.
Grosman3,
Adriana G.
Prat1,2,
Steven C.
Borkan4,
Yihan
Wang4,
Ignacio L.
Reisin3,
Catherine R.
O'Riordan5, and
Dennis A.
Ausiello1,2
1 Renal Unit, Massachusetts
General Hospital East, Charlestown 02129;
2 Department of Medicine, Harvard
Medical School, Boston 02115;
4 Renal Section, Boston Medical
Center, Boston 02118;
5 Genzyme Corporation, Framingham,
Massachusetts 01701; and
3 Departamento de Química
General e Inorgánica, Facultad de Farmacia y Bioquímica,
Universidad de Buenos Aires, 1113 Buenos Aires, Argentina
 |
ABSTRACT |
Expression of the
cystic fibrosis transmembrane conductance regulator (CFTR), and of at
least one other member of the ATP-binding cassette family of transport
proteins, P-glycoprotein, is associated with the electrodiffusional
movement of the nucleotide ATP. Evidence directly implicating CFTR
expression with ATP channel activity, however, is still missing. Here
it is reported that reconstitution into a lipid bilayer of highly
purified CFTR of human epithelial origin enables the permeation of both
Cl
and ATP. Similar to
previously reported data for in vivo ATP currents of CFTR-expressing
cells, the reconstituted channels displayed competition between
Cl
and ATP and had multiple
conductance states in the presence of Cl
and ATP. Purified
CFTR-mediated ATP currents were activated by protein kinase A and ATP
(1 mM) from the "intracellular" side of the molecule and were
inhibited by diphenylamine-2-carboxylate, glibenclamide, and anti-CFTR
antibodies. The absence of CFTR-mediated electrodiffusional ATP
movement may thus be a relevant component of the pleiotropic cystic
fibrosis phenotype.
adenosine 5'-triphosphate channels; adenosine
5'-triphosphate-binding cassette transporters; nucleotide
transport
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INTRODUCTION |
THE CYSTIC FIBROSIS transmembrane conductance regulator
(CFTR) supports ATP-dependent
Cl
channel activity (3,
14). However, controversy still exists as to whether this functional
feature of CFTR is sufficient to explain the complete pathophysiology
of this disease (7). The cellular epithelial cystic fibrosis (CF)
phenotype, for example, is associated both with a dysfunctional
phospholipase A2 activity and a
deranged arachidonic acid metabolism (10, 24) and with an abnormally
increased apical epithelial Na+
permeability (6), which is regulated by CFTR (26). Expression of CFTR
is also associated with the recovery of outwardly rectifying Cl
channel activity of CF
cells (12) by an autocoid mechanism entailing the release of cellular
ATP, which, in turn, helps modulate other cell responses (23). These
disparate observations are unlikely to stem from an altered
Cl
conductance. Alternate
paradigms to explain the various features of the CF phenotype are
currently being sought.
CFTR has been implicated in the release of cellular ATP (18, 23) and
electrodiffusional ATP movement (17, 22, 23); however, other studies
have failed to detect electrodiffusional ATP movement in preparations
containing a functional CFTR (13, 21). In this report, we have
revisited the question of whether CFTR is capable of permeating ATP.
Highly purified and functional human epithelial recombinant CFTR (14)
was reconstituted into a lipid bilayer in which addition of protein
kinase A (PKA) and ATP elicited diphenylamine-2-carboxylate
(DPC)-inhibitable Cl
- and
ATP-permeable ion channel activity, in agreement with our previous
findings in intact cells (9, 22, 23). The present data indicate that
ATP transfer by CFTR may not require an "adjacent" transmembrane
structure to allow permeation of the nucleotide.
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MATERIALS AND METHODS |
Purification of CFTR.
Recombinant human epithelial CFTR was obtained from
baculovirus-infected Sf9 (insect) cell membranes according to
O'Riordan et al. (14). Briefly, CFTR was purified with a small (2.6 × 6 cm, 30 ml) ceramic hydroxyapatite column and an equilibration buffer containing 10 mM sodium phosphate buffer, pH 6.4, 0.15% sodium
dodecyl sulfate (SDS), and 5 mM dithiothreitol (DTT) and was eluted
with a linear gradient of 100-600 mM sodium phosphate, 0.15% SDS,
and 5 mM DTT as originally described (3). A Superdex column and
chromatography in 0.1% SDS, rather than 0.25% lithium dodecyl
sulfate, were also used to facilitate purification of larger quantities
(mg) of insect CFTR (14). In cases in which purified CFTR was obtained
from CFTR-expressing Chinese hamster ovary (CHO) cells, cells grown on
microcarriers were harvested, washed with sodium phosphate-buffered
saline (PBS), and lysed in buffer containing protease inhibitors (for
more details, see Ref. 14). The supernatant was processed to obtain a
membrane suspension, which was pelleted at 100,000 g for 60 min. The supernatant containing the solubilized CFTR was further purified by immunoaffinity resins prepared with monoclonal antibody (MAb) 13-1 and MAb 24-1 (Genzyme, Framingham, MA), activated with sodium periodate, and coupled
to Hydrazide Avidgel (Unisyn Technologies) in 50 mM sodium acetate, pH
5.0. Immunoaffinity chromatography was performed by incubation of
CFTR-containing membranes (25 ml) with 15 ml of resin for at least 3 h
at 4°C. After collection of the flow-through, the resin was rinsed
with 100 ml of wash buffer containing 150 mM NaCl, 50 mM
tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 8.0, 1 mM EDTA, and 1% sodium cholate to remove nonspecifically bound
proteins. CFTR was further eluted from the resin using elution buffer
(150 mM NaCl, 50 mM Tris · HCl, pH 7.4, 1 mM EDTA,
10% glycerol, and 0.5% sodium cholate) containing 5 mg/ml of the
appropriate peptide antigen, synthesized by QCB (Hopkinton, MA).
