From the S. C. Johnson Medical Research Center, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259
Received for publication, January 18, 2001
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ABSTRACT |
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After phosphorylation by protein kinase A, gating
of the cystic fibrosis transmembrane conductance regulator (CFTR)
chloride channel is regulated by the interaction of ATP with its
nucleotide binding domains (NBDs). Models of this gating regulation
have proposed that ATP hydrolysis at NBD1 and NBD2 may drive channel opening and closing, respectively (reviewed in Nagel, G. (1999) Biochim. Biophys. Acta 1461, 263-274). However, as yet
there has been little biochemical confirmation of the predictions of
these models. We have employed photoaffinity labeling with 8-azido-ATP, which supports channel gating as effectively as ATP to evaluate interactions with each NBD in intact membrane-bound CFTR. Mutagenesis of Walker A lysine residues crucial for azido-ATP hydrolysis to generate the azido-ADP that is trapped by vanadate indicated a greater
role of NBD1 than NBD2. Separation of the domains by limited trypsin
digestion and enrichment by immunoprecipitation confirmed greater and
more stable nucleotide trapping at NBD1. This asymmetry of the two
domains in interactions with nucleotides was reflected most
emphatically in the response to the nonhydrolyzable ATP analogue, 5'-adenylyl- The cystic fibrosis transmembrane conductance regulator
(CFTR)1 has two nucleotide
binding domains (NBDs) that are believed in some manner to regulate
permeation of chloride ions (1). Whereas the two NBDs of another ABC
protein, the P-glycoprotein multidrug transporter, are highly
homologous and believed to be functionally equivalent (2), the domains
in members of the ABCC subfamily to which CFTR belongs show less
sequence similarity (3). This asymmetry is reflected functionally in
two members of this subfamily, SUR1 (4, 5) and MRP1 (6-8). Both ATP
and ADP act on SUR1 to regulate the Kir6.2 potassium channel; ATP is
bound at NBD1 and also bound and hydrolyzed at NBD2, and the ADP
produced stabilizing ATP binding to NBD1 (9). Similarly in the case of
MRP1, which transports conjugated anions, ATP binding was detected
exclusively at NBD1 and was enhanced allosterically by the trapping of
ADP produced by hydrolysis at NBD2 (6).
Schemes of ATP binding and hydrolysis by CFTR have been proposed solely
on the basis of channel-gating responses (10-12). Implicit in these
models is the idea that channel opening is driven by ATP binding and
hydrolysis at one NBD and closing at the other (13). Intact CFTR (14)
and bacterial fusion proteins containing either of the NBDs (15, 16)
hydrolyze ATP. It has been reported also that photoaffinity labeling of
CFTR by 8-azido-ATP at NBD1 occurred in the absence of orthovanadate,
whereas NBD2 labeling required its presence (17). Here we show that
8-azido-ATP, which supports CFTR channel activity as well as ATP,
labels both NBDs in a vanadate-stimulatable manner indicating that
hydrolysis and trapping occurs at both domains.
Furthermore, the nonhydrolyzable ATP analogue AMP-PNP, which has been
relied upon heavily in the formulation of models of the action of the
NBDs in gating (18, 19), was found to have entirely opposite effects on
the two domains. Strong competitive inhibition of nucleotide trapping
at NBD1 without prevention of channel opening (1) makes it unlikely
that hydrolysis at NBD1 drives channel opening. Similarly stimulation
rather than inhibition of trapping at NBD2 does not support the idea
that AMP-PNP prevents channel closing by inhibition of hydrolysis there.
Materials--
BHK-21 cells stably expressing wild-type human
CFTR were prepared and maintained as described previously (20) as were
cells expressing the K464A and K1250A mutants. The mouse monoclonal antibodies, L12B4 and M3A7, used for immunoprecipitation and Western blotting, were those described by Kartner et al. (21). The
antibody MM13-4 recognizes an epitope between residues 24 and
35.2
8-Azido-[ Membrane Isolation--
CFTR-expressing BHK cells were
homogenized in an ice-cold hypotonic buffer containing 10 mM HEPES, pH 7.2, 1 mM EDTA, and a protease
inhibitor mixture consisting of leupeptin (1 µg/ml), aprotinin
(µg/ml), E64 (3.5 µg/ml), and benzamidine (120 µg/ml). Nuclei and
unbroken cells were removed by centrifugation for 15 min at 600 × g. Membranes were pelleted by centrifugation for 30 min at
100,000 × g and then resuspended in 40 mM
Tris-HCl, pH 7.4, 5 mM MgCl2, 0.1 mM EGTA.
