From the Research Institute, Hospital for Sick Children and the
Physiology Department, University of Toronto,
Toronto M5G 1XB, Canada
Received for publication, November 16, 2000, and in revised form, December 15, 2000
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ABSTRACT |
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Mutations in the cystic fibrosis gene coding for
the cystic fibrosis transmembrane conductance regulator (CFTR) lead to
altered chloride (Cl Cystic fibrosis (CF)1 is
a genetic, lethal disease that affects epithelial cells lining the
duct-like passages of the respiratory tract, gastrointestinal tract,
and the biliary and reproductive systems (1). Although the protein
coded by the CF gene, the cystic fibrosis transmembrane conductance
regulator (CFTR), has been implicated in multiple functions in
epithelial tissues (2), it acts primarily as a nucleotide- and
phosphorylation-regulated chloride channel (3-5).
CFTR belongs to the ATP-binding cassette (ABC) superfamily of membrane
proteins (6). Other clinically important members of this family include
P-glycoprotein (MDR1), the phosphatidylcholine translocase (MDR3), the
multidrug resistance-related protein (MRP1), and the sulfonylurea
receptor (SUR1; Refs. 7-10). Like CFTR, these family members possess
two nucleotide binding domains (NBDs), which bind and hydrolyze ATP,
and membrane-spanning domains (transmembrane domains (TMDs)), which
form (or regulate, in the case of SUR1) a pathway for the movement of
certain substrates through the membrane (3, 11).
Although the mechanism underlying CFTR channel activity remains
unclear, it is generally thought that MgATP binding and hydrolysis by
phosphorylated CFTR is required for normal regulation of channel gating
from the open and closed conductance states (12-23). This current
model is based on electrophysiological studies of channel gating and
biochemical studies of ATPase activity. Several electrophysiological studies of the chloride channel function of phosphorylated CFTR show
that MgATP, but not nonhydrolyzable ATP analogues, cause channel
activation (3, 20-22). Furthermore, nonhydrolyzable ATP analogues and
ATPase transition-state analogues like orthovanadate and berillium
chloride disrupt normal transitions from the open to the closed
configuration (20, 24). Our studies of purified, reconstituted CFTR
revealed that CFTR, like other ABC proteins, possesses intrinsic ATPase
activity and that, like channel gating, this catalytic activity is
activated by phosphorylation (18, 25). Furthermore, chelation of
magnesium, an essential cofactor of catalytic activity by other
ATPases, inhibited CFTR ATPase activity and altered the rate of channel
gating (18, 23). Moreover, the disease-causing mutation in the
conserved Walker C motif of the first NBD of CFTR, i.e.
CFTR-G551D, impaired both ATPase activity and the rate of channel
opening (18). These studies show that the NBDs can transmit signals
(conferred by nucleotide occupancy and/or hydrolysis) to the channel
pore and regulate the gate that controls chloride flux through the pore of CFTR.
For the mammalian ABC membrane proteins P-glycoprotein, MDR3 and MRP1,
there is evidence supporting long-range signal transduction from the
membrane-spanning domains to the nucleotide binding domains. Substrates
that bind to residues in the transmembrane domains of these
transporters induce changes in the ATPase activity by the NBDs
(26-29). In fact, ATPase activity of P-glycoprotein stimulated by
certain substrates has been shown to correlate well with transport, since the turnover numbers are similar (26). Inhibitors that bind to
the membrane domain to block the transport of these substrates also
cause a change in the catalytic activity (30, 31). Furthermore, modification of certain cysteine residues strategically engineered into
the transmembrane domains of P-glycoprotein inhibited its catalytic
activity, and this effect could be protected by substrates of
P-glycoprotein (30-32). Therefore, cross-talk between the pore and
NBDs is an important means of regulation of these ABC transporters.
To date, there are several clues that signals originating from the
transmembrane domains of CFTR may regulate the catalytic activity of
this protein. For example, diphenylamine-2-carboxylate (DPC), an
inhibitor that permeates the chloride channel pore of CFTR, not only
causes a change in the rate of chloride ion flux but also the rate of
channel gating (33). Furthermore, certain disease-causing mutations in
the transmembrane domains of CFTR have been shown to cause a change in
gating (33-35).
In the present work, we found that perturbations of the pore either by
the channel blocker DPC or through mutagenesis inhibited ATP turnover
rate by purified CFTR protein. These results provide direct biochemical
evidence suggesting that pore properties can signal feedback inhibition
to the NBDs. Furthermore, low millimolar concentrations of GSH appear
to inhibit CFTR ATPase activity via a similar pathway, revealing the
potential physiological significance of this regulatory mechanism in
health and disease.