Fractions containing CFTR were pooled and concentrated using a
Centricell 20 to a final concentration of 1 mg/ml (500 µl). CFTR was
further purified using either gel filtration or ion exchange
chromatography (14). Gel filtration chromatography was performed on a
Superdex 200 HR 10/30 column preequilibrated with 150 mM NaCl, 50 mM
Tris · HCl, 1 mM EDTA, 10% glycerol, and 0.5%
sodium cholate, pH 7.5. Immunoaffinity-purified CFTR (200 µl) was
applied to the column, fractions were collected, and CFTR was detected
by SDS-polyacrylamide gel electrophoresis followed by silver staining.
These fractions were pooled and concentrated, and CFTR was quantitated
by an enzyme-linked immunosorbent assay (for details, see Ref. 14). In
cases in which ion exchange chromatography was used, a 50-ml column of DEAE-Sepharose was equilibrated with 10 mM
K2HPO4,
10% glycerol, and 0.05%
-lysophosphatidylcholine, pH
7.5. Stripped membranes (100 mg) were solubilized in the above buffer,
and the resulting 100,000-g
supernatant was applied to the column. CFTR bound to DEAE-Sepharose
under these conditions. After extensive washing of the resin with
equilibration buffer, a linear gradient of 10-150 mM
K2HPO4
was applied. CFTR eluted from the column at ~60-85 mM K2HPO4
(14).
Assessment of CFTR purity.
Purified CFTR obtained from insect Sf9 cells was subjected to
4-20% SDS gradient gel electrophoresis to identify potential protein contaminants ranging in size from <1 to 300 kDa.
Electrophoresis was stopped before the bromophenol blue dye front
reached the bottom of the gel. The gel was then overstained with silver
to enhance sensitivity. Molecular size was estimated using markers obtained from GIBCO BRL (Grand Island, NY).
Detection of CFTR by immunoblot analysis.
Mouse mammary carcinoma (C127I) cells transfected with human epithelial
CFTR (WT1 cells) were harvested by washing with ice-cold Ca2+-free PBS (5 ml), scraped with
a rubber policeman, centrifuged, and resuspended in ice-cold lysis
buffer [containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris · HCl, pH 7.5, 1 mM ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 mM EDTA, 0.25 mM sodium vanadate, 10 µg/ml
phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin] and then
sonicated. Purified CFTR from Sf9 cells was prepared as indicated
above. Protein determination was performed using the Bio-Rad assay
(Bio-Rad, Richmond, CA). Briefly, each sample was mixed with dilution
buffer (final concentrations 62.5 mM Tris · HCl, pH
6.5, 2% SDS, 10% glycerol, and 50 mM DTT) and heated for 5 min at
60°C. Proteins were separated on a 7.5% SDS-polyacrylamide gel and
then transferred to polyvinylidene difluoride membranes prewetted with
methanol and soaked in transfer buffer for 15 min. Membranes were
blocked with 5% dried milk and 0.5% nonimmune goat serum in TBST (50 mM Tris · HCl, pH 7.6, 141 mM NaCl, and 0.2% Tween
20) for 1 h. Blots were probed with one of two CFTR MAb (0.5-1
µg/ml; MAb 13-1 or MAb 24-1, Genzyme) in TBST containing 1% bovine
serum albumin for 36-48 h at 4°C. After three washings with
TBST, membranes were incubated with peroxidase-labeled goat anti-mouse
antibody (Sigma) for 1 h at 25°C. Immunoreactive bands were
detected by enzyme-linked chemiluminescence (Kirkegaard and Perry,
Gaithersburg, MD).
Reconstitution of CFTR into phospholipid vesicles.
Immunoaffinity-purified CFTR was concentrated as described above and
reconstituted using a modification of the procedure described by Bear
et al. (3). An aliquot containing a known amount of CFTR (15-20
µg/ml) was added to 100 µl of a solution containing 15 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES; pH 7.4) and 0.5 mM EGTA and also containing 1 mg of a
sonicated phospholipid mixture of phosphatidylethanolamine,
phosphatidylserine, phosphatidylcholine, and ergosterol (5:2:1:2 molar
ratio) and 1% sodium cholate. After a 40-min incubation on ice, the
mixture was dialyzed at 4°C for 24 h against 15 mM HEPES, 0.5 mM
EGTA, pH 7.4, and 1.5% sodium cholate. Dialysis continued with daily changes of buffer for an additional 3 days against the same buffer without sodium cholate. The sample was further dialyzed against 15 mM
HEPES, 0.5 mM EGTA, and 150 mM sodium isethionate, pH 7.4, for 24 h.
The resulting proteoliposomes were quickly frozen at
80°C,
thawed, and sonicated for 5 s in a bath sonicator (Lab Supplies,
Hicksville, NY). In some cases, reconstitution of CFTR-containing proteoliposomes was conducted without freezing and thawing to avoid the
potential denaturing of the protein.
Planar lipid bilayer studies.
Purified CFTR from either transfected Sf9 or CHO cells was
reconstituted into a lipid bilayer reconstitution system that has been
previously used to assess CFTR-mediated
Cl
channel activity (3,
14). The reconstituted protein was either fused into the lipid bilayer
by reconstitution of proteoliposomes with techniques previously
described (3, 14) or applied directly to the lipid bilayer after
elution through a Sephadex G-50 (fine) column to remove denaturing SDS.
Reconstitution of CFTR under these conditions was conducted as follows.
The glass rod used for painting the lipid bilayer was "dipped"
consecutively in the CFTR-containing eluent and then into the lipid
mixture used for forming the bilayer. This CFTR-containing mixture was
used to break and repaint the lipid bilayer. To test the lack of
detergent contamination as a potential source of "spurious" ion
channel activity, equivalent samples of SDS were also eluted through a Sephadex G-50 column and/or incorporated into the bilayer as
indicated above. None of the eluate SDS fractions tested showed a
"nonspecific" leak conductance, either in the absence or presence
of PKA. Thus reconstituted CFTR channel activity was only correlated
with the presence of the protein and not with either contaminant SDS or protein degradation products that eluted before the protein.
