Departments of Internal Medicine and Physiology and Biophysics, Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, Iowa 52242
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ABSTRACT |
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While studying the regulation of the cystic
fibrosis transmembrane conductance regulator (CFTR), we found that
addition of F to the
cytosolic surface of excised, inside-out membrane patches reversibly
increased Cl
current in a
dose-dependent manner. Stimulation required prior phosphorylation and the presence of ATP.
F
increased current even in
the presence of deferoxamine, which chelates
Al3+, suggesting that stimulation
was not due to A
. F
also stimulated current
in a CFTR variant that lacked a large part of the R domain, suggesting
that the effect was not mediated via this domain. Studies of single
channels showed that F
increased the open-state probability by slowing channel closure from
bursts of activity; the mean closed time between bursts and single-channel conductance was not altered. These results suggested that F
influenced
regulation by the cytosolic domains, most likely the nucleotide-binding
domains (NBDs). Consistent with this, we found that mutation of a
conserved Walker lysine in NBD2 changed the relative stimulatory effect
of F
compared with
wild-type CFTR, whereas mutation of the Walker lysine in NBD1 had no
effect. Based on these and previous data, we speculate that
F
interacts with CFTR,
possibly via NBD2, and slows the rate of channel closure.
nucleotide-binding domain; adenosine 5'-triphosphate; patch clamp; channel gating
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INTRODUCTION |
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THE CYSTIC FIBROSIS transmembrane conductance regulator
(CFTR) is an epithelial Cl
channel with novel structure and regulation (for review, see Refs. 31,
41). CFTR is composed of two sets of membrane-spanning domains that
contribute to the formation of the ion-conducting pore and three
intracellular domains that regulate channel activity: two
nucleotide-binding domains (NBDs) and the R domain. Loss of CFTR
Cl
channel function causes
cystic fibrosis, a common lethal genetic disease (42).
Regulation of CFTR Cl
channel activity is complex. Phosphorylation of the R domain by an
adenosine 3',5'-cyclic monophosphate (cAMP)-dependent
protein kinase (PK) catalytic subunit (PKA) is necessary
but not sufficient for channel activity. Once the R domain has been
phosphorylated, ATP is required to open the channel (1). Functional
studies of ATP regulation of variant channels and nucleotide binding to
full-length CFTR and to NBD peptides suggest that ATP interacts with
both NBDs (3, 9, 19, 25, 35, 38-40). The inability of
nonhydrolyzable analogs to open the channel suggests that ATP
hydrolysis is required for activity (1, 6, 9, 16) and that isolated
NBD1 and whole CFTR hydrolyze ATP (24, 27). However, several studies
indicate that the two NBDs may have different roles in controlling
channel activity (3, 9, 35). Such observations have led to the development of models in which hydrolysis at NBD1 opens the channel and
hydrolysis at NBD2 closes the channel (9, 22).
While studying the regulation of CFTR, we found that
F stimulated CFTR channel
activity. The purpose of this study was to investigate the mechanism by
which F
activates CFTR to
better understand regulation of the channel. We were also interested in
studying the effect of F
because in patch-clamp studies of CFTR,
F
is often added to the
cytosolic bathing solution to inhibit phosphatase activity (28, 29, 32,
33).
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EXPERIMENTAL PROCEDURES |
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Chemicals and solutions.
PKA was obtained from Promega (Madison, WI). ATP, guanosine
5'-O-(3-thiotriphosphate)
(GTPS), and all other reagents were obtained from Sigma (St. Louis,
MO).
Site-directed mutagenesis. CFTR mutants were constructed in the vaccinia virus expression plasmid pTM-CFTR4 (13) by the method of Kunkel (26). Mutants were verified by restriction enzyme analysis, DNA sequencing around the sites of mutation, and in vitro transcription and translation assays (15). Mutants are named by the amino acid residue number preceded by the wild-type amino acid and followed by the amino acid to which the residue was changed using the single-letter amino acid code.
Cells and CFTR expression systems.