Production and Purification of CFTR-His Proteins--
Procedures
describing productions and purification of CFTR-His proteins were
published previously (36). Briefly, Sf9-baculovirus expression
system was used for large scale production of wild type or mutant CFTR
proteins. Crude plasma membranes from frozen Sf9 cell pellets
expressing recombinant CFTR-His proteins were obtained and solubilized
in 8% pentadecafluorooctanoic acid, 25 mM phosphate, pH
8.0. Purification of CFTR-His proteins was performed using nickel
affinity chromatography. The solubilized and filtered samples were
applied to a freshly generated nickel column at a rate of 2 ml/min. A
pH gradient (pH 8.0-6.0) was then applied to the column using fast
protein liquid chromatography, and 5-ml fractions were collected.
Identification and Analysis of Immunopositive Fractions--
Dot
blot was used for analyzing the fractions eluted from the column for
the presence of CFTR protein. Immunopositive fractions were selected,
and each was concentrated separately in a Centricon YM-100 concentrator
(molecular mass cut off 100,000 Da; Millipore Corp., Bedford, MA) to a
final volume of ~100 µl. Concentrated samples in 4%
pentadecafluorooctanoic acid (w/v), 25 mM phosphate, and
100 mM NaCl were diluted 1:10 in buffer containing 8 mM Hepes, 0.5 mM EGTA, pH 7.2, and further
concentrated to a final volume of 100 µl to reduce the
pentadecafluorooctanoic acid concentration to 0.4%. Three microliters
from each concentrated fraction were subjected to 6%
SDS/polyacrylamide electrophoresis gels (Novex, Carlsbad, CA) and
analyzed for the quantity and purity of CFTR proteins by Western blot
and silver-stained protein gel, as described previously (37, 38). For
immunoblotting, the protein was transferred to a nitrocellulose
membrane and probed with an anti-CFTR polyclonal antibody generated
against a fusion protein corresponding to the predicted NBD2 and C
terminus of CFTR, amino acids N1197-L1480. Immunopositive bands were
visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).
CFTR-His Reconstitution into Liposomes--
Procedures
describing liposome preparation are reported elsewhere (36).
Concentrated fractions containing purified CFTR-His in 0.4%
pentadecafluorooctanoic acid (~100 µl) were mixed with an excess (1 mg) of a sonicated liposome preparation containing phosphatidylethanolamine:phosphatidylserine:phosphatidylcholine:ergosterol (5:2:1:1 by weight; phospholipids were from Avanti Polar Lipids Inc., Birmingham, AL). A lipid control was generated by diluting 1 mg
of liposome mixture containing
phosphatidylethanolamine:phosphatidylserine:phosphatidylcholine:ergosterol into 100 µl of a buffer containing 8 mM Hepes, 0.5 mM EGTA, pH 7.2. The concentrated CFTR fractions and lipid
control were dialyzed in a Spectra/Por dialysis membrane (Spectrum
Laboratories Inc., Rancho Dominguez, CA; molecular mass cut off 50,000 Da) overnight at 4 °C against 4 liters of a buffer containing 8 mM Hepes, 0.5 mM EGTA, pH 7.2.
Phosphorylation of CFTR-His--
Half of the reconstituted CFTR
protein (200-500 µg of total purified CFTR) and the lipid control
were phosphorylated by the catalytic subunit of PKA (Promega, Madison,
WI) for 1 h at room temperature. Phosphorylation reaction mixture
contained 250 nM catalytic subunit of PKA, 1 mM
MgCl2, and 500 µM ATP in 50 mM Tris-HCl, 50 mM NaCl, pH 7.5. The other half of the
reconstituted protein and lipid control were mock-phosphorylated
(i.e. catalytic subunit of PKA omitted from reaction). PKA
was removed after phosphorylation by dialyzing all samples in a
Specta/Por dialysis membrane (molecular mass cut off 50,000 Da)
overnight at 4 °C against 4 liters of a buffer containing 8 mM Hepes, 0.5 mM EGTA, and 0.025%
NaN3, pH 7.2. The next day the buffer was changed to one
containing no NaN3, and the samples were further dialyzed
at 4 °C overnight.
Assay of the Catalytic Activity of CFTR Protein--
The
catalytic activity was measured as the production of
[
ATPase reaction mixtures were incubated at 33 °C for 2 h and
then stopped by the addition of 14 µl of 10% SDS, 88% formic acid
(v/v). One-microliter samples from the ATPase reaction were spotted
onto polyethyleneimine-cellulose plates (VWR, Mississauga, ON) and
developed in 1 M formic acid, 0.5 M LiCl, as
described previously (39). A STORM840 Molecular Dynamics PhosphorImager was used to visualize ADP production by phosphorylated and
nonphosphorylated CFTR samples. The quantity of ATP hydrolyzed was
determined using the ImageQuant software package (Molecular Dynamics,
Sunnyvale, CA).