Experiments were also conducted with membrane preparations of
CFTR-expressing WT1 cells (22) (data not shown). Either proteoliposomes
containing purified CFTR or CFTR mixed with lipid components was fused
to planar lipid bilayers by painting onto a 0.1-mm hole in a 13-mm polystyrene cuvette (Warner Instrument, Hamden, CT), as described by
Alvarez (2). The phospholipid composition of the lipid bilayers was
1-palmitoyl-2-oleyl-sn-glycero-3-phosphoethanolamine,
1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine, and
oleyl-sn-glycero-3-phosphatidylserine
(7:2:1, vol/vol/vol; Avanti Polar Lipids, Alabaster, AL) in
n-decane (Aldrich Chemical, Milwaukee,
WI) to a final concentration of 14, 6, and 0.5 mM, respectively. CFTR
eluted through Sephadex G-50 (fine) was collected at a final
concentration of 1.0 µg/ml in saline. The initial
cis and
trans solutions usually contained,
respectively, 10 mM 3-(N-morpholino)propanesulfonic acid
(MOPS)-20 µM
CaCl2 (pH 7.0) and 100 mM MgATP
(pH 7.0). However, the solutions varied for the different experiments
as indicated below.
Single-channel recordings of purified CFTR.
Input signals were acquired with a PC-501A patch/whole cell clamp
amplifier via a 10-G
head stage for lipid bilayers (Warner Instrument). The output currents were low-pass filtered at 500 Hz with
an eight-pole Bessel filter (Frequency Devices, Haverhill, MA),
digitized at 37 kHz and 14 bits with a VR-10B digital data recorder
(Instrutech, Great Neck, NY), and stored on a videocassette recorder
(JVC, Fairfield, NJ) until further analysis with pCLAMP 6.0.3 (Axon
Instruments, Foster City, CA). Single-channel tracings were
Gaussian-filtered at 100-200 Hz for display purposes, and analyzed
as per patch-clamp protocols. Unless otherwise stated, values of
n given in the text are the numbers of
individual current measurements and do not necessarily reflect the
numbers of experiments analyzed.
Calculation of the perm-selectivity ratio under asymmetrical
ATP/Cl
conditions.
To calculate the ATP-to-Cl
perm-selectivity ratio
(PATP/PCl)
under asymmetrical conditions
(ATP/Cl
),
single-channel conductances
[
= current
(I) divided by holding potential
(Vh)]
were best fitted to the Goldman-Hodgkin-Katz (GHK) equation, such that
where
i (species in
trans compartment) and
j (species in
cis compartment) represent
Cl
or ATP, depending on
their locations on either side of the membrane, Vh is in mV,
zi and
zj are the
charges for species i and
j, respectively, and
Ci and
Cj are the concentrations of
i and j, respectively.
Pi and
Pj represent the
permeability coefficients for the species
i or
j, respectively, and
= ziFVh/RT
and
= zjFVh/RT,
where F,
R, and
T are Faraday's constant, the gas
constant, and absolute temperature, respectively. All cases in which
MgATP was used were fitted with z =
2, and those in which Tris·ATP was used instead were fitted
with z =
4.
Drugs and chemicals.
ATP salts (MgATP and Tris·ATP; Sigma) were added from stock solutions
(100 mM) in distilled water. The catalytic subunit of the adenosine
3',5'-cyclic monophosphate (cAMP)-dependent protein kinase
(PKA; Sigma) was used at final concentrations ranging from 75 to 252 nM. The Cl
channel blocker
DPC (Fluka Chemical, Ronkonkoma, NY) was kept in a 100-fold stock
solution (20 mM) in 50% water-ethanol.
4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (Sigma)
and glibenclamide (RBI, Natick, MA) were kept in 10 mM stock solutions
in 100% dimethyl sulfoxide. The MAb raised against amino acids
729-736 of the regulatory domain (R-domain) of CFTR
(MAb 13-1, Genzyme) was directly diluted 1:100 in the intracellular or
bathing solution from a stock solution (292 µg/ml). An inactive
antibody was obtained by preheating the CFTR antibody for 30 min at
100°C.
 |
RESULTS |
Cl
and ATP currents of highly
purified human epithelial CFTR.
To assess the role of CFTR in electrodiffusional ATP transfer, human
epithelial CFTR was expressed and purified from either Sf9 insect or
mammalian CHO cells (14) and functionally reconstituted into a lipid
bilayer system to measure ion channel activity as reported previously
(3, 14). The summarized data of the various experimental conditions
and preparations used are shown in Table 1. Purified
material from either CHO (data not shown) or Sf9 cells (Fig.
1) (14) was
reconstituted in a chamber containing either 50 mM KCl and 100 mM MgATP
(data not shown) or 75 mM MgCl2 and 100 mM Tris·ATP (Fig.
2A) in the
cis and
trans compartments, respectively. Both
compartments contained 10 mM MOPS, pH 7.4, and the
cis compartment also contained 200 nM
PKA. Eight of nine experiments in KCl/MgATP and seven of nine
experiments in
MgCl2/Tris · ATP
displayed anion-selective channel activity after PKA addition from the
cis side of the chamber. Under the
latter conditions, currents were observed in both directions (Fig.
2A), with at least two distinct
(discernible) single-channel conductances of 8.57 ± 0.16 pS
(n = 10) and 34.3 ± 0.32 pS
(n = 7), respectively (Fig. 2B). The reversal potentials
(Er ) were
2.17 ± 1.83 mV (n = 10) and
0.35 ± 0.29 mV (n = 7) and
were thus consistent with a
PATP/P Cl of 0.1 and 0.2, for the smaller and
larger conductances, respectively. These values are in close agreement
with the
PATP/PCl
values previously obtained from whole cell and single-channel currents
of CFTR-expressing cells and calculated from the respective
conductances with symmetrical concentrations of the two ions, in the
range of 0.2-0.4 (22). A further indication that the CFTR molecule
itself may be implicated in ATP transport is suggested by the
observation that addition of the active, but not the heat-inactivated,
anti-CFTR R-domain antibody 13-1 (Genzyme) inhibited
simultaneously and almost completely both
Cl
and ATP currents
of highly purified CFTR (Fig. 2C) in
five of five experiments tested. These data further suggest that CFTR incorporation into the lipid reconstitution chamber was indeed functional and oriented as expected, with the PKA activation sites on
the cis side of the membrane.

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Fig. 1.
