From the The function of the human cystic fibrosis
transmembrane conductance regulator (CFTR) protein as a chloride
channel or transport regulator involves cellular ATP binding and
cleavage. Here we describe that human CFTR expressed in insect
(Sf9) cell membranes shows specific,
Mg2+-dependent nucleotide occlusion,
detected by covalent labeling with 8-azido-[ Mutations in the cystic fibrosis transmembrane conductance
regulator (CFTR)1 protein
result in the development of cystic fibrosis, the most frequent lethal
hereditary disease among Caucasians (1, 2). CFTR belongs to the
superfamily of the ATP-binding cassette (ABC) transporters and resides
in the apical plasma membrane of epithelial cells. The CFTR protein
contains a tandem repeat of transmembrane domains and conserved
nucleotide binding domain (NBD) motifs, connected by a large linker
region, the so called regulatory (R) domain (1, 3, 4). In contrast to
several homologous proteins that work as ATP-dependent
pumps, CFTR forms chloride channels and modulates the function of other
membrane transport proteins (4-8). The function of CFTR is regulated
by protein kinase-dependent phosphorylation, as well as by
direct ATP binding and/or hydrolysis (9-13).
It has been shown that both NBDs of the CFTR can bind cellular ATP, and
although with a low rate, ATP is hydrolyzed by the CFTR protein (14,
15). In the ABC transporters working as pumps (e.g. MDR1,
MRP1, TAP), ATP hydrolysis and transport are inhibited by vanadate,
while vanadate was shown to stabilize the open state of the CFTR
chloride channel (16, 17). Covalent SH-group modification by
N-ethylmaleimide inhibits the ATP-dependent function of those ABC transporters, which have cysteine residues in the
NBDs, including MDR1 and CFTR (18-20).
It has been shown for several ABC transporters that in the presence of
vanadate they occlude (trap) an adenine nucleotide, and this trapped
nucleotide in the MDR1 protein was found to be exclusively ADP
(21-24). Experimentally this nucleotide occlusion can be followed by
incubating the membranes with 8-azido-[ There are numerous models to explain the functional characteristics and
the adenine nucleotide regulation of CFTR (10, 12, 13), while due to
the low expression level and strongly membrane-embedded nature of this
protein, direct biochemical data are relatively scarce in this regard.
Eukaryotic heterologous expression systems may help in overcoming such
difficulties, and isolated membranes of baculovirus-infected insect
cells were shown to be suitable for performing functional assays of
various ATP-dependent transport proteins (see Refs. 25, 28,
and 29). In the present experiments we have expressed the human CFTR in
a baculovirus-Sf9 (Spodoptera frugiperda) cell system
and characterized the adenine nucleotide occlusion in this protein in
isolated Sf9 cell membranes. As documented earlier, human CFTR
expressed in this system (see Refs. 28 and 30) is phosphorylated by
endogenous protein kinases and CFTR forms active chloride channels
without the need of additional protein kinase activation (31, 32).
Our present results suggest that the two NBDs of CFTR have different
nucleotide occlusion characteristics, and only the occlusion in the
C-terminal NBD2 requires vanadate. According to these data, although
the formation of an occluded nucleotide can occur in both NBDs, the
N-terminal NBD1 seems to have a rate-limiting role in the full ATP
hydrolysis cycle. These results may significantly help our
understanding of the molecular mechanism and regulation of the CFTR protein.
Chemicals--
8-Azido-[ hCFTR Expression and Membrane Preparation--
Sf9 cells
were cultured and infected with baculovirus vectors as described in
Ref. 33. Recombinant baculovirus for CFTR expression was obtained from
E. M. Price and R. C. Boucher, University of North Carolina,
Chapel Hill, NC, and the correct cDNA sequence was established by
resequencing with the help of Izabella Klein. Baculovirus Sf9
cell CFTR expression and chloride channel characterization were
described in Refs. 28 and 29. Sf9 cells were infected, membrane
fractions isolated, and the membrane protein concentrations determined
as described in Ref. 29. For the electrophoresis and immunoblotting of
CFTR, see Ref. 29. Quantitative estimation of the expression of human
CFTR was performed using polyclonal antibodies. Ab D4 (CFTR-EC1,
affinity-purified antipeptide antibody, kindly provided by Dr. Mary C. Rose, Children's National Medical Center, Washington, D. C.)