For ATPase reactions carried out in the presence of DPC,
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or
glibenclamide, the respective drug was first added to 30 µl of
freshly dialyzed proteoliposomes followed by a 2-h incubation at
4 °C. The remaining reaction mixture containing 1 mM
nonradioactive ATP, 20 mM Tris, 40 mM NaCl, 4 mM MgCl2, pH 7.5, and 2 µCi of
[ Statistical Analysis--
Results are expressed as the mean ± S.E. Statistical significance was assessed using one way analysis of
variance (ANOVA; with or without trend, as appropriate) or by
two-tailed Student's t test. For comparison of drug
treatment to 100% control, one-population Student's t test
was used.
The Pore Blocker DPC Inhibits ATPase Activity of Wild Type
CFTR--
As reported previously (18), purified and reconstituted CFTR
protein displays low level ATPase activity unless phosphorylated by
exogenous PKA (Fig. 1A).
Electrophysiological studies have shown that PKA phosphorylation is
also required for the chloride channel function of CFTR (4, 40). As
seen in Fig. 1B, the open channel blocker DPC inhibited the
catalytic activity of phosphorylated CFTR at concentrations of 2 and 10 mM. This inhibitory effect was evident only for
phosphorylated protein (Fig. 1C), as expected if DPC acts
inside the open channel pore. A modest (but statistically insignificant) decrease in ATPase activity (~6%) in phosphorylated CFTR samples was also observed with
5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), another channel
blocker from the arylaminobenzolate family (data not shown). Kinetic
analyses revealed that DPC inhibits CFTR ATPase activity through an
effect on nucleotide turnover (Fig. 2).
As shown in Fig. 2, DPC treatment caused a 4-5-fold decrease in the
Vmax of CFTR-ATPase, from 0.84 (nmol of ATP
hydrolyzed/2 h) to 0.18 (nmol of ATP hydrolyzed/2 h). On the other
hand, DPC did not have a major effect on ATP binding to the NBDs of
phosphorylated CFTR, with a Km (MgATP) (0.4 mM) close to that typically observed in the absence of drug
(0.3 mM; (18)). These data suggest that DPC is unlikely to
interact directly with the NBDs but, rather, decreases catalytic
activity through its effect on the pore.
Both DIDS and glibenclamide are large organic anions thought to bind
and occlude the CFTR pore. However, the site of action of these
inhibitors is less clearly defined than for DPC (41). In contrast to
our findings with DPC, interaction of these blockers with CFTR failed
to cause a significant change in the ATPase activity of either the
unmodified or PKA-phosphorylated CFTR protein at concentrations
typically used to block CFTR chloride channel activity (Refs. 41 and
42; Fig. 3). In fact, both DIDS and
glibenclamide tended to enhance (though not significantly) the ATPase
activity of unmodified and PKA-phosphorylated forms of the protein
relative to the effect of the vehicle alone (2.5% Me2SO).
These data suggest that the sites of action of DIDS and glibenclamide
on CFTR differ from that of DPC.
Electrophysiological evidence supporting the claim that DPC binds in
the open pore of CFTR was obtained in part through chloride competition
studies (43, 44). McDonough et al. (44) have shown that the
avidity of DPC blockade was dependent on the concentration of chloride
ions on the external side of the membrane, with the IC50
for blockade by DPC decreasing with a concomitant decrease in chloride
ion concentrations (44). To determine whether the inhibitory effect of
DPC on CFTR ATPase activity was also sensitive to changes in chloride
ion concentrations, we initially assessed the effect of varying NaCl
concentrations on CFTR ATPase activity. Surprisingly, we found that the
catalytic activity was affected by changes in NaCl concentration alone
in the absence of DPC. These findings made the above competition
studies problematic given that the protein is randomly oriented in the
inside-out and outside-out conformations in our proteoliposomes. We
found that increasing the concentration of NaCl from 14 to 144 mM caused a gradual decrease in the ATPase activity of the
PKA-phosphorylated but not the unphosphorylated CFTR protein
(p = 0.002, ANOVA with linear trend; Fig.
4A). A similar trend was
observed when phosphorylated CFTR was exposed to increasing
concentrations (14 to 144 mM) of sodium gluconate or
NMDG-Cl salts (Fig. 4B). Furthermore, there was no
significant difference between absolute ATPase measurements obtained
with NaCl, sodium gluconate, or NMDG-Cl salts at any given
concentration (Fig. 4B). As all of these salt solutions evoked similar concentration-dependent effects on CFTR
ATPase, we suggest that the response is not specific to chloride and
may relate to changes in the ionic strength, affecting the conformation and function of phosphorylated CFTR protein. However, this hypothesis remains to be rigorously tested.