We used NIH/3T3 fibroblasts that stably express CFTR after infection
with a retrovirus expressing wild-type human CFTR (2). The cells were
maintained as previously described (7). Functional studies performed on
these cells have revealed hundreds of CFTR Cl channels per excised,
inside-out cell-free patch (1, 7). We transiently expressed wild-type
or mutant CFTR in HeLa cells using the vaccinia virus-T7 hybrid
expression system described previously (30).
Patch-clamp technique.
The methods used for patch-clamp recording are similar to those
previously described (17). The excised, inside-out configuration was
used in all patch-clamp experiments. All cells and baths were maintained at 34-36°C by a temperature-controlled microscope
stage (Brook Industries, Lake Villa, IL) except when ~25°C bath
conditions are specifically indicated. The bath electrode consisted of
an Ag-AgCl pellet connected to the bathing solution via an agar bridge filled with 1 M KCl. The junction potentials introduced after addition
of 20 mM NaF were less than +1 mV. Pipette resistance was 2-6
M, and seal resistance was 2-25 G
. An Axopatch 200-A amplifier (Axon Instruments, Foster City, CA) was used for voltage clamping and current amplification. A Gateway 2000 computer, system P5-90 (N. Sioux City, SD), and the pClamp 6.0.3 software package (Axon
Instruments) were used for data acquisition and analysis. Data were
recorded on digital audiotape on a DTR-1203 digital tape recorder
(Biologic Science Instruments, Molecular Kinetics, Pullman, WA). Some
of the data acquisition and analysis required use of the Indec Systems
laboratory computer system (Sunnyvale, CA) as previously described (8).
[-32P]8-azidoadenosine
5'-triphosphate photolabeling.
Photolabeling of membrane-associated CFTR was performed as described
previously (40). Monolayer cultures of Spodoptera
frugiperda cells were infected with a baculovirus
containing the entire coding sequence for wild-type human CFTR (gift of
R. J. Gregory and A. E. Smith, Genzyme, Framingham, MA). Membranes were
prepared by differential centrifugation and resuspended in 20 mM HEPES,
pH 7.5, 50 mM NaCl, and 3 mM
MgSO4, with 2 µg/ml each of
leupeptin, aprotinin, and pepstatin. Membranes were collected by
centrifugation and resuspended at a concentration of 2.5 mg protein/ml
in the same buffer.
Statistical analysis. Values are presented as means ± SE. The unpaired Student's t-test was used when comparing values. Values were considered statistically significant at P < 0.05.
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RESULTS |
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Effect of F on CFTR
Cl
current in excised patches of
membrane.
While studying phosphorylation-dependent regulation of CFTR, we
observed that F
stimulated
CFTR Cl
current. Figure
1A shows
that PKA and ATP activated CFTR
Cl
channels in excised
patches of membrane and that addition of F
further increased
current. The increase in current was concentration dependent, as shown
in Fig. 1, A and
B.
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Effect of deferoxamine on stimulation by
F.
Previous work by Baukrowitz et al. (6) has shown that
BeF3 and orthovanadate
(VO4) can stimulate CFTR channel
activity by slowing the rate of closure from bursts of activity, i.e., by "locking" CFTR open. They proposed that these agents adopt a
conformation similar to the
-phosphate of ATP associated with an
active CFTR channel. Therefore, we considered the possibility that the
effects of F
may have been
due to AlF3 because solutions are
frequently contaminated by Al3+.
We found that our bath solution contained up to 10 µM
Al3+. To chelate the
Al3+, we added 1 mM deferoxamine
(44) to the bathing solution. Figure 3
shows that deferoxamine had no effect on the current stimulated by
F
(n = 4). Moreover, when deferoxamine
was added first, F
still
stimulated current (n = 3, data not
shown). In the solutions we used, it can be calculated that with 10 µM AlCl3 and 20 mM NaF, the free
Al3+ concentration is 3.2 × 10
27 M and the
A
concentration is 5.1 × 10
10 M (44). For
comparison, Sternweis and Gilman (36) reported that in the absence of
deferoxamine, 1 µM Al3+ with 5 mM F
was required to begin
to observe G protein stimulation of adenylate cyclase. In CFTR,
Baukrowitz et al. (6) found that 5 mM CsF added with 0.5 mM
BeSO4 stimulated CFTR. These
considerations and our results suggest that
F
stimulated CFTR
independently of Al3+.