Purity of cystic fibrosis transmembrane conductance regulator (CFTR) in
reconstituted preparations. A:
purified CFTR material from Sf9 cells, obtained as indicated in
MATERIALS AND METHODS and used in
reconstitution studies, was subjected to silver staining following
electrophoresis on a 4-20% gradient SDS-polyacrylamide gel. Dye
front was preserved to retain low-molecular-mass proteins. Presence of
a band complex running at ~140 kDa was observed for 0.7 µg
(lane 1) and 0.3 µg
(lane 2) purified CFTR. This is in close
agreement with previously reported data (14).
Left lane, molecular mass markers.
B: immunolabeling of CFTR was
conducted with monoclonal antibody (MAb) 13-1 against CFTR on either
purified material from Sf9 cells or membranes of either subconfluent or
fully confluent CFTR-expressing WT1 cells. Top arrow
indicates fully "mature" (i.e., glycosylated) CFTR.
Lower arrow indicates nonglycosylated CFTR. Higher molecular
mass CFTR complexes were also observed in Sf9 material.
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Fig. 2.
Cl and ATP currents through
purified human epithelial CFTR. A:
purified CFTR was reconstituted into a lipid bilayer chamber containing
10 mM MOPS, pH 7.4, 5.0 mM MgATP, and 75 mM
MgCl2 in
cis side of chamber;
trans side contained 100 mM Tris·ATP
and same buffer composition. CFTR was always incorporated from
cis chamber, which also contained
protein kinase A (PKA; 200 nM). Holding potentials
(Vh), as
indicated at top right of each tracing, were driven
from trans chamber. c, Closed state.
B: current-voltage
(I-V)
relationships of most common conductance states of channels observed in
presence of 75 mM MgCl2 and 100 mM
Tris·ATP. Both Cl and ATP
were readily permeable under these conditions. Solid lines, best fits
to experimental points using Goldman-Hodgkin-Katz (GHK) equation.
Dashed lines, linear fits of experimental data, from the slopes of
which single-channel conductances ( ) were calculated (see Table 2).
Smaller conductance represented 25% of larger one, and thus at least
four levels could be recognized, in particular at 40 mV.
C: both
Cl (positive potentials;
left) and ATP currents (negative
potentials; right) were inhibited by
addition to cis side of monoclonal
antibody (MAb) 13-1 against regulatory domain of human epithelial CFTR.
After antibody addition, open probability of single channels changed
from 0.8 to 0.5 for positive potentials
(left) and from 0.15 to 0.02 for
negative potentials (right).
Histograms beside tracings indicate increment in
frequency of closed state (indicated with "c" and arrow) after
antibody addition. Channels observed displayed characteristics
remarkably similar to previously published data showing CFTR-associated
channel activity under cell-attached conditions (9).
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Single-channel currents seemed to correspond to the simultaneous gating
of smaller conductance states of the channel (in this case, four; Fig.
3A). Subconductance
states of the ATP currents of purified human epithelial CFTR were
observed (3 of 3 experiments) as indicated above, for currents driven
by 100 mM Tris·ATP. At least four identical current levels
could be recognized, although the channel gated as a complex of the
four levels. Expanded tracings indicated that channel levels "crept
up" (Fig. 3B) from seemingly even
smaller channel currents, which, as indicated in the third tracing,
were consistent with a conductance of 4.5-5.6 pS (see dashed
lines).

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Fig. 3.
Subconductance states of ATP currents of purified human epithelial
CFTR. A: purified CFTR was
reconstituted into a lipid bilayer chamber containing 10 mM MOPS, pH
7.4, 5.0 mM MgATP, and 75 mM MgCl2
in cis side of chamber;
trans side contained 100 mM Tris·ATP
and same buffer composition. CFTR was always incorporated from
cis chamber, which also contained PKA
(200 nM). Vh was
40 mV, thus indicating ATP currents. At least 4 identical
current levels could be recognized, although channel gated as a complex
of the 4 levels. B: expanded tracing
of that indicated in A by solid bar.
Channel levels "crept up" (asterisks) from seemingly even smaller
channel currents, which, as indicated in third tracing, were consistent
with a conductance of 4.5-5.6 pS (dashed lines). Data are
representative of 3 experiments.
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Effect of low ATP on the Cl
conductance of purified CFTR.
To further determine the role of
Cl
in the single-channel
permeability to ATP, experiments were also conducted in the presence of
low cis (5-10 mM) MgATP and high
N-methyl-D-glucamine chloride (300 mM) in the
trans compartment (Fig.
4A).
Both compartments also contained 10 mM MOPS, pH 7.4, and the
cis compartment contained 200 nM PKA.
Ion channel activity was observed with unitary conductances of 22.2 ± 1.87 pS (n = 11) and 54.9 ± 3.19 pS (n = 20; Fig.
4B). Interestingly, ion channel
activity was also observed at positive potentials, indicating a sizable
permeability to 10 mM MgATP (identical results were obtained either in
the absence of cis MOPS or by its
replacement with HEPES). The
Er values of
these two channel conductances were highly similar,
15.0 ± 3.54 mV (n = 11) and
18.4 ± 1.0 mV (n = 20, P < 0.3), indicating a
PATP/PCl
on the order of 9.1-12.9, as obtained when fitting the data with
the GHK equation (Fig. 4B). Under
similar conditions, a higher conductance state of CFTR, 511 ± 58.1 pS (n = 7), was also observed, albeit rarely, with similar
Er and
PATP/PCl
of
15.6 mV and 12.6, respectively. In the presence of 300 mM
cis
Cl
and only 6 mM
trans MgATP, with the ionic gradients
thus reversed, the single-channel conductance of purified CFTR was 26.6 ± 3.50 pS (n = 16), with an
Er of 16.2 ± 3.98 mV (n = 16) and a
PATP/PCl of 17. The data suggest that CFTR may be highly sensitive to external ATP, a phenomenon that will require further examination.

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Fig. 4.
Electrodiffusional Cl and
ATP movement through purified CFTR. A:
purified CFTR, incorporated from cis
chamber, was reconstituted into a lipid bilayer chamber containing, on
cis side, 10 mM MOPS, pH 7.4, 10 mM
MgATP, and 75 nM PKA; trans side of
chamber contained 300 mM N-methyl-D-glucamine
chloride (NMGCl) and same buffer composition.