The Pore Mutant CFTR-R347D Exhibits Altered ATPase
Activity--
Direct evidence for communication between the chloride
channel pore and the catalytic domains of CFTR came from assessments of
the ATPase activity of a disease-causing variant of CFTR bearing an
amino acid substitution (Arg to Asp) at position 347 within the
putative pore region. This disease-causing variant is associated with
mild-disease phenotype and has been shown to have altered chloride ion
conduction through the pore of CFTR (45). As shown in Fig.
5A, basal specific ATPase
activity (i.e., nonphosphorylated preparations) was
essentially abolished in this mutant, but activity could be stimulated
by PKA phosphorylation, as previously reported for wild type protein
(18), suggesting that the purified mutant is not grossly misfolded and
can undergo phosphorylation-dependent conformational
changes important for catalysis. However, the specific activity of
PKA-phosphorylated CFTR-R347D was clearly defective, exhibiting only
5-10% of the activity of phosphorylated wild type protein (1.17 ± 0.14, n = 3 and 15.8 ± 5.7, n = 9, p = 0.01, unpaired Student's t test).
The increase in ATPase activity associated with PKA-treated CFTR-R347D
samples was not due to PKA itself, since treatment with this enzyme
conferred less than 8% of the total activity measured for the
phosphorylated mutant (see legend to Fig. 5A for
details).
The MgATP dependence of catalysis by phosphorylated mutant protein was
well described using the Michaelis-Menten equation (r2 = 0.95; Fig. 5B), and this
analysis revealed that the decrease in specific ATPase activity caused
by this mutation was primarily due to a decrease in
Vmax of the enzyme. Whereas the
Vmax of phosphorylated wild type CFTR protein is
about 50 nmol/mg/min (18), the Vmax determined
for phosphorylated R347D protein is 1.1 nmol/mg/min (Fig.
5B, Table I). This reduced
ATPase activity is comparable with that obtained for mutations of
conserved residues thought to reside in the nucleotide binding pocket
in the NBDs (e.g. G551D; (18)). Defective ATPase activity by
CFTR-R347D mutant was not due to global misfolding of the nucleotide
binding folds, as the apparent affinity of the phosphorylated mutant
for MgATP was comparable, even somewhat higher, than previously
reported for phosphorylated wild type protein (Km = 0.1 mM for CFTR-R347D versus Km = 0.3 mM for wild type protein,
respectively (18)).
The pore blocker DPC exerted a similar inhibitory effect on the ATPase
activity of phosphorylated CFTR-R347D, as it did on the ATPase activity
of wild type protein (67.3 ± 6.1% of control versus
66.8 ± 3.6% of control, respectively). These results support previous reports suggesting that DPC interacts with a pore-lining residue other than Arg-347, probably Ser-341 (44), and that the R347D
mutation does not disrupt DPC binding to this site by inducing global
misfolding. Moreover, a similar inhibitory trend was observed for
increasing salt concentration on the ATPase activity of R347D protein
(Fig. 5C), as compared with the wild type protein. However,
there was a significantly greater inhibition for the mutant protein at
144 mM NaCl, supporting the notion that electrostatic interactions are altered in this pore mutant (35) and that these interactions may be important in signaling from the membrane domains to
the NBDs.
Glutathione Inhibits the ATPase Activity of Wild Type and Mutant
CFTR Proteins--
Linsdell and Hanrahan (46) have shown that GSH, the
most potent antioxidant in cells, could permeate through the CFTR
channel pore and block chloride flux. Oxidized glutathione (GSSG) could also interact with the pore but with lower affinity. Consequently, we
reasoned that GSH and possibly GSSG may alter the catalytic activity of
CFTR through modification of the pore. Furthermore, if this regulation
occurs at physiological concentrations of GSH (1-10 mM),
it is likely to be biologically important. As shown in Figs.
6, A and B, GSH
within the concentration range of 5-10 mM significantly
decreased the ATPase activity of phosphorylated CFTR protein
(one-population Student's t test, p < 0.03). We have several pieces of evidence to support the notion that
GSH is interacting with the open channel pore. First, the effect of GSH
was only seen when the protein was phosphorylated, the form of the
protein known to gate to the open channel configuration. Furthermore, physiological and high nonphysiological concentrations of GSSG (20 µM and 10 mM, respectively) did not
significantly affect the catalytic activity of either phosphorylated or
nonphosphorylated CFTR, consistent with its reported weaker affinity
with the channel pore (46). Finally, we observed a significant
difference in the inhibitory effect of GSH (10 mM) on the
ATPase activity of wild type and CFTR-R347D proteins, 39 and 63% of
untreated controls, respectively (Fig. 6C, p = 0.04). These results suggest that the mutated pore either exhibits
altered interaction with GSH and/or an altered glutathione-mediated
signal from the pore to the catalytic domains. However, our results do
not rule out the possibility that GSH may also have a direct effect on
the NBDs of CFTR, and we plan to address this possibility in our future
studies.