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Effect of GTPS on CFTR
Cl
current.
Because F
can have some
stimulatory effects on G proteins that do not require
Al3+ (4, 5, 20), we tested the
possibility that the stimulatory effects might have resulted from G
protein activation. It was possible that
F
may have activated a G
protein that stimulated CFTR. Therefore, we asked whether GTP
S would
stimulate CFTR. Figure 4 shows that addition of up to 250 µM GTP
S did not stimulate CFTR current (1.5 ± 4.5% increase, n = 5, P = 0.77). When 20 mM
F
was added to the same
membrane patches, CFTR current increased. These data suggesting that
the effect of F
was not via
a G protein are consistent with the report of Ismailov et al. (23), who
found that 100 µM GTP
S had no effect on CFTR channels incorporated
into planar lipid bilayers.
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Effect of F on
CFTR
R-S660A current.
Because CFTR requires phosphorylation for activation and because
F
is known to inhibit
several protein phosphatases (34), we asked whether
F
might stimulate CFTR
activity by inhibiting a phosphatase. To test this possibility, we
studied a CFTR variant (CFTR
R-S660A) in which part of the R domain
(amino acids 708-835) is deleted and a remaining phosphorylation
site (serine 660) is mutated to alanine (30). This channel shows
constitutive activity in the presence of ATP and is not further
activated by PKA. Figure 5 shows that
addition of F
increased the
current in CFTR
R-S660A (n = 3),
suggesting that F
did not
stimulate the channel by inhibiting a phosphatase.
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Effect of F on
8-N3 ATP photolabeling.
The stimulation by F
is
similar to the effects of some other agents that stimulate CFTR. For
example, both inorganic pyrophosphate (PPi) and
F
stimulate CFTR only after
phosphorylation and in the presence of ATP (12, 16).
PPi increased photolabeling of
CFTR by 8-N3ATP, suggesting that
PPi modulates channel gating by
altering the interaction with nucleotides (12). However, in contrast to
the effect of PPi, 10 mM
F
had no measurable effect
on 8-N3ATP photolabeling (Fig.
6). This result suggests that
F
modulates channel gating
by a mechanism distinct from that of PPi.
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Kinetic effects of F on CFTR
single-channel activity.
These data suggest that F
might interact directly with CFTR.
F
could stimulate current
by increasing single-channel conductance, Po, or the number
of channels. To evaluate these possibilities, we examined the effect of
F
on single-channel
currents; an example is shown in Fig.
7A. The single-channel current-voltage relationships are shown in Fig. 7B. The channels were
Cl
selective as indicated
by the reversal potentials (reversal potential = 24 mV, expected
Cl
reversal potential = 27 mV). Single-channel conductance in the absence of
F
was 9.87 ± 0.39 pS
and in the presence of 20 mM NaF was 8.45 ± 0.44 pS
(P = 0.055). This small decrease in
single-channel conductance is not unexpected, since
F
is less conductive than
Cl
(2, 37).
F
stimulation did not
change the linear current-voltage relationship of single CFTR channels
or macroscopic CFTR current (Fig.
7B) and showed no voltage dependence
(n = 8, data not shown). These data
indicate that the increase in current produced by
F
could not be explained by
an increase in conductance.
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Effect of F on CFTR with Walker
lysine mutations.
Previous work had suggested that NBD2 is a primary determinant of burst
duration (9, 11, 22). This suggested that
F
might interact with CFTR
at NBD2. To investigate this possibility further, we studied channels
in which the conserved Walker lysine in NBD2 (lysine 1250) was mutated.
We found that F
stimulates
CFTR-K1250M channels to a greater extent than wild-type CFTR (Fig.
8). In contrast, channels containing a
mutation of the conserved Walker lysine of NBD1 (lysine 464) had a
response similar to that of wild-type CFTR. These data suggest that
F
may have altered gating
by NBD2.