Photoaffinity Labeling with
8-N3[ Limited Proteolysis and
Immunoprecipitation--
Photoaffinity-labeled membranes were
incubated with TPCK-treated trypsin for 15 min on ice at an enzyme to
membrane protein mass ratio of 1:180. Digestion was terminated with
excess soybean inhibitor, and membranes were solubilized in RIPA buffer
(50 mM Tris-HCl, pH 7.4, 1% deoxycholate, 1% Triton,
0.1% SDS, 150 mM NaCl) and immunoprecipitated with either
of the monoclonal antibodies L12B4 or M3A7 (21). The immunoprecipitates
were subjected to SDS-PAGE and transferred to nitrocellulose for
standard (x-ray film) and electronic (Packard Instant Imager) autoradiography.
Photolabeling of CFTR by
8-N3[ Influence of Walker A Lysine Mutagenesis and Domain-specific
Antibody Binding--
In P-glycoprotein (2) and MRP1 (6)
mutagenesis of the Walker A lysine residues in either NBD1 or NBD2
prevents ATP hydrolysis and hence trapping of
8-azido-[
As an additional indirect means of assessing the role of the two NBDs
in the photolabeling of the whole protein the influence of the binding
of different monoclonal antibodies was measured. L12B4, which binds
near the N terminus of NBD1 (21), reduced labeling to a very low level
again suggesting that much of the labeling occurred at NBD1. There was
a lesser reduction of labeling by M3A7 that binds near the C terminus
of NBD2. A third antibody, MM13-4, binding away from the NBDs near the
N terminus of CFTR had no effect on photolabeling.
Localization of Labeling Sites--
To assess hydrolysis and
trapping at each of the NBDs more directly, membranes were treated with
trypsin to cleave between the domains. After detergent solubilization
the same two antibodies employed above were then used to
immunoprecipitate polypeptide fragments containing each domain. It was
first necessary to identify these fragments by immunoblotting after
their separation by SDS-PAGE. These results are shown in Fig.
3A. The L12B4 antibody
detected undigested CFTR at the top of the gel, a tight set of bands in the 70-80 kDa range, and a band at ~40 kDa. The same procedure using
the M3A7 antibody detected in addition to undigested CFTR a strong band
at 95-105 kDa that is actually a doublet and a set of bands in the
30-35 kDa range. The large fragments detected by each antibody reflect
trypsin cleavage(s) in the R-domain (Fig. 2C). The sites
responsible for the 40-kDa band detected by L12B4 are not clearly
established. Digestion near the N terminus of NBD2 is responsible for
the 30-35 kDa bands detected by M3A7. The assignment of the bands was
confirmed by additional antibodies recognizing epitopes within NBD1 and
NBD2 (data not shown).
When immunoprecipitations were carried out after digestion to enrich
the NBD-containing fragments, the results shown in Fig. 3B
were obtained. These blots confirmed that the L12B4 antibody has
immunoprecipitated the NBD1-assigned fragments, and the M3A7 antibody
has immunoprecipitated the NBD2 fragments (Fig. 3B,
lanes 2 and 3). However, unexpectedly this
experiment also showed that the large fragments containing the two NBDs
remained strongly associated after trypsin digestion. That is, probing
the NBD2 immunoprecipitate with antibody to NBD1 revealed the presence of the large NBD1 bands (Fig. 3B, lane 1).
Similarly probing the NBD1 immunoprecipitate with antibody to NBD2
detected the lower member of the large NBD2 doublet (Fig.
3A, lane 4). These immunoprecipitations were done
in RIPA buffer with its multiple detergents, but even when the trypsin
digests were incubated in 2% SDS prior to dilution to RIPA conditions
these same co-immunoprecipitations occurred. These data do not
absolutely prove that the strong interaction between these fragments
are exclusively between the NBDs, because the large NBD2-containing
fragment also contains all of the second membrane-associated domain and
part of the R-domain. Other studies with the domains expressed
separately have found interactions between NBD1 and NBD2 (24), but the
observations reported here are the first suggesting that the strong
binding occurs in the intact functional protein. This is not crucial to
the main point of this paper, which is to demonstrate the asymmetry of
nucleotide interactions at the two NBDs and their relevance to channel
regulation, but it will be important in more detailed future studies of
the mechanism. The positions of the reduced immunoglobulin bands
present in the immunoprecipitates are shown in Fig. 3C and
account for the bands of these mobilities in Fig. 3B.