Although it has been known for quite some time that the catalytic
function of other ABC family members, e.g. P-glycoprotein and MRP1, is regulated by compounds that specifically interact with
their membrane domains (26-29), this particular regulatory mechanism
has not been previously directly assessed for CFTR. The previous lack
of such studies relates primarily to the requirement to purify and
reconstitute large amounts of CFTR protein to study its low specific
catalytic activity. Second, CFTR is known to possess very different
"transport" functions than P-glycoprotein and MRP1. Unlike these
related proteins, which are thought to act as pumps, CFTR has been
shown to function as an anion channel that utilizes ATP to operate a
gate through which permeant anions can flux. There is sparse evidence
in the ion channel literature documenting the regulation of channel
gating mechanisms by permeant ions. However, for certain members of the
ClC family of chloride channels such as ClC-0, it has been
determined that voltage-dependent gating may be due in part
to concentration of the permeant anion and its translocation through
the pore (47-49). With regards to CFTR, there is indirect evidence
supporting interaction between permeation and gating, in that certain
mutations in the transmembrane segments of CFTR, namely S1118F in TM11
(33) and the disease-causing mutant R117H (50), exhibit altered channel
open times. In the present paper, we provide the first direct evidence
that perturbation of pore-lining residues of CFTR by either open pore
blockers or by mutagenesis can lead to long range conformational
changes in NBDs affecting nucleotide hydrolysis. Furthermore, as this
mechanism of interdomain signaling can be mediated via pore blockade by low millimolar concentrations of the potent antioxidant glutathione, we
suggest that it is biologically important.
Our work shows that only certain anions that bind to the pore-lining
residues of CFTR can significantly alter its catalytic activity. The
synthetic inhibitor DPC has been previously shown to interact with the
open pore to block chloride ion flux (33, 44, 46). Our current data
demonstrate that DPC can also inhibit CFTR ATPase activity. However,
other open channel blockers such as DIDS, glibenclamide, or NPPB do not
affect CFTR ATPase activity, at least at the concentrations employed in
this study. Due to the nature of these studies and the requirement for
large concentrations of purified protein, we did not perform complete
dose response curves for each of these channel blockers. However, it is
feasible that we may have seen significant effects on CFTR ATPase
activity at higher concentrations of certain drugs. In fact, the
incomplete inhibition of CFTR ATPase activity by DPC at 2 mM may reflect nonspecific sequestration of this lipophilic
compound onto the liposomes (51). Furthermore, as there is no clear
consensus regarding their relative potencies of blockade in the
literature, with the available data it is difficult to assess whether a
correlation exists between the efficacy of chloride channel block by
these synthetic compounds and inhibition of CFTR ATPase activity.
Future studies of the physical basis for the cross-talk from the pore to the NBDs of CFTR will benefit from a better understanding of the
molecular basis for blockade by these inhibitors. Such detailed molecular mapping studies have been initiated by McCarty and co-workers (52) in studies of the voltage-dependent block by
DPC and NPPB in wild type and mutant (S341A and T1134F) CFTR. These
studies show that although both drugs block the open pore, each
inhibitor shows differential sensitivity to mutations in putative
pore-lining residues, suggesting that the sites of interaction with the
pore are overlapping but not identical (52).
Certain organic anions including GSH, GSSG, and gluconate have been
previously shown to block chloride ion flux through CFTR. However, in
the present studies only GSH exerted a significant and specific
inhibitory effect on the catalytic activity of CFTR. The relative
effectiveness of GSH for blockade of CFTR ATPase activity appears to
relate to its relatively high efficacy for blockade of chloride ion
flux (46).
We found further evidence for direct communication between the pore
domain and NBDs of CFTR when investigating the catalytic activity of
CFTR-R347D, a disease-causing variant with a mutation in TM6 associated
with mild disease (45). The R347D mutation led to inhibition of the
ATPase activity of CFTR, suggesting that the region in which this
arginine resides participates in the physical communication between the
pore and the NBDs. This residue is thought to reside in the inner
vestibule of the CFTR channel and has been implicated in anion binding
within the pore (45, 50). Alternately, Cotten and Welsh (35) proposed
recently that Arg-347 has an important role in stabilizing the pore
architecture. Future studies are required to determine whether
alterations in anion binding site occupancy and/or pore architecture
caused by this mutation provide the trigger for long range changes in
the NBDs.