recognizes the first extracellular loop of human CFTR (34), while Ab 50 and Ab 858 react with the R-domain and the C terminus of CFTR,
respectively (28). As a secondary antibody, anti-rabbit,
peroxidase-conjugated IgG (10,000 × dilution, Jackson
ImmunoResearch) was used. Horseradish peroxidase-dependent luminescence (ECL, Amersham) was determined by luminography and quantitated by the Bio-Rad phosphoimager system.
Nucleotide Trapping--
Isolated Sf9 cell membranes
(100-200 µg of protein) were incubated for 2 min at 37 °C in a
Tris-KCl-EGTA reaction buffer (see Ref. 25), with or without 2 mM MgCl2 or 500 µM sodium
orthovanadate, in a final volume of 50 µl, in the presence of 5 µM final concentration of 8-azido-ATP, containing 0.2 MBq
of 8-azido-[ Limited Proteolysis--
Isolated Sf9 cell membranes
(100-200 µg of protein) were subjected to nucleotide occlusion as
described above, and the labeled nucleotides were covalently bound to
CFTR by UV irradiation. The samples were treated with different amounts
of trypsin (1, 5, or 10 µg/ml) for 1, 2, 5, or 10 min at 4 °C, in
a reaction buffer containing 40 mM MOPS-Tris, pH 7.0, 50 mM KCl, 0.5 mM EGTA, 2 mM
dithiothreitol. The reaction was stopped by the addition of excess
soybeen trypsin inhibitor, and the membranes were washed in a
Tris-KCl-EGTA reaction buffer as above, collected in the electrophoresis buffer, and run on 4-15% SDS-gradient gels (Bio-Rad). The proteins were electroblotted and subjected to autoradiography and
immunostaining as described above.
Fig. 1 demonstrates the expression
of CFTR in Sf9 cell membranes and the nucleotide occlusion of
this protein from 8-azido-[ In the experiments shown in Fig. 1, isolated Sf9 cell membranes
were incubated for 2 min at 37 °C with 5 µM
8-azido-[ As shown in Fig. 1A, a significant amount of 32P
labeling was observed in CFTR even after washing the membranes in high
ATP-containing cold buffers before photo cross-linking. Only a very
small amount of 8-azido-nucleotide occlusion could be observed in the
corresponding region of Fig. 2 demonstrates the effect of sodium
orthovanadate on 8-azido- National Institute of
Haematology and Immunology,
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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-32P]ATP.
Nucleotide occlusion in CFTR requires incubation at 37 °C, and the
occluded nucleotide can not be removed by repeated washings of the
membranes with cold MgATP-containing medium. By using limited tryptic
digestion of the labeled CFTR protein we found that the adenine
nucleotide occlusion preferentially occurred in the N-terminal
nucleotide binding domain (NBD). Addition of the ATPase inhibitor
vanadate, which stabilizes an open state of the CFTR chloride channel,
produced an increased nucleotide occlusion and resulted in the labeling
of both the N-terminal and C-terminal NBDs. Protein modification with
N-ethylmaleimide prevented both
vanadate-dependent and -independent nucleotide occlusion in
CFTR. The pattern of nucleotide occlusion indicates significant
differences in the ATP hydrolyzing activities of the two NBDs, which
may explain their different roles in the CFTR channel regulation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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-32P]ATP,
washing with high concentrations of MgATP, and separating the proteins
by SDS-polyacrylamide gel electrophoresis after UV light treatment. In
the case of MDR1, nucleotide occlusion is entirely
vanadate-dependent and
N-ethylmaleimide-sensitive (21, 22). The addition of
transported MDR1 drug substrates significantly increases the rate of
nucleotide trapping (25), thus nucleotide occlusion seems to reflect a
drug-stimulated partial reaction of the MDR1 ATPase catalytic cycle. In
this cycle only one nucleotide is occluded by one MDR1 protein (23),
although both NBDs have to be functional to obtain this partial
reaction (25-27). To our knowledge no experimental data are available
as yet for nucleotide occlusion in the CFTR protein.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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-32P]ATP (666 GBq/mM) was obtained from ICN Biomedicals.