Immunoprecipitation with M3A7 was employed to detect fragments
labeled with N3[ AMP-PNP Inhibits Labeling of NBD1 and Increases Labeling of
NBD2--
The nonhydrolyzable ATP analogue, AMP-PNP, has been used
extensively in attempts to determine the role of nucleotide binding and
hydrolysis in CFTR channel gating (1, 25). Its ability when present
together with ATP to lock the channel open has been attributed to its
suggested binding to NBD2 and prevention of hydrolysis there proposed
to be necessary for channel closing (13). Incubation of increasing
concentrations of AMP-PNP together with
8-azido-[
To explore these reciprocal effects of AMP-PNP further we had the
compound 8-azido-AMP-PNP synthesized. Because 8-azido-ATP was far more
effective than ATP in competing for photolabeling by
8-azido-[ The advantage gained by utilization of two rather than one NBD by
ABC proteins is not obvious. In the case of CFTR, which is unique among
these molecules as an ion channel, a possible correspondence of this
duality with the two discrete steps in channel gating, i.e.
opening and closing, is an attractive idea. Indeed this concept has
been central in prevalent models of CFTR channel gating (25).
Consistent with this possible separation of function between the
domains, nucleotide interactions at the NBDs of other ABCC family
proteins are markedly asymmetric (6-9). As we show here this also
seems to be the case for CFTR, i.e. ATP binding and ADP
trapping is tighter at NBD1 than NBD2, and AMP-PNP has opposite effects
on the two domains. This latter observation is significant, because
studies of the effect of AMP-PNP on CFTR channel gating in the presence
of ATP have been interpreted on the basis of the assumption that it
binds with high affinity to NBD2 (1). By so doing the nonhydrolyzable
nucleotide was proposed to maintain the channel in an open state until
it slowly dissociated (18). Explicit in this proposal was the idea that
the hydrolysis of ATP at this site would normally be necessary for
channel closing. Our present observations show that AMP-PNP competes
for nucleotide trapping at NBD1 and at the same time enhances the
interaction at NBD2 probably as an allosteric response to the higher
affinity binding at NBD1. The fact that the two domains seem firmly
associated in the intact protein supports the feasibility of such
allosteric interactions. In this regard it is significant that major
conformational changes have been observed in P-glycoprotein on binding
AMP-PNP (26).
Although most of the attention has been paid to the locking-open effect
of AMP-PNP (1, 13, 25) Weinreich et al. (27) more recently
have shown that it also slows channel opening. On this basis these
authors suggested that there may be two binding sites for AMP-PNP on
CFTR. Our data also indicate that the compound has two different types
of impact on the protein. It should be noted also that the CFTR channel
can be gated in the absence of ATP by high concentrations of AMP-PNP
alone (28). However, this requires much larger concentrations ( In summary our present findings indicate that the two NBDs of
CFTR are quite asymmetrical in terms of their ability to bind and
occlude nucleotide, the interaction at NBD1 being more stable than that
at NBD2. The influence of AMP-PNP is quite different than that
predicted from its effect on channel gating and hence has implications
for models based on these. There is a high affinity competitive
interaction at NBD1 and a concomitant stimulatory influence on NBD2
arguing directly against the notion that it inhibits channel closing by
preventing hydrolysis at NBD2 (1, 25). These and other data showing
that mutagenesis of crucial residues in either NBD does not influence
channel opening and closing predictably (11) makes it difficult to
attribute each transition separately to nucleotide interactions at one
or other of the domains. Instead it may be more likely that opening and closing occur as a consequence of nucleotide binding and dissociation or hydrolysis at a functional unit involving both domains. Such an
interpretation is consistent with the fact that the domains are
physically associated in the intact protein and with proposals that the
NBDs of other ABC proteins may participate jointly in ATP binding and
hydrolysis utilizing amino acid residues from each (2, 29).