Vh were driven
from trans chamber and kept at 150 mV
until channel activation was evident. Tracing is representative of
Cl channel activity
observed in 29 of 52 experiments. B:
I-V
relationship of 2 of the most common conductance states of channel.
Solid lines, fits of experimental values
(n = 4 different experiments) with
Goldman-Hodgkin-Katz equation. Dashed lines, best linear fits of
experimental data.
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ATP channel activity in the absence of
Cl
.
Single ATP channel activity of CFTR was also determined in the complete
absence of Cl
(Fig.
5). With
cis 10 mM MgATP and
trans 100 mM MgATP (and similar buffer
conditions as before), ATP channel currents were observed (Fig. 5,
A and
B), with single-channel conductance
of 32.4 ± 3.10 pS (n = 33; Fig.
5C). Addition of
cis
Cl
, however, reduced the
conductance of the ATP channel currents (Fig.
5D). Occasionally, a channel
conductance of 235 ± 40.8 pS (n = 5) was also observed. The
Er ranged from
10.5 ± 2.30 mV (n = 33) to
6.3 ± 1.4 mV (n = 5), which
was statistically different from the theoretical
Er for
MgATP2
,
(RT/zF) · ln([ATP]cis/[ATP]trans) = 29.1 mV (where
[ATP]cis and
[ATP]trans are the
cis and trans concentrations of ATP,
respectively; see Fig. 5C,
bottom). Thus it is possible either
that ATP4
(theoretical
Er = 14 mV) makes
a contribution to the ionic conductance of CFTR in the presence of
MgATP or that CFTR is also permeable to
Mg2+. In either case, the CFTR
preparation was permeable to ATP, which was further determined by
replacing ATP salts. In the presence of
trans 100 mM Tris·ATP
(cis 10 mM MgATP), the single ATP
channel conductance was 38.3 ± 6.1 pS
(n = 13), similar to that seen in the
presence of MgATP. The
Er shifted,
however, to
52.6 ± 14.0 mV
(n = 13), consistent with a strong
difference in the permeabilities of both free and
Mg2+-complexed ATP. These findings
indicate that, although CFTR is capable of
Cl
permeation, its ability
to preferentially move Cl
or ATP may depend not only on the driving force for the intracellular nucleotide ATP but also on the ATP species, the
PATP/PCl,
and the intracellular Cl
concentration. Under physiological intracellular conditions (5 mM ATP
and 30 mM Cl
), ATP
transport may be favored, however, as the electrochemical gradient for
the multivalent ATP will be 1,000-fold higher than that for
Cl
(18, 22).

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Fig. 5.
Electrodiffusional ATP movement through purified CFTR in absence of
Cl .
A: purified CFTR, incorporated from
cis chamber, was reconstituted into a
lipid bilayer chamber containing, on
cis side, 10 mM MOPS, pH 7.4, 10 mM
MgATP, and 75 nM PKA and, on trans
side, 100 mM MgATP, with same buffer composition.
Vh were driven
from trans chamber and kept at 150 mV
until channel activation was evident. Channel activity was also present
at positive Vh,
thus indicating ATP currents from 10 mM MgATP. Tracing at
bottom left shows expansion of segment marked
with a bold line in tracing at top.
Histogram on bottom right corresponds to expanded section.
Tracings are representative of 16 of 25 experiments.
B: purified CFTR single ATP channels
often displayed subconductance states (compare
a and
b,
middle and
bottom). N/div indicates the number
of 128-sample point bins per division.
C,
top left:
I-V
relationships of single-channel currents in asymmetrical MgATP. Two
distinct main conductance states were observed under this condition.
Solid lines, fits of experimental data to GHK equation. Dashed lines,
best linear fits to experimental points with slopes as indicated.
C, top
right: replacement of
trans MgATP with an equimolar
concentration of Tris·ATP shifted
Er but not value
of single-channel conductance, indicating that both free and
Mg2+-complexed ATP may have
different permeabilities (7 of 7 experiments).
C,
bottom:
I-V
relationship is an expansion of C,
top left, to more clearly indicate
predicted Er for
z = 2
(right),
z = 3
(middle), and
z = 4
(left) obtained from fitting with
GHK equation of experimental data using asymmetrical MgATP (10 vs. 100 mM) for larger (235 pS) and smaller (32.4 pS) conductances.
D: in presence of 10 mM
cis MgATP and 100 mM
trans MgATP, further addition of
cis NMGCl (50 mM) reduced
single-channel currents. Change is shown for 2 channels
(top and
middle) whose main conductance state
was defined as current amplitude at which channel closed
(bottom). Single-channel opening
(bottom left,
a) decreased its main conductance
state from 25 pS to 11.7 pS (bottom right,
b) after addition of
cis
Cl . Data are representative
of 2 experiments.
|
|
 |
DISCUSSION |
The expression of CFTR has been recently associated with the otherwise
unapparent permeation of ATP. This phenomenon has been observed by the
cAMP-induced release of ATP from CFTR-expressing cells under
physiological conditions (18), as well as by electrophysiological studies under highly nonphysiological conditions (22), which determined, nevertheless, the channellike nature of the PKA-induced ATP
movement at the single-molecule level (17, 22, 23). This is in
agreement with our observation that cAMP activation is associated with
extrusion of ATP in wild-type (18) but not in
508 CFTR-expressing
cells (8). The molecular steps associated with ATP movement in
CFTR-expressing cells, however, have not been unequivocally determined.
Although the simplest possible explanation for these findings is to
implicate CFTR itself as the channel responsible for the ATP-permeable
pathway, direct evidence to support this claim is still lacking.