-32P]ATP. The reaction was stopped by the
addition of 500 µl of ice-cold Tris-EGTA + 10 mM MgATP
(with or without 500 µM vanadate) buffer, the membranes
washed twice in the same ice-cold buffer, and the pellet resuspended in
20 µl of Tris-EGTA. The membranes were UV-irradiated at 4 °C after
these washing steps, collected in the electrophoresis buffer, and the
samples run on 6% Laemmli-type SDS-polyacrylamide gel electrophoresis
as described in Ref. 25. The proteins were electroblotted onto
polyvinylidene difluoride membranes, and the blots were dried and
subjected to autoradiography in a phosphoimager (Bio-Rad). The identity
of the 32P-azido-nucleotide-labeled bands was assured by
immunostaining on the same blot.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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-32P]ATP. As shown earlier
(28, 30), the CFTR protein expressed in insect cell membranes has an
underglycosylated form with an apparent molecular mass of 140 kDa. It
has been documented that CFTR expressed in Sf9 cells is
phosphorylated by endogenous protein kinases and produces active
chloride channels without further kinase action (see Refs. 31 and 32).
In parallel patch-clamp experiments we could reproduce the described
channel forming properties of the CFTR expressed in our
baculovirus-Sf9 cell
system.2
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Fig. 1.
Nucleotide trapping from
8-azido-[ -32P]ATP in the human
CFTR, expressed in Sf9 membranes. A,
autoradiogram of the isolated Sf9 cell membranes labeled with
8-azido-
-32P-nucleotide, followed by electrophoresis and
electroblotting; B, immunostaining of the same blot of the
isolated Sf9 cell membranes with the anti-CFTR antibody 858. Nucleotide trapping and covalent photoaffinity labeling were performed
as described under "Materials and Methods." Membranes expressing
-galactosidase (
-gal) (lane 6) or the human
CFTR (lanes 1-5) were incubated at 37 °C for 2 min in
the presence of 5 µM 8-azido-[
-32P]ATP.
The reaction buffers contained 2 mM MgCl2
(lanes 1 and 3-6) or 2 mM EDTA
(lane 2). In some experiments membranes were preincubated
with 200 µM N-ethylmaleimide (lane
5), 1 mM ATP (lane 3), or 1 mM
AMP (lane 4). The arrows indicate the position of
human CFTR on the blots.
-32P]ATP, in the presence of various
additions as indicated in the figure legend. The membranes were washed
twice in the dark in ice-cold solutions containing 10 mM
MgATP, and the collected membrane fragments were irradiated by UV light
after these washing steps. SDS-gel electrophoresis was applied to
separate the 32P-labeled bands (Fig. 1A), and
immunoblotting (Fig. 1B) was used to identify the CFTR protein.
-galactosidase expressing Sf9 cell
membranes, while a nonspecific labeling occurred in all membrane
preparations at about 120 kDa (see later). Nucleotide occlusion in CFTR
was abolished by excess (1 mM) MgATP, but not by 1 mM MgAMP. This occlusion required the presence of
Mg2+ (addition of EDTA blocked 32P labeling),
and preincubation of the isolated membranes with 200 µM
N-ethylmaleimide, a covalent SH-group reagent, strongly inhibited this reaction (see Fig. 1A). Nucleotide occlusion
also required at least 2 min of incubation at 37 °C (there was no
significant occlusion at 4 °C), while washings at 37 °C instead
of 4 °C resulted in the loss of labeling (data not shown). These
data indicate that the occluded nucleotide in CFTR is formed in a
Mg2+-dependent partial reaction of the
temperature-dependent ATPase cycle of this protein.