,
-imidodiphosphate (AMP-PNP), which in the gating models was proposed to bind with high affinity to NBD2 causing inhibition of ATP hydrolysis there postulated to drive channel closing.
Instead we found a strong competitive inhibition of nucleotide hydrolysis and trapping at NBD1 and a simultaneous enhancement at NBD2.
This argues strongly that AMP-PNP does not inhibit ATP hydrolysis at
NBD2 and thereby questions the relevance of hydrolysis at that domain
to channel closing.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was purchased from ICN
Biomedicals. AMP-PNP and other nucleotides and TPCK-treated trypsin
were from Sigma as were all protease inhibitors except Pefabloc, which
was from Roche Molecular Biochemicals. 8-Azido-AMP-PNP was synthesized
by Affinity Labeling Technologies, Inc.
-32P]ATP--
The photoactivable
radionucleotide at the concentration indicated in each experiment was
incubated with the resuspended membranes (15-30 µg of protein for
direct analysis by SDS-PAGE and 150-200 µg of protein for
immunoprecipitation) for 10 min at 37 °C in the presence or absence
of sodium orthovanadate (0.5 mM). After transfer to ice the
membrane suspension was irradiated for 2 min in a Stratalinker UV
cross-linker (
= 254 nm) either before or after pelleting to
wash out unbound 8-azido-[
-32P]ATP and
resuspension in 40 mM Tris-HCl, pH 7.4, 0.1 mM
EGTA. The membranes then were solubilized directly in gel loading
buffer for SDS-PAGE or digested with trypsin prior to immunoprecipitation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP--
A previous study
showed photolabeling of CFTR expressed in insect Sf9 cells with
8-azido-ATP (17). Fig. 1, A
and B, shows that CFTR in mammalian cell (BHK) membranes is
photolabeled by 8-azido-[
-32P]ATP in a
vanadate-stimulatable manner and that no labeling occurs in the absence
of UV irradiation. Fig. 1C shows that unlabeled N3ATP competes for photolabeling by
N3[
-32P]ATP much more effectively than ATP
suggesting that there is higher affinity for the derivatized nucleotide
than for ATP. We also find that N3ATP is more effective on
a dose basis than ATP in supporting CFTR single-channel gating (data
not shown). This may be a common feature of ABCC subfamily members,
because N3ATP also supports organic anion transport by MRP1
more effectively than ATP (6). This provides assurance that 8-azido-ATP
is a functionally relevant probe of nucleotide interactions with these proteins.
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Fig. 1.
Photoactivation-dependent
radiolabeling of CFTR with
8-azido-[ -32P]ATP.
A, membranes (30 µg of protein) from BHK-21 cells
expressing CFTR were incubated with 5 µM
8-azido- [
-32P]ATP in the absence (
) or presence
(+) of vanadate as described under "Experimental Procedures." After
irradiation, samples were subjected to SDS-PAGE and autoradiography.
Lane m had membranes from mock-transfected BHK cells not
expressing CFTR. B, results of an experiment identical to
the one in A but with no UV irradiation. C,
inhibition of photolabeling by increasing concentrations of ATP (
)
and 8-azido-ATP (
) present during incubation prior to
photoactivation. 32P radioactivity associated with the CFTR
band was determined by electronic autoradiography (Packard Instant
Imager).
-32P]ADP. Because neither CFTR channel gating
(22) nor hydrolysis of ATP (23) is prevented completely by substitution
of these residues, it was of interest to determine their effect on
photolabeling. Fig. 2A shows
that labeling of the NBD2 mutant (K1250A) is reduced only a small
amount compared with wild type indicating that normally there may be
little hydrolysis and trapping at NBD2. In contrast the comparable
substitution in NBD1 (K464A) greatly diminished labeling compared with
wild type indicating that the majority of labeling and trapping may
occur at NBD1. However, the effects of this mutation have to be
considered in light of the fact that it also interferes with
biosynthetic maturation of the protein as is reflected in the much
lower ratio of the larger mature band to the smaller immature band in
the Western blot shown below the autoradiogram. Nevertheless the amount
of labeling is reduced substantially relative to the amount of mature
protein present.
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Fig. 2.