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DISCUSSION |
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Our results show that F
stimulated CFTR Cl
channels
but only after they had been phosphorylated and only in the presence of ATP. The effect of F
was
likely independent of phosphatase inhibition, since
F
increased current in
CFTR
R-S660A. Moreover, stimulation by
F
was probably not via G
protein activation, since GTP
S had no effect on CFTR. The evidence
suggests that F
was
interacting with CFTR.
Some clues about how F
stimulated CFTR came from studies of single-channel currents and mutant
CFTR. We cannot exclude the possibility that
F
stimulated CFTR through
an interaction with the membrane-spanning domains; however, the minimal
effects on single-channel conductance and the lack of voltage
dependence suggest that this was unlikely. The findings that
F
stimulated activity when
added to the cytoplasmic surface of the patch and that
F
affected the gating of
CFTR suggested an interaction with the cytoplasmic domains. It seemed
unlikely that F
stimulated
through an interaction with the R domain because
F
stimulated
CFTR
R-S660A. Instead, the data suggest that
F
interacts with the NBDs.
Models of CFTR gating suggest distinct functions for each NBD, with
burst duration influenced primarily by NBD2 (3, 6, 9, 22, 35). The
effect of F
on burst
duration suggested the possibility of an interaction with NBD2. This
suggestion was supported by the finding that mutation of the conserved
Walker lysine of NBD2 but not NBD1 enhanced the effect of
F
.
Baukrowitz et al. (6) found that
BeF3 stimulated CFTR and suggested
that BeF3 stabilized the
open-channel state. They found that
BeF3 prolonged bursts of activity
by locking the channel in the open state. However, they found that
F alone was ineffective.
Those studies led us to test the possibility that the effect of
F
was, in fact, due to
AlF3. However, our studies suggest
that even in the absence of Al3+
or beryllium, F
stimulated
activity. There are several possible explanations for differences
between the two studies. Although most of our studies were performed at
37°C, a difference in temperature is not likely to explain the
difference because we found that
F
stimulated current at
25°C, the temperature used by Baukrowitz et al. (6). Another
possible explanation is that different measurements were made in the
two studies. Baukrowitz et al. (6) measured closing rate after removal
of the stimulating agent, whereas we measured current in the presence
of F
under equilibrium
conditions. Another, but less likely, possibility is that
Baukrowitz et al. (6) studied CFTR from guinea pig cardiac
myocytes. This cardiac CFTR is alternatively spliced, deleting 30 amino
acids from exon 5 (18, 21). It is possible that this structural
difference may have accounted for the different effects of
F
.
The suggestion that F might
stimulate CFTR by interacting with NBD2 prompts a comparison with the
effects of other agents predicted to have an effect on the NBDs (Table
1). Previous studies showed that
PPi increased burst duration,
increased the rate of channel opening (i.e., decreased the long closed
time), and increased 8-N3ATP
photolabeling (12, 16). Like PPi,
F
increased burst duration
but did not increase channel-opening rate or
8-N3ATP photolabeling. More
strikingly, PPi stimulated less
current in K1250M-CFTR than in wild-type CFTR (14), whereas F
stimulated more current
in K1250M-CFTR than in wild-type CFTR. Therefore, there are distinct
differences between the effects of
F
and
PPi.
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Like F,
5'-adenylylimidodiphosphate (AMP-PNP) prolonged the burst
duration, had no effect on the long closed time, and has been proposed
to bind to NBD2 (9, 16, 22). But unlike
F
, AMP-PNP decreased
8-N3ATP photolabeling and required
the continued presence of PKA to have an effect (9, 22, 40). Therefore, although AMP-PNP and F
may
both affect NBD2 to prolong the burst duration, there are differences
between the two that suggest they interact differently. The effect of
F
on CFTR was very
different from the effect of inorganic phosphate (Pi).
Pi decreased the long closed time,
decreased 8-N3ATP photolabeling, and had no effect on burst duration (10).
The effect of F was
qualitatively most similar to that reported for
VO4 and
BeF3 (6, 16). These agents prolong
the burst duration; 8-N3ATP
photolabeling was not evaluated.