-32P-nucleotide occlusion in
the human CFTR and MDR1 proteins, expressed in Sf9 cell
membranes. As shown, nucleotide occlusion by CFTR was significantly
increased (about doubled) in the presence of vanadate. In the control
(
-galactosidase expressing) membranes there was very little labeling
in the molecular mass range corresponding to CFTR, either in the
presence or absence of vanadate. As mentioned above, in all Sf9
cell membrane preparations a vanadate-independent nucleotide occlusion
was observed in a protein band of about 120 kDa, and a
vanadate-dependent occlusion in a protein of about 180 kDa
(see Figs. 1 and 2). These bands may correspond to endogenous ATP
binding or hydrolyzing proteins in the Sf9 membranes, not detected by the human CFTR-specific antibodies. As shown in Fig. 2, it
is important to note that nucleotide trapping in MDR1 could only be
observed in the presence of vanadate (see also Refs. 23 and 25).
Preincubation with N-ethylmaleimide inhibited nucleotide occlusion in both the CFTR and MDR1 proteins (see Fig. 2).
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Fig. 2.
Effects of vanadate on nucleotide trapping
from 8-azido-[ -32P]ATP in the
human MDR1 and CFTR, expressed in Sf9 membranes.
Autoradiogram of the isolated Sf9 cell membranes labeled with
8-azido-
-32P-nucleotide. Nucleotide trapping and
covalent photoaffinity labeling were performed as described under
"Materials and Methods." Membranes expressing
-galactosidase
(
-gal) (lanes 1-3), human CFTR (lanes
4-6), or human MDR1 (lanes 7-9) were incubated at
37 °C for 2 min in the presence of 5 µM
8-azido-[
-32P]ATP and 2 mM
MgCl2. Additional reagents were 500 µM sodium
orthovanadate (lanes 2, 3, 5,
6, 8, and 9, indicated by "Va
+") or 200 µM N-ethylmaleimide
(lanes 3, 6, and 9). The
arrows indicate the position of human CFTR and human MDR1 on
the blots.
Both in CFTR and MDR1, vanadate-induced nucleotide occlusion required Mg2+ and was eliminated by 1 mM ATP and not by 1 mM AMP. Nucleotide occlusion in CFTR with or without sodium orthovanadate at 5 µM 8-azido-ATP only slightly increased when the incubation period at 37 °C was increased up to 10 min, and labeling showed an apparent saturation above 50 µM 8-azido-ATP (data not shown in detail).
Nucleotide occlusion in MDR1 reflects a Mg2+-dependent and drug substrate-stimulated partial reaction of the ATPase cycle, with the formation of a transitory state intermediate that is not exchangeable by ATP or ADP (21-23, 25). The absolute vanadate dependence of the nucleotide occlusion in MDR1 indicates that ATP hydrolysis by this protein is relatively fast, and only the full inhibiton of the ATPase cycle by this transitory state phosphate analog can stabilize a trapped nucleotide. As shown above, in contrast to MDR1, nucleotide occlusion in CFTR could be observed without vanadate, simply by cooling down the incubation medium after the formation of the occluded nucleotide at 37 °C. These results indicate that the overall rate of ATP hydrolysis by CFTR is much slower than by MDR1, but involves basically the same Mg2+-dependent transition state intermediate(s).
In the following experiments we have examined the formation of the occluded nucleotides in the two different nucleotide binding domains of CFTR. In order to separate these domains, we performed limited proteolysis of the CFTR in the Sf9 cell membranes after the nucleotide occlusion was achieved and the labeled nucleotides were covalently bound to CFTR by UV irradiation.
As shown in Fig. 3, in agreement with the
data in the literature (see Ref. 35), a properly executed limited
trypsin cleavage of CFTR yielded two major fragments. These represented
the C-terminal (about 65 kDa) and the N-terminal (about 40-45 kDa)
parts, each without the R-domain of CFTR. As identified by antibodies
recognizing either the N-terminal part, the C-terminal part, or the
R-domain of the CFTR, at shorter time intervals or lower trypsin
concentration, fragments of both the C-terminal (about 80 kDa) and the
N-terminal (about 65-75 kDa) halves, containing most of the R-domain,
also appeared on the blots. Further trypsinolysis resulted in the
formation of degraded C-terminal (about 35 kDa) and N-terminal (about
25 kDa) fragments as well (all these fragments are still slightly visible on the overexposed immunoblots shown in Fig.