ATP-permeable channels in CFTR-expressing cells may be a reflection of
endogenous functional channel proteins that are regulated by, but are
distinct from, CFTR. For example, expression of both mammalian (1) and
Drosophila melanogaster (5)
P-glycoproteins, CFTR congeners of the ATP-binding cassette (ABC)
family of transport proteins, has been associated with the expression
of ATP channel activity, thus raising the possibility that transport
proteins other than CFTR are present in certain preparations. At least one recent report demonstrated an osmotically stimulated
electrodiffusional ATP movement in human hepatoma cells seemingly
devoid of CFTR (28). A direct role of CFTR in ATP movement has also
been questioned by recent reports that failed to detect ATP-permeable
channel activity in cells or preparations containing CFTR (13, 21). However, these studies also failed to detect "any" other ATP
channel activity, which is inconsistent with the ATP release associated with CFTR expression (18, 23). This raises the possibility that the
presence of putative ATP-permeable pathways in CFTR-expressing preparations may largely depend on the cellular (tissue) model under
study and/or the nature of the ATP complexes driven. Alternate possibilities, however, may also entail more critical experimental conditions for determining ATP movement than previously expected. A
previous study that failed to detect ATP release from CFTR-expressing cells (27), for example, may have simply failed to meet the technological parameters suitable for detecting the expected amounts of
ATP released to the extracellular milieu (18). Furthermore, the
possibility that under certain conditions, electroneutral ATP complexes
are driven through channel structures cannot yet be ruled out.
In this report, we revisited the question as to whether CFTR is
responsible for ATP movement. Reconstituted CFTR displayed ATP channel
activity either in the absence or presence of
Cl
. Several (including
large) conductance states were also observed (see summary of results in
Table 2), in agreement with our original in
vivo observations (9, 22). In those studies, both a 5-pS and a 50-pS
ATP channel were observed. At least two other groups have observed
single-channel ATP currents associated with CFTR expression with a
similar "low" (5-pS) conductance (16, 17, 23). The issue of the
single ATP channel conductance of CFTR may be confounded by the
observation that both Cl
and ATP modify each other's movement, thus changing the single-channel conductance of the ions in either direction (22). In the presence of
extracellular 100 mM MgATP and 140 mM intracellular
Cl
, the single ATP channel
conductances rectified with a limiting conductance of 24.8 pS (Fig. 9 of Ref. 22), which is different from the observed single-channel
conductance for either ion alone (22). Furthermore, a recent study
also detected Cl
channel rectification by CFTR, which is a striking departure from the
established paradigm of CFTR behavior (15). This report also summarizes
previous data from the literature indicating that single-channel
conductances from 7 to 13 pS have been previously attributed to CFTR
(Ref. 15, see references within).
In the present report, addition of cis
Cl
(N-methyl-D-glucamine chloride, 50 mM) in the
presence of 10 mM MgATP reduced the CFTR single-channel conductance
from 25 to 11.7 pS (Fig. 5D), a
condition similar to previously reported values for CFTR-associated Cl
movement (13). This
finding further suggests that both
Cl
and ATP may compete with
each other for the conductance pore of the channel, as was originally
reported in intact cells (22).
Reconstituted CFTR ATP channel activity was readily inhibited by the
anti R-domain CFTR antibody MAb 13-1 (Genzyme), in agreement with
previous reports in intact cells (19). The possibility that a
functional CFTR is required to regulate ATP channels other than CFTR,
however, cannot be easily ruled out, since, as indicated above, recent
studies failed to detect any ATP-permeable channel activity in
preparations containing CFTR (13, 21). At least one such study was
conducted with a purified CFTR preparation similar to the one presented
in this report (13). Thus a likely possibility is that activated CFTR,
which is indeed permeable to
Cl
, may behave as an
ATP-selective ion channel only under certain circumstances (see below).
It should also be noted that these reports did not exactly mimic our
present or previous studies. We have found that PKA activation of CFTR,
for example, was readily observed (15 of 18 experiments) in experiments
tested with daily fresh batches of PKA. However, under certain
experimental conditions, namely the use of "older" PKA (prepared
>24 h before the experiments), CFTR activation under
Cl
-free conditions also
required the presence of sustained holding potentials (between +100 and
+150 mV; 41 of 82 experiments). This finding may actually explain
previous failed attempts to obtain single ATP channel currents from
reconstituted CFTR in which PKA was absent (21) and further
suggests the possibility that adjacent structures other than CFTR may
also be required for proper channel activation in vivo (19).
Although the reason for the required voltage activation of CFTR in the
presence of PKA is as yet unknown, it is not unprecedented for ATP
transport by other ABC transporters. Concerning the electrodiffusional ATP movement by the murine mdr1 gene
product, P-glycoprotein (1), for example, it was observed
that a double mutation of this channel protein in either
nucleotide-binding domain rendered the mutated P-glycoprotein
completely unable to spontaneously release cellular ATP. However,
voltage activation reestablished ATP transport and, interestingly,
Cl
channel activity
associated with this protein as well (1). Similar findings have also
been recently observed with the D. melanogaster mdr gene
products Mdr65 and Mdr49 P-glycoprotein homologues, which were also
voltage activated (5). Although it is unlikely that this activation
process has any physiological significance, it raises the interesting
possibility that other relevant intracellular factors that are absent
in the purified CFTR preparation may be required for a proper PKA
activation of CFTR. Under no circumstances, however, did voltage
stimulation induce any ATP-permeable channel activity in the absence of
PKA.
The present data also dispel a common misconception about the
size of ATP as it is transported through this channel molecule. In
solution, ATP takes various conformations by interacting with counterions other than Mg2+ (11).
However, only one conformation has been observed for the
MgATP2
complex, the
relevant moiety associated with molecules that bind and/or
hydrolyze ATP (4, 11). The average planar diameter of the MgATP
complex under these conditions is <5 Å (see Fig. 6), at least similar to, if
not smaller than, the expected diameter of
I
, a CFTR-permeable anion
(for details, see Fig. 6). Thus ATP "size" is not a constraint to
movement through CFTR, provided that it is either free or complexed to
Mg2+. Counterion-ATP complexes of
larger sizes, however, may reflect different interactions with CFTR. A
recent paper (13), reporting that purified CFTR was tested and failed
to detect ATP transport, suggested that the
K2ATP salt used in that study
actually blocked Cl
movement through CFTR. Because K+
is almost nine times larger than
Mg2+, the larger
K2ATP moiety is likely to be a
blocking agent of CFTR channel activity. This is in agreement with an
expected, undetectable, 0.14-pS
K2ATP single-channel conductance
(assuming a monovalent salt as the minimum size) calculated by
extrapolating the single-channel conductance of 26.2 pS obtained in the
present study, as a function of the counterionic radius of the MgATP
complex.