Both the interaction of DPC with the pore and mutation of the putative
pore-lining residue, R347D, induced similar changes in the catalytic
activity of phosphorylated CFTR, namely, both of these perturbations
caused a 3-5-fold decrease in the Vmax of the
enzyme. Previous studies indicate that NBD1 and NBD2 of CFTR
functionally interact in the whole protein, and in this context, NBD2
exhibited a higher rate of hydrolysis than NBD1 (16). Hence, we suggest
that pore perturbation may decrease overall catalytic activity of CFTR
by decreasing the relative contribution by NBD2 to this overall
function. This hypothesis remains to be tested in our future studies.
Earlier studies by Kopito and co-workers (53) show that oxidizing and
reducing reagents alter the gating kinetics of CFTR channels. For
example, treatment with the strong reducing agent dithiothreitol
increased both the opening and closing rates of the channel. Such data
imply that the redox potential in cells may also alter the catalytic
activity of CFTR. We showed that GSH effectively inhibited the
catalytic activity of CFTR in concentrations that are biologically
relevant, supporting the notion that cellular redox potential might
determine the catalytic function of this protein. However, the
mechanism of action of dithiothreitol differs from that of glutathione,
as we found that dithiothreitol caused a slight stimulation rather than
inhibition of CFTR ATPase activity (data not shown), consistent with
its stimulatory effect on channel gating reported by Harrington
et al. (53). The site of action of dithiothreitol was not
indicated in their studies. On the other hand, our data specifically
support the suggestion originally proposed by Linsdell and Hanrahan
that GSH interacts with the open pore of CFTR (46). First, GSH only
inhibited the fully phosphorylated protein, known to be associated with
channel activity with a high open probability. Second, mutation of the
pore-lining residue R347D led to a change in the extent of blockade of
CFTR ATPase activity by GSH.
Our findings show that the ATPase activity of CFTR is inhibited by
certain small molecules like GSH, which interact with its membrane
domain. The catalytic activity of CFTR has been correlated to channel
open probability and conductance, i.e. the rate of chloride
flux through the pore (16, 18). Hence, a reduction in catalytic
function will be expected to lead to a net reduction in chloride ion
flux across biological membranes. Since cellular glutathione levels can
vary dramatically, e.g., during the cell cycle (increasing
before cell division) and in response to inflammation and oxidative
burden (54-57), cellular glutathione may constitute an important
mechanism for the regulation of CFTR channel function in
vivo.
Previous studies by Linsdell and Hanrahan (46, 58) show that organic
anions such as gluconate, GSSG, and GSH can only permeate the CFTR pore
from its cytosolic vestibule. The uni-directionality and energy
dependence of this process prompted these authors to suggest that CFTR
might mediate transport of certain large anions via a pump-like
mechanism, similar to that proposed for P-glycoprotein, MDR3, and MRP1
(46, 58). As mentioned previously, substrates that are transported by
these ABC proteins are thought to first bind to the membrane domain
from which signals are transduced to the NBDs. This causes primarily,
but not exclusively, the stimulation of ATPase activity by the NBDs in
P-glycoprotein or to inhibition of ATPase activity, as in the case of
MDR3. In our present studies, we found that GSH significantly inhibited
ATPase activity by purified CFTR. Therefore, we suggest that this
modulation may be consistent with the proposed role of CFTR in the
energy-dependent transport of this anion across epithelial
membranes. Clearly, further studies of this putative function using our
reconstitution system for purified CFTR are required to substantiate
this claim.
Finally, we determined by direct measurement of the ATPase activity of
purified and reconstituted wild type and mutant CFTR proteins that
perturbation of the pore signals long distance conformational changes
to the NBDs and causes a change in the function of these domains. These
findings support a novel mechanism for the regulation of this protein
and potentially a novel molecular mechanism wherein disease-causing
mutations may affect function.
) flux in affected epithelial
tissues. CFTR is a Cl
channel that is regulated by
phosphorylation, nucleotide binding, and hydrolysis. However, the
molecular basis for the functional regulation of wild type and mutant
CFTR remains poorly understood. CFTR possesses two nucleotide binding
domains, a phosphorylation-dependent regulatory domain, and
two transmembrane domains that comprise the pore through which
Cl
permeates. Mutations of residues lining the channel
pore (e.g. R347D) are typically thought to cause disease by
altering the interaction of Cl
with the pore. However, in
the present study we show that the R347D mutation and
diphenylamine-2-carboxylate (an open pore inhibitor) also inhibit CFTR
ATPase activity, revealing a novel mechanism for cross-talk from the
pore to the catalytic domains. In both cases, the reduction in ATPase
correlates with a decrease in nucleotide turnover rather than affinity.