3B).
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Fig. 3A demonstrates that when nucleotide occlusion was
performed in the presence of 5 µM
8-azido-[-32P]ATP and 2 mM
Mg2+, 32P labeling was observed only in the
N-terminal half of CFTR that is in the region of NBD1. In contrast,
when labeling and washings were performed in the presence of sodium
orthovanadate, both halves of the CFTR were labeled, that is the
C-terminal NBD(2) domain could also occlude adenine nucleotide. The
absence of free Mg2+ or the addition of
N-ethylmaleimide eliminated labeling in both NBDs, and there
were no corresponding labeled fragments in the
-galactosidase-expressing membranes (not shown).
In the case of MDR1, an active drug pump, it has been established that the protein can bind two ATP molecules, while only one occluded ADP is formed. In MDR1, ATP hydrolysis and vanadate-dependent nucleotide occlusion in each NBD occur randomly, in an alternating way, with a cooperative but equivalent role of the two NBDs (21-23, 25, 36).
The present experiments with CFTR suggest that the two NBDs of this protein have different catalytic properties. These studies do not allow as yet to estimate the number of occluded nucleotides or to establish their actual form (ATP or ADP). Still, they strongly indicate that in the presence of MgATP, the N-terminal NBD1 in the CFTR rapidly forms an occluded nucleotide, while the complete hydrolysis of ATP (with the release of ADP and inorganic phosphate) is a much slower process. In contrast, in the C-terminal NBD2 ATP hydrolysis and ADP release can be much faster, unless vanadate blocks this release by stabilizing the transition state of the enzyme.
In contrast to MDR1, the amino acid sequences in the two NBDs of the CFTR are strikingly different, and this may be the basis of their different ATP hydrolyzing properties. A recent report indicated differences in the nucleotide binding features of the two NBDs of another ABC transporter, the ATP-dependent K+ channel regulator SUR1 protein, and nucleotide occlusion by SUR1 could be observed even in the absence of Mg2+ and vanadate (37). It is tempting to speculate that channel-forming or -regulating ABC transporters allow nucleotide occlusion for the production of a functionally effective conformational change, but prevent unnecessary, rapid ATP hydrolysis.
Current models of CFTR function (13) indicate that the frequency of ATP
hydrolysis in the N-terminal NBD1 determines how often the chloride
channel opens, while the rate-limiting step in channel closing is the
ATP hydrolysis in the C-terminal NBD2. These steps may involve
consecutive or parallel reactions, and the co-operation of the two
NBDs, probably modified by protein kinase phosphorylation, may have a
key role (38, 39). Further nucleotide occlusion experiments, in
connection with channel or regulatory function studies by using mutant
CFTR proteins, should help in deciphering the molecular mechanism of
this clinically important protein.
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ACKNOWLEDGEMENTS |
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We are grateful for obtaining the wild type CFTR baculovirus from E. Price, J. M. Stutts, S. E. Gabriel, and R. C. Boucher, University of North Carolina, Chapel Hill, NC, as well as for their help in characterizing CFTR chloride channels in our baculovirus-infected Sf9 cells. We thank Izabella Klein for the help in resequencing hCFTR cDNA in the recombinant baculovirus and Dr. M. C. Rose and S. E. Earp for providing the purified antibodies. The technical help of Ilona Zombori is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported by research grants from OMFB, NWO-OTKA, FKFP, and ETT (Hungary).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.
¶ Howard Hughes International Research Scholar. To whom correspondence should be addressed: National Institute of Haematology and Immunology, 1113 Budapest, Daróczi u. 24, Hungary. Tel.:/Fax: 36-1-372-4353; E-mail: b.sarkadi{at}ohvi.hu.
2
S. E. Gabriel, M. J. Stutts, and T. Hegeds, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; ABC transporters, ATP-binding cassette transporters; MDR1, human multidrug resistance protein, P-glycoprotein; NBD, nucleotide binding domain; Sf9 cells, Spodoptera frugiperda ovarian cells; SUR1, sulfonylurea receptor protein; R, regulatory; MOPS, 4-morpholinepropanesulfonic acid.
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