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|
Fig. 6.
Models of free and Mg2+-complexed
ATP. Scaled color models of
Mg2+-complexed ATP
(MgATP2 ;
B and
D) and free ATP
(A and
C) were created with
Chem3DPro
molecular modeling system (version 3.0, 1990, Cambridge Scientific
Computing, Cambridge, MA). C atoms, solid purple; O atoms, solid red; P
atoms, striped red; H atoms, light blue. Mg atom is indicated as a
green striped ball at bottom left center of
B, and extreme
left of
D. A
and B show phosphate groups covered by
their respective oxygen atoms to left
of molecule. C and
D are 3-dimensional perspectives
indicating pyridine ring toward front of molecule. Molecule (of either
model) is largely oblong. Shorter diameter is drawn from steric
distance between N1 and N3 of pyridine ring (2.46 Å), and
longer diameter is drawn from C2 and P17 (5.19 Å). Thus
phosphates "encircle" adenine moiety (using ribose as the hinge),
such that their position is coordinated not only by nucleoside but also
by their surrounding oxygens. In comparison, N1-N3 distance in
MgATP2 was found to be
identical to that of free ATP. However, arrangement of phosphates
surrounding adenine moiety was completely different, as distance
between C4 and O25, which is 0.719 Å in
ATP4 , becomes 6.86 Å in MgATP2 . In
addition, distance between C2 and P17, 5.19 Å in free ATP,
becomes 6.18 Å (C2-P18) in
MgATP2 , thus suggesting
that, although both models have similar diameters, MgATP seems to be
"longer" than free ATP. Both structures were "minimized"
for structural errors but not by their minimal energy conformations.
Furthermore, it is also important to realize that ether bonds for
either model were computed as 1.0, despite the fact that bond orders
for high-energy phosphates are significantly shorter (on the order of
0.36-0.41), thus suggesting much smaller 3-dimensional structures
(20). Model of free ATP, however, is almost identical (although without
structural minimization) to that of Stryer's Biochemistry
(Ref. 25, p. 329), although other possible conformations for
MgATP2 (Ref. 25, p. 352)
were not explored in above molecules.
|
|
Reconstituted CFTR mediated the electrodiffusional movement of both
Cl
and ATP and displayed
several conductance states, which may be related to "clustering"
of CFTR in complexes that have the same perm-selectivity properties.
Each ion seems to modify the permeability properties of the other,
which may be due to either competition for one another at the
conductance pore of the channel or the possibility that both ions have
distinct, but mutually interactive, permeable pathways. This is a
phenomenon that will require further investigation. Interestingly,
however, both the cis concentration of
Cl
and the
trans concentration of ATP modified
the single-channel conductance of CFTR.
In conclusion, the present data are consistent with the most likely,
but not exclusive, possibility that CFTR permeates ATP. This is in
close agreement with previous reports indicating that wild-type CFTR
(9, 17, 22, 23), but not
508 CFTR (8), may be involved in ATP
movement in vivo. "Contaminant" channels that are permeable
to ATP but are distinct from CFTR cannot be ruled out, although the
finding that preparations with different degrees of purification of
CFTR, including the purified protein, show functional characteristics
similar if not identical to those observed in vivo strongly suggest a
direct role of this channel protein in ATP movement. Additional
studies, including mutational analysis of this channel protein, will be
required, however, to further assess the relevance of the CFTR-mediated
ATP transport in the context of
Cl
vs. ATP transport and,
most importantly, in the onset and reversal of the CF phenotype.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Dr. Yi Wang for assistance in the
analysis of the structural models of ATP.
 |
FOOTNOTES |
Address for reprint requests: H. F. Cantiello, Renal Unit,
Massachusetts General Hospital East, 149 13th St., Charlestown, MA
02129.
Received 16 May 1997; accepted in final form 26 November 1997.
 |
REFERENCES |
1.
Abraham, E. H.,
A. G. Prat,
L. Gerweck,
T. Seneveratne,
R. J. Arceci,
R. Kramer,
G. Guidotti,
and
H. F. Cantiello.
The multidrug resistance (mdr1) gene product functions as an ATP channel.
Proc. Natl. Acad. Sci. USA
90:
312-316,
1993[Abstract].
2.
Alvarez, O.
How to set up a bilayer system.
In: Ion Channel Reconstitution, edited by C. Miller. New York: Plenum, 1986, p. 115-130.
3.
Bear, C. E.,
C. Li,
N. Kartner,
R. J. Bridges,
T. J. Jensen,
M. Ramjeesingh,
and
J. R. Riordan.
Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR).
Cell
68:
809-818,
1992[Medline].
4.
Berry, M. B.,
B. Meador,
T. Bilderback,
P. Liang,
M. Glaser,
and
G. N. Phillips, Jr.
The closed conformation of a highly flexible protein: the structure of E. coli adenylate kinase with bound AMP and AMPPNP.
Proteins
19:
183-198,
1994[Medline].
5.
Bosch, I.,
G. R. Jackson, Jr.,
J. M. Croop,
and
H. F. Cantiello.
Expression of Drosophila melanogaster P-glycoproteins is associated with ATP channel activity.
Am. J. Physiol.
271 (Cell Physiol. 40):
C1527-C1538,
1996[Abstract/Free Full Text].
6.
Boucher, R. C.,
C. U. Cotton,
J. T. Gatzy,
M. R. Knowles,
and
J. R. Yankaskas.
Evidence for reduced Cl
and increased Na+ permeability in cystic fibrosis human primary cell cultures.
J. Physiol. (Lond.)
405:
77-103,
1988[Abstract].
7.
Boucher, R.,
M. Knowles,
C. Cotton,
M. Jackson Stutts,
J. Yankaskas,
and
J. Gatzy.
On a unified theory of cystic fibrosis lung disease.
