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(Received for publication, August 9, 1996)
From the Divisions of Cell Biology and § Genetics,
Research Institute, Hospital for Sick Children,
Toronto, Canada M5G 1X8
ABSTRACT The gene mutated in cystic fibrosis codes for the
cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic
AMP-activated chloride channel thought to be critical for salt and
water transport by epithelial cells. Plausible models exist to describe
a role for ATP hydrolysis in CFTR channel activity; however,
biochemical evidence that CFTR possesses intrinsic ATPase activity is
lacking. In this study, we report the first measurements of the rate of
ATP hydrolysis by purified, reconstituted CFTR. The mutation CFTRG551D
resides within a motif conserved in many nucleotidases and is known to
cause severe human disease. Following reconstitution the mutant protein
exhibited both defective ATP hydrolysis and channel gating, providing
direct evidence that CFTR utilizes ATP to gate its channel
activity.
INTRODUCTION The cystic fibrosis transmembrane conductance regulator
(CFTR)1 is an integral membrane protein
that resides on the apical membrane of several types of epithelial
cells, and its absence or dysfunction in cystic fibrosis is thought to
impair severely the salt and water transport capacity of these cells
(1). CFTR shares structural similarities with other members of the
ATP-binding cassette (ABC) superfamily of traffic ATPases; namely, it
possesses two repeats, each consisting of a membrane-spanning domain
followed by a nucleotide-binding domain (2, 3). Furthermore, CFTR
possesses a unique cytoplasmic domain, the R domain, which possesses
several putative sites for PKA and PKC phosphorylation and links the
two halves of the molecule (2).
Unlike other members of the ABC superfamily, CFTR exhibits chloride
channel activity. The chloride channel function of CFTR was implicated
in mutagenesis studies in which substitution of certain charged
residues within the putative transmembrane-spanning domains caused
changes in single channel conductance or anion selectivity (4, 5).
Further convincing evidence of the chloride channel activity of CFTR
came from our electrophysiological studies of purified, reconstituted
CFTR (6). Fusion of liposomes containing purified CFTR with planar
lipid bilayers resulted in the appearance of chloride-selective
channels that exhibited biophysical features identical to those
associated with CFTR expression in epithelial cell membranes (7,
8).
Phosphorylation by PKA is required but not sufficient to activate CFTR
chloride channel function. Electrophysiological evidence supports the
hypothesis that hydrolysis of ATP is essential for normal opening and
closing or gating of the channel (9, 10, 11, 12). First, the addition of
Mg-ATP, but not nonhydrolyzable ATP analogs, to PKA-phosphorylated CFTR
causes channel gating (10). Second, the subsequent addition of
nonhydrolyzable ATP analogs to ATP-activated CFTR channels interferes
with normal channel gating kinetics (13). Third, chelation of magnesium
ion, an essential cofactor in most reactions involving nucleotide
binding and/or hydrolysis, causes alterations in the rates of channel
opening and closing (14, 15). However, other research groups have
reported electrophysiological studies with conflicting results, namely
that nonhydrolyzable analogs of ATP fail to compete with the opening or
closing of CFTR channels (16) and that gating persists, albeit at a
slower rate, after removal of magnesium (17, 18). As it has been widely
hypothesized that CFTR is functionally coupled to other membrane
proteins through direct protein interactions (19), it is possible that
the sensitivity of CFTR channel gating to ATP is conferred or modulated
by associated membrane proteins. Hence, contradictory reports such as
those described above may reflect variabilities in the membrane
environment for CFTR.
To eliminate possible modulatory effects of associated membrane
proteins, we examined the role of ATP hydrolysis in CFTR channel gating
using purified, reconstituted CFTR protein. We present novel
biochemical evidence that the CFTR protein possesses intrinsic ATPase
activity and that this catalytic activity is coupled directly to the
channel function of this protein.
EXPERIMENTAL PROCEDURES Two liters of Sf9 cells
were infected with recombinant baculovirus containing the complete
coding sequence for CFTR as described previously (6). CFTR purification
was performed according to our published procedure (6) with the
following modifications. Infected cells were harvested after 48 h,
and the pellet was treated with a phosphate-buffered saline containing
2% Triton X-100, 2 units/ml DNase, 5 mM MgCl2,
2 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 1 mM benzamidine, and 10 µM E64.
The mixture was stirred for 2 h at 15 °C after which the
insoluble material was centrifuged at 100,000 × g for
2 h. The resulting pellets were treated with 180 ml of 2% SDS,
3% mercaptoethanol in 10 mM sodium phosphate, pH 7.2, and
the mixture was stirred overnight at 4 °C. Insoluble material was
centrifuged at 60,000 × g, and the supernatant was
filtered through a 0.22-µm filter before being applied at 1 ml/min to
a ceramic hydroxyapatite column. Washing, elution, and identification
of the CFTR-containing peaks were performed as described previously
(6). The CFTR-containing fractions from the ceramic hydroxyapatite
column were concentrated and applied to a Superose 6 column as the
final purification step. The purified protein was quantitated by amino
acid analysis. NH2-terminal sequencing and amino acid
analysis were performed by the HSC-U of T-Pharmacia Biotechnology
Centre. An identical procedure was used for the purification of the
CFTR variant CFTRG551D.
SDS-polyacrylamide gel electrophoresis of
purified CFTR and CFTRG551D protein was performed using 6% acrylamide
gels as described previously (20). Total protein was detected with
silver stain as described previously. For immunoblotting, protein was
transferred to nitrocellulose, and CFTR was probed with an anti-CFTR
monoclonal antibody as described previously (20). Immunoreactive bands
were visualized by chemiluminescence using the ECL system (Amersham
Corp.)
The CFTR
sample (5 µg) in 1 ml of solution containing 10 mM Tris
buffer, pH 7.8, containing 0.25% lithium dodecyl sulfate and 100
mM NaCl was concentrated in a Centricon concentrator
(molecular mass cutoff, 100 kDa) to a volume of 100 µl. The protein
sample was then diluted to 1,000 µl with 8 mM Hepes
buffer containing 0.5 mM EGTA, pH 7.2 (buffer A), and
reconcentrated to a 100-µl volume. The final lithium disulfate
concentration was 0.025%. Liposomes, 10 mg/ml, were prepared in buffer
A as described previously using a lipid mixture of PE:PS:PC:ergosterol
5:2:1:1 by weight. 100 µl of liposomes containing 1 mg of lipid was
added to 100 µl of the CFTR sample