Walker A lysine mutagenesis and antibody
binding inhibit photolabeling of NBD1. A, membranes
from BHK cells expressing wild-type and the K1250A (15 µg of protein)
and K464A variants (60 µg of protein) were incubated with 20 µM 8-azido-[ -32P]ATP in the presence of
0.5 mM orthovanadate and processed as under "Experimental
Procedures." A Western blot of the same gel probed with M3A7 is shown
below this autoradiogram. Note that 4 times more K464A membrane protein
was used than wild type and K1250. The cells expressing K464A had also
been grown at 26 °C to promote maturation of the mutant CFTR.
B, 15 µg of BHK membrane protein containing wild-type CFTR
was incubated at 0 °C for 1 h with 50 µg/ml purified
immunoglobulin from each of the mouse monoclonal antibodies. Prior to
photolabeling membranes were pelleted and rinsed with binding buffer
before incubation with 8-azido-ATP. C, a sketch indicating
locations of epitopes recognized by antibodies L12B4, M3A7, and MM13-4
and approximate major trypsin digestion sites (T) is
shown.
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Fig. 3.
Co-immunoprecipitation of CFTR NBD-containing
fragments produced by limited trypsin digestion. A,
membranes from CFTR-expressing BHK cells were digested with trypsin
(mass ratio, 1:180) for 15 min at 0 °C. After SDS-PAGE Western blots
were probed with monoclonal antibodies recognizing epitopes near the N
terminus of NBD1 (L12B4) and C terminus of NBD2 (M3A7; Ref. 21).
B, similarly trypsin-digested membranes were solubilized
with RIPA buffer and immunoprecipitated with each of the antibodies.
The immunoprecipitates were electrophoresed. Western blots were probed
with each of the antibodies as indicated. C, Western blots
showing mobilities of reduced immunoglobulin bands after mock
immunoprecipitations in which no solubilized membranes were present are
shown.
-32P]ATP. Fig.
4A reveals that both domains
are labeled, but there is more labeling of NBD1 than NBD2. In this
experiment the free 8-azido-ATP incubated with membranes was not
removed prior to photoactivation. When the nucleotides were washed out
before irradiation labeling of NBD1 was not altered, but labeling at
NBD2 was reduced greatly (Fig. 4B). This was evident
particularly in the large decrease in intensity of the small NBD2 bands
but was seen also in the large NBD2 bands. This result suggests there
may be lesser affinity for the nucleotide at NBD2 than at NBD1. This is
substantiated by the concentration dependence shown in Fig.
4C, which also indicates saturation of labeling at NBD1 at a
lower 8-azido-[
-32P]ATP than at NBD2.
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Fig. 4.
Photolabeling of NBDs with
8-azido-[ -32P]ATP.
A, CFTR-containing membranes (200 µg) were incubated with
50 µM 8-azido-[
-32P]ATP in the presence
of vanadate. After irradiation, digestion with trypsin and
solubilization in RIPA buffer CFTR fragments were immunoprecipitated
with M3A7. After SDS-PAGE the gel was dried and exposed to x-ray film
to yield the autoradiogram shown. B, a similar experiment
was performed with UV irradiation before (lane 1) or after
(lane 2) washout of free nucleotide. C,
concentration dependence (in µM) of
8-azido-[
-32P]ATP labeling without or with washout of
free nucleotide before photoactivation is shown.
-32P]ATP prior to photoactivation strongly
inhibited labeling at NBD1 but not at NBD2 (Fig.
5A). In fact there was
increased labeling of the NBD2 bands at the lower AMP-PNP
concentrations. In contrast to the differential effects of AMP-PNP,
other nucleotides including ATP, ADP, and dATP competed for labeling at
either domain approximately equally (Fig. 5B). dATP was more
potent than ATP and ADP, consistent with other observations in which
the deoxynucleotide had a lower Km for hydrolysis by
CFTR and a strong influence on channel gating (data not shown).
However, the opposite effects of AMP-PNP on NBD1 and NBD2 were most
striking, because it is the only compound we have found yet to
influence the two domains differentially. This observation is
important, because it is the first direct test of AMP-PNP interactions
predicted on the basis of the its locking-open of the channel in the
presence of ATP (18). The enhancement of nucleotide trapping at NBD2
would not seem to be in agreement with the predicted inhibition of
hydrolysis there. The pronounced reduction in trapping at NBD1 could
reflect an inhibition of hydrolysis at that domain.
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Fig. 5.