In: Cystic Fibrosis: Horizons, edited by D. Lawson. Brighton, UK: Wiley, 1984, p. 167-177.
8.
Cantiello, H. F.
Nucleotide transport through the cystic fibrosis transmembrane conductance regulator.
Biosci. Rep.
17:
147-171,
1997[Medline].
9.
Cantiello, H. F.,
A. G. Prat,
I. L. Reisin,
E. H. Abraham,
L. B. Ercole,
J. F. Amara,
R. J. Gregory,
and
D. A. Ausiello.
External ATP activates the cystic fibrosis transmembrane conductance regulator.
J. Biol. Chem.
269:
11224-11232,
1994[Abstract/Free Full Text].
10.
Carlstedt-Duke, J.,
M. Bronnegard,
and
B. Strandvik.
Pathological regulation of arachidonic acid release in cystic fibrosis: the putative basic defect.
Proc. Natl. Acad. Sci. USA
83:
9202-9206,
1986[Abstract].
11.
Cohn, M.
Structural and chemical properties of ATP and its metal complexes in solution.
In: Biological Actions of Extracellular ATP, edited by G. R. Dubyak,
and J. S. Fedan. New York: NY Acad. Sci., 1990, p. 151-164.
12.
Egan, M.,
T. Flotte,
S. Afione,
R. Solow,
P. L. Zeitlin,
B. J. Carter,
and
W. B. Guggino.
Defective regulation of outwardly rectifying Cl
channels by protein kinase A corrected by insertion of CFTR.
Nature
358:
531-536,
1992[Medline].
13.
Li, C.,
M. Ramjeesingh,
and
C. Bear.
Purified cystic fibrosis transmembrane conductance regulator (CFTR) does not function as an ATP channel.
J. Biol. Chem.
271:
11623-11626,
1996[Abstract/Free Full Text].
14.
O'Riordan, C.,
A. Erickson,
C. Bear,
C. Li,
P. Manavalan,
K. Wang,
J. Marshall,
R. Scheule,
J. McPherson,
S. Cheng,
and
A. Smith.
Purification and characterization of recombinant cystic fibrosis transmembrane conductance regulator from Chinese hamster ovary and insect cells.
J. Biol. Chem.
270:
17033-17043,
1995[Abstract/Free Full Text].
15.
Overholt, J.,
A. Saulino,
M. Drumm,
and
R. Harvey.
Rectification of whole cell cystic fibrosis transmembrane conductance regulator chloride current.
Am. J. Physiol.
268 (Cell Physiol. 37):
C636-C646,
1995[Abstract/Free Full Text].
16.
Pasyk, E. A.,
and
J. K. Foskett.
Mutant (delta F508) cystic fibrosis transmembrane conductance regulator Cl
channel is functional when retained in endoplasmic reticulum of mammalian cells.
J. Biol. Chem.
270:
12347-12350,
1995[Abstract/Free Full Text].
17.
Pasyk, E. A.,
and
J. K. Foskett.
Cystic fibrosis transmembrane conductance regulator-associated ATP and adenosine 3'-phosphate 5'-phosphosulfate channels in endoplasmic reticulum and plasma membranes.
J. Biol. Chem.
272:
7746-7751,
1997[Abstract/Free Full Text].
18.
Prat, A. G.,
I. L. Reisin,
D. A. Ausiello,
and
H. F. Cantiello.
Cellular ATP release by the cystic fibrosis transmembrane conductance regulator.
Am. J. Physiol.
270 (Cell Physiol. 39):
C538-C545,
1996[Abstract/Free Full Text].
19.
Prat, A. G.,
Y.-F. Xiao,
D. A. Ausiello,
and
H. F. Cantiello.
cAMP-independent regulation of CFTR by the actin cytoskeleton.
Am. J. Physiol.
268 (Cell Physiol. 37):
C1552-C1561,
1995[Abstract/Free Full Text].
20.
Pullman, B.,
and
A. Pullman.
Quantum Biochemistry. New York: Interscience, 1963, p. 367.
21.
Reddy, M.,
P. Quinton,
C. Haws,
J. Wine,
R. Grygorczyk,
J. Tabcharani,
J. Hanrahan,
K. Gunderson,
and
R. Kopito.
Failure of the cystic fibrosis transmembrane conductance regulator to conduct ATP.
Science
271:
1876-1879,
1996[Abstract].
22.
Reisin, I. L.,
A. G. Prat,
E. H. Abraham,
J. F. Amara,
R. J. Gregory,
D. A. Ausiello,
and
H. F. Cantiello.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a dual ATP and chloride channel.
J. Biol. Chem.
269:
20584-20591,
1994[Abstract/Free Full Text].
23.
Schwiebert, E.,
M. Egan,
T.-H. Hwang,
S. Fulmer,
S. Allen,
G. Cutting,
and
W. Guggino.
CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.
Cell
81:
1063-1073,
1995[Medline].
24.
Strandvik, B.,
M. Bronnegard,
H. Gilljam,
and
J. Carlstedt-Duke.
Relation between defective regulation of arachidonic acid release and symptoms in cystic fibrosis.
Scand. J. Gastroenterol. Suppl.
143:
1-4,
1988.
25.
Stryer, L.
Biochemistry. New York: Freeman, 1988.
26.
Stutts, M. J.,
C. M. Canessa,
J. C. Olsen,
M. Hamrick,
J. A. Cohn,
B. C. Rossier,
and
R. C. Boucher.
CFTR as a cAMP-dependent regulator of sodium channels.
Science
269:
847-850,
1995[Medline].
27.
Takahashi, T.,
K. Matsushita,
M. J. Welsh,
and
J. B. Stokes.
Effect of cAMP on intracellular and extracellular ATP content of Cl
-secreting epithelia and 3T3 fibroblasts.
J. Biol. Chem.
269:
17853-17857,
1994[Abstract/Free Full Text].
28.
Wang, Y.,
R. Roman,
S. D. Lidofsky,
and
J. G. Fitz.
Autocrine signaling through ATP release represents a novel mechanism for cell volume regulation.
Proc. Natl. Acad. Sci. USA
93:
12020-12025,
1996[Abstract/Free Full Text].
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