Finally, we demonstrate that glutathione (GSH) inhibits CFTR ATPase and
that this inhibition is altered in the CFTR-R347D variant. These
findings suggest that cross-talk between the pore and nucleotide
binding domains of CFTR may be important in the in vivo
regulation of CFTR in health and disease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ADP from [
-32P]ATP by purified,
reconstituted CFTR, as described previously (18, 36). Radiolabeled ADP
and ATP were separated by polyethyleneimine chromatography. Correction
for spontaneous hydrolysis of ATP in reactions containing
proteoliposomes was done by subtracting the [
-32P]ADP/[
-32P]ATP ratio of control
liposomes. Unless otherwise stated, the ATPase assay was carried out in
a reaction mixture containing 30 µl of freshly dialyzed
proteoliposomes or liposomes, 1 mM nonradioactive ATP, 20 mM Tris, 40 mM NaCl, 4 mM
MgCl2, pH 7.5, and 2 µCi of [
-32P]ATP
(10 µCi/µl; Amersham Pharmacia Biotech). In few ATPase reactions, NaCl was substituted for sodium gluconate or NMDG-Cl. When the final
concentration of chloride, gluconate, or NMDG-chloride was other than
48 mM, the concentration of the inorganic anion was adjusted accordingly to obtain either 14 or 144 mM.
However, the concentration of all other components was unchanged, and
osmolarity was maintained constant by adjusting with mannitol.
-32P]ATP was added to the proteoliposomes followed by
a 2-h incubation at 33 °C.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ATPase activity of purified, reconstituted
wild type CFTR protein in the absence and presence of the channel
blocker DPC. Catalytic activity was measured as the production of
[ -32P]ADP from [
-32P]ATP after
incubation with [
-32P]ATP for 2 h at 33 °C.
ADP and ATP were separated by thin layer chromatography (TLC). The
quantity of radioactive ADP and ATP was assayed using ImageQuant
software (Molecular Dynamics). Panel A, a representative TLC
plate showing ATP hydrolyzed by PKA-phosphorylated and
nonphosphorylated CFTR protein. Panel B, ATPase activity of
phosphorylated CFTR protein in the absence and presence of DPC (2 and
10 mM). Panel C, quantitation of the effect of
DPC (2 and 10 mM) on the catalytic activity of
phosphorylated (closed bars) and nonphosphorylated
(open bars) CFTR protein relative to untreated control. The
effect of the treatment relative to protein with no drug added
(control) was analyzed by one-population Student's t test.
The asterisk represents statistically significant changes in
ATPase activity (*, p < 0.05; **, p < 0.002). Each value is represented as mean ± S.E.,
(n = 4). At least two different preparations of protein
were assessed for each treatment group.
View larger version (14K):
[in a new window]
Fig. 2.
MgATP dependence of ATP hydrolysis by
purified phosphorylated wild type CFTR protein in the presence and
absence of DPC. Each point represents the mean activity for a set
of duplicate values. Data were analyzed according to the
Michaelis-Menten equation to yield parameters for CFTR protein in the
absence of DPC : Vmax = 0.84, Km = 0.98 mM, and
r2 = 0.98 or, in the presence of DPC :
Vmax = 0.18, Km = 0.37 mM, r2 = 0.92.
View larger version (21K):
[in a new window]
Fig. 3.
Effect of the channel blockers glibenclamide
or DIDS on the catalytic activity of PKA-phosphorylated and
nonphosphorylated CFTR. Me2SO (DMSO) was
used as a solvent for glibenclamide. The effect of the treatment in
each group was calculated as a percentage of no-drug control. Mean ± S.E. are shown. Two different protein preparations were studied
independently with respect to the effect of glibenclamide or DIDS. For
the Me2SO control, each point represents the mean activity
for a set of duplicate values.
View larger version (18K):
[in a new window]
Fig. 4.
Effect of salt concentration on the ATPase
activity of phosphorylated and nonphosphorylated wild type CFTR
protein. Panel A, changes in CFTR ATPase activity in
the presence of different concentrations of NaCl relative to 48 mM NaCl control were analyzed by one-way ANOVA
(p = 0.005), with a post-test for linear trend
(p = 0.002). Each value is shown as the mean ± S.E. Three protein preparations, studied in duplicates, are shown for
each treatment group, except for 30 mM chloride, where each
value represents a mean of duplicate values. Panel B,
comparison of the effect of 14 mM (hatched bar)
or 144 mM (open bar) NaCl, sodium gluconate
(Na-Glc), or NMDG-chloride on the catalytic activity of
phosphorylated CFTR. The effect of the different salts with the same
concentration relative to each other on the catalytic activity of CFTR
was analyzed by ANOVA. No statistical differences were found among the
different treatment groups with the same salt concentration. However,
inhibition of ATPase was seen in all groups at high salt concentration
(*, unpaired Student's t test, p < 0.02).
Three different protein preparations were studied independently with
respect to the effect of NaCl (14 and 144 mM) or Na-Glc
(144 mM). For Na-Glc (14 mM) and NMDG-Cl (14 and 144 mM), each value represents the mean activity for a
set of duplicate values.