VO4 has been proposed to be a
phosphate analog and to bind tightly at a site where the
-phosphate is released after ATP hydrolysis, resulting in a
stable conformation and prolonging CFTR burst duration (6). BeF3 is thought to have a similar
effect. Similarity between the effects of
VO4 and
BeF3 and those which we observed
with F
raises the question
of whether they might share a similar mechanism. There are several
reports demonstrating an
Al3+-independent activation of
heterotrimeric G proteins by
F
(4, 5, 20). Higashijima
et al. (20) found that millimolar concentrations of
F
plus
Mg2+ were able to increase G
protein fluorescence, suggesting that MgF2 could stimulate G proteins.
In addition, Antonny et al. (4) found that in the presence of
Mg2+,
F
concentrations >3 mM
could activate a G protein (transducin-guanosine diphosphate) in the absence of
Al3+ or beryllium. However, in
contrast to BeF3, the inactivation rate was 100-1,000 times faster for
F
alone than for
AlF3 or
BeF3, suggesting that
F
(MgF2) was less stable or that
it bound less tightly to the protein than did
AlF3 or
BeF3. Nuclear magnetic resonance
spectroscopy of G proteins revealed that activation by
MgF2 was chemically distinct from
that obtained with AlF3 (5),
although both MgF2 and
AlF3 were thought to mimic the
-phosphate of a GTP. Because our solutions contained
Mg2+ out of necessity (MgATP is
required for channel activity), we speculate that
F
, perhaps
MgF2, is interacting with CFTR in
a manner analogous to that observed in G proteins. This scenario might
also explain, in part, the difference in burst duration in the presence
of F
(~450 ms) compared
with that in the presence of VO4
[bursts lasted for minutes (6, 16)]. Perhaps,
as proposed for VO4,
F
or
MgF2 functions as a phosphate
analog, binding at a site where the
-phosphate is released after ATP
hydrolysis, resulting in a stable conformation and prolonging CFTR
burst duration (6). However, burst duration may be shorter with
F
than with
BeF
or
VO4 because those agents bind more
tightly.
There is also the interesting possibility that
F may interact with a site
in CFTR that is different from that to which
VO4 and
BeF3 bind. Such a mechanism could
be analogous to the inhibitory effect of
F
on guanosine
5'-triphosphatase (GTPase)-activating proteins (GAP) (43).
F
-dependent inhibition of
GTPase activity leads to prolonged GTP binding and activation of GTP
binding proteins such as Rac (43). F
inhibition of GAP
activity was independent of Al3+,
and F
did not affect
guanosine diphosphate release from Rac. Perhaps F
modification of an
allosteric site on CFTR might regulate its activity in an analogous
manner. Future studies with site-directed mutations will be needed to
determine the residues that are important for
F
interaction and
stimulation of CFTR.
Finally, our results have practical implications for studies of CFTR
function. Because F is a
nonspecific phosphatase inhibitor, it is sometimes added to solutions
bathing CFTR in an attempt to prevent dephosphorylation. Our data
suggest that F
should be
added to the solutions with caution because it can affect channel
gating.
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ACKNOWLEDGEMENTS |
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We thank Pary Weber, Phil Karp, Theresa Mayhew, and Deb Gilmere for
excellent technical assistance and our laboratory colleagues for
critical comments and discussions. We thank Dr. Mark Arnold, Dr. Johna
Leddy, and Carolyn Green (Dept. of Chemistry, Univ. of Iowa) for
calculations of Al3+ and
A in our solutions. We thank
John Marshall and Seng Cheng (Genzyme) for the M13-1 antibody and the
baculovirus encoding CFTR.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-42385.
H. A. Berger was a Parker B. Francis Fellow, and M. J. Welsh is an Investigator of the Howard Hughes Medical Institute.
Address for reprint requests: M. J. Welsh, Howard Hughes Medical Institute, Univ. of Iowa College of Medicine, 500 EMRB, Iowa City, IA 52242.
Received 13 May 1997; accepted in final form 21 November 1997.
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