AMP-PNP inhibits photolabeling of NBD1 with
8-azido-[ -32P]ATP.
A, membranes were incubated with 25 µM
8-azido-[
-32P]ATP in the presence of the indicated
concentrations of AMP-PNP. After irradiation and trypsin digestion
membranes were solubilized in RIPA buffer, immunoprecipitated with
L12B4, and electrophoresed. B, similar experiments were
performed using increasing concentrations of ATP, ADP, or dATP. The
amount of 32P radioactivity associated with NBD1 and NBD2
was determined by electronic autoradiography and plotted as the
histograms show.
-32P]ATP (Fig. 1C) we reasoned
that 8-azido-AMP-PNP also might have a more potent influence than
AMP-PNP on the interactions of 8-azido-[
-32P]ATP. Fig.
6 shows that this was indeed the case.
Qualitatively, the effect was the same as that of AMP-PNP,
i.e. there was inhibition of labeling at NBD1 and
enhancement of that at NBD2. Quantitatively however these effects
occurred at nearly 10-fold lower concentrations and were more
clear-cut. The data points relating labeling at NBD1 and
8-azido-AMP-PNP concentration (Fig. 6B) fit very well with a
strictly competitive inhibition curve with a Ki in
the range of 1-2 µM. The curve for labeling at NBD2 is
almost the mirror image of that at NBD1 at concentrations up to 100 µM. The basis of this effect cannot be interpreted so
readily, but it may reflect an indirect consequence of the
high affinity interaction at NBD1. At concentrations between 100 µM and 1 mM this effect is overcome probably
by direct competition for binding to the lower affinity site on
NBD2.
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Fig. 6.
8-Azido-AMP-PNP inhibits photolabeling of
NBD1 and stimulates labeling of NBD2. The experiment was performed
as in Fig. 5A but 8-azido-AMP-PNP replaced AMP-PNP.
A, autoradiogram; B, plots of the relative
amounts of radioactivity associated with NBD1 and NBD2 as determined by
electronic autoradiography. The NBD2 curve is that of strictly
competitive inhibition using a Ki of 2 µM. The experimental points fit this curve well.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5
mM) than those that elicit the two effects described in
this paper.
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ACKNOWLEDGEMENTS |
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We thank Susan Bond and Marv Ruona for preparation of the manuscript and figures, respectively.
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FOOTNOTES |
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* This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (DK51619).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Mayo Clinic
Scottsdale, S. C. Johnson Medical Research Center, 13400 E. Shea
Blvd., Scottsdale, AZ 85259. Tel.: 480-301-6206; Fax: 480-301-7017;
E-mail: riordan@mayo.edu.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M100515200
2 L. Aleksandrov, A. Mengos, X. Chang, A. Aleksandrov, and J. R. Riordan, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
CFTR, cystic
fibrosis transmembrane conductance regulator;
NBD, nucleotide binding
domain;
AMP-PNP, 5'-adenylyl-,
-imidodiphosphate;
BHK, baby
hamster kidney;
TPCK, tosylphenylalanyl chloromethyl ketone;
PAGE, polyacrylamide gel electrophoresis;
RIPA, radioimmune
precipitation;
8-N3ATP, 8-azidoadenosine
5'-triphosphate.
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REFERENCES |
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1. | Gadsby, D. C., and Nairn, A. C. (1999) Physiol. Rev. 79, S77-S107[Medline] [Order article via Infotrieve] |
2. |
Urbatsch, I. L.,
Gimi, K.,
Wilke-Mounts, S.,
and Senior, A. E.
(2000)
J. Biol. Chem.
275,
25031-25038 |
3. | Klein, I., Sarkadi, B., and Varadi, A. (1999) Biochim. Biophys. Acta 1461, 237-262[Medline] [Order article via Infotrieve] |
4. |
Ueda, K.,
Inagaki, N.,
and Seino, S.
(1997)
J. Biol. Chem.
272,
22983-22986 |
5. |
Ueda, K.,
Komine, J.,
Matsuo, M.,
Seino, S.,
and Amachi, T.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
1268-1272 |
6. |
Hou, Y.-X.,
Cui, L.,
Riordan, J. R.,
and Chang, X.-B.
(2000)
J. Biol. Chem.
275,
20280-20287 |
7. |
Gao, M.,
Cui, H. R.,
Loe, D. W.,
Grant, C. E.,
Almquist, K. C.,
Cole, S. P.,
and Deeley, R. G.
(2000)
J. Biol. Chem.
275,
13098-13108 |
8. |
Nagata, K.,
Nishitani, M.,
Matsuo, M.,
Kioka, N.,
Amachi, T.,
and Ueda, K.
(2000)
J. Biol. Chem.
275,
17626-17630 |
9. | Ueda, K., Matsuo, M., Tanabe, K., Morita, K., Kioka, N., and Amachi, T. (1999) Biochim. Biophys. Acta 1461, 305-313[Medline] [Order article via Infotrieve] |
10. | Hwang, T.-C., Baukrowitz, G., Nagel, G., Horie, A. C., and Gadsby, D. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4698-4702[Abstract] |
11. |
Ikuma, M.,
and Welsh, M. J.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
8675-8680 |
12. |
Carson, M. R.,
Travis, S. M.,
and Welsh, M. J.
(1995)
J. Biol. Chem.
270,
1711-1717 |
13. | Nagel, G. (1999) Biochim. Biophys. Acta 1461, 263-274[Medline] [Order article via Infotrieve] |
14. |
Li, C.,
Ramjeesingh, M.,
Wang, W.,
Garami, E.,
Hewryk, M.,
Lee, D.,
Rommens, J. M.,
Galley, K.,
and Bear, C. E.
(1996)
J. Biol. Chem.
271,
28463-28468 |
15. |
Ko, Y. H.,
and Pedersen, P. L.
(1995)
J. Biol. Chem.
270,
22093-22096 |
16. | Randak, C., Neth, P., Auerswald, E. A., Eckerskorn, C., Assfalg-Machleidt, I., and Machleidt, W. (1997) FEBS Lett. 410, 180-186[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Szabo, K.,
Szakacs, G.,
Hegeds, T.,
and Sarkadi, B.
(1999)
J. Biol. Chem.
274,
12209-12212 |
18. | Baukrowitz, T., Hwang, T. C., Nairn, A. C., and Gadsby, D. C. (1994) Neuron 12, 473-482[Medline] [Order article via Infotrieve] |
19. |
Mathews, C. J.,
Tabcharani, J. A.,
Chang, X. B.,
Jensen, T. J.,
Riordan, J. R.,
and Hanrahan, J. W.
(1998)
J. Physiol. (Lond)
508,
365-377 |
20. |
Chang, X.-B.,
Tabcharani, J. A.,
Hou, Y.-X.,
Jensen, T. J.,
Kartner, N.,
Alon, N.,
Hanrahan, J. W.,
and Riordan, J. R.
(1993)
J. Biol. Chem.
268,
11304-11311 |
21. | Kartner, N., Augustinas, O., Jensen, T. J., Naismith, A. L., and Riordan, J. R. (1992) Nat. Genet. 1, 321-327[Medline] [Order article via Infotrieve] |
22. | Anderson, M. P., and Welsh, M. J. (1992) Science 257, 1701-1704[Medline] [Order article via Infotrieve] |
23. | Ramjeesingh, M., Li, C., Garami, E., Huan, L. J., Galley, K., Wang, Y., and Bear, C. E. (1999) Biochemistry 38, 1463-1468[CrossRef][Medline] [Order article via Infotrieve] |
24. | Ostedgaard, L. S., Rich, D. P., DeBerg, L. G., and Welsh, M. J. (1997) Biochemistry 36, 1287-1294[CrossRef][Medline] [Order article via Infotrieve] |
25. | Sheppard, D. N., and Welsh, M. J. (1999) Physiol. Rev. 79, S23-S45[Medline] [Order article via Infotrieve] |
26. | Julien, M., and Gros, P. (2000) Biochemistry 39, 4559-4568[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Weinreich, F.,
Riordan, J. R.,
and Nagel, G.
(1999)
J. Gen. Physiol.
114,
55-70 |
28. |
Aleksandrov, A. A.,
Chang, X.,
Aleksandrov, L.,
and Riordan, J. R.
(2000)
J. Physiol. (Lond.)
528,
259-265 |
29. | Jones, P. M., and George, A. M. (1999) FEMS Microbiol. Lett. 179, 187-202[CrossRef][Medline] [Order article via Infotrieve] |