View larger version (16K):
[in a new window]
Fig. 5.
Characterization of the effect of CFTR-R347D
mutation on the ATPase activity. Panel A, catalytic
activity of phosphorylated and nonphosphorylated wild type
(WT) and mutant CFTR proteins at 1 mM ATP. The
mean ± S.E. is shown for nine phosphorylated and
nonphosphorylated wild type CFTR preparations. For CFTR-R347D
preparations, each bar represents the activity of duplicate
values. Differences between either phosphorylated or nonphosphorylated
wild type and mutant preparations were analyzed by unpaired Student's
t test. The asterisk represents a statistically
significant difference in ATPase activity of phosphorylated mutant
relative to phosphorylated wild type CFTR (p = 0.014).
PKA phosphorylation of liposomes alone, treated in the same manner as
CFTR-R347D preparations, accounted for less than 8% of the ATPase
activity observed for the phosphorylated CFTR-R347D protein (0.16 nmol
of ATP hydrolyzed/2 h versus 2.03 nmol of ATP hydrolyzed/2
h). The effect of PKA phosphorylation was calculated by subtracting the
basal ATPase activity of nonphosphorylated liposomes from PKA-treated
liposomes. Panel B, MgATP dependence of the catalytic
activity of purified and either phosphorylated or nonphosphorylated
CFTR-R347D protein. Curve fitting was performed by nonlinear regression
analysis (Prism software, San Diego, CA) using the Michaelis-Menten
equation to yield parameters for the phosphorylated protein of
Vmax = 1.1, Km = 0.1 mM and, for the nonphosphorylated protein, of
Vmax = 0.2, Km = 1.1 mM. The mean ± S.E. is shown for each treatment
group. Each sample contained ~8 µg of purified, reconstituted
CFTR-R347D protein, and duplicate or triplicate samples were assessed.
Panel C, effect of ionic strength on the ATPase activity of
phosphorylated wild type CFTR and CFTR-R347D proteins. Differences in
catalytic activity between the mutant (hatched) and wild
type (open) proteins at the same salt concentration were
assessed by unpaired Student's t test. The
asterisk represents statistically significant difference
(p = 0.02). Duplicate samples of wild type protein and
triplicate samples of CFTR-R347D were studied.
Kinetic parameters of PKA-phosphorylated and non-phosphorylated
wild-type and R347D proteins
View larger version (20K):
[in a new window]
Fig. 6.
Effect of glutathione on the catalytic
activity of wild type and CFTR-R347D proteins. Panel A,
effect of reduced glutathione (GSH) or oxidized glutathione (GSSG) on
the ATPase activity of phosphorylated (closed bars) or
nonphosphorylated (open bars) CFTR. Differences in catalytic
activity relative to no drug control were analyzed by one-population
Student's t test. The asterisk (*) represents
significant changes in ATPase activity (p < 0.03). The
mean ± S.E. is shown for two to four protein preparations. For 10 mM GSSG, each point represents the mean activity for a set
of duplicate values. Panel B, effect of increasing GSH
concentrations on the catalytic activity of phosphorylated CFTR as a
percentage of control (no GSH). Data were analyzed by ANOVA
(p = 0.0003), with a post-test for linear trend
(p < 0.001). Mean ± S.E. is shown for three to
four protein preparations. Panel C, effect of 10 mM GSH on the catalytic activity of phosphorylated wild
type (WT, open) and CFTR-R347D
(hatched) proteins relative to control (no GSH). Differences
in ATPase activity were analyzed by Student's t test.
Statistical significance (p < 0.05) is marked by
asterisk (*). Mean ± S.E. for quadruplicate samples is
shown for each treatment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Mary Corey (Research Institute, Hospital for Sick Children) for the assistance with the statistical analysis and Dr. Johanna Rommens for providing us with cDNA coding for the mutant R347D (Research Institute, Hospital for Sick Children).
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FOOTNOTES |
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* This work was supported by the Canadian Cystic Fibrosis Foundation (to C. E. B.).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.
§ Supported by Natural Sciences and Engineering Research Council of Canada and Canadian Cystic Fibrosis Foundation studentship awards.
¶ To whom correspondence should be addressed: Research Institute, Hospital for Sick Children, 555 University Ave., Toronto M5G 1XB, Canada. Tel.: 416-813-5981; Fax: 416-813-5028; E-mail: bear@sickkids.on.ca.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M010403200
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ABBREVIATIONS |
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The abbreviations used are: CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator; NBD, nucleotide binding domain; ABC, ATP binding cassette; DPC, diphenylamine-2-carboxylate; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; GSH, reduced glutathione; GSSG, oxidized glutathione; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoic acid; PKA, protein kinase A; NMDG-Cl, N-methyl-D-glucamine chloride; ANOVA, analysis of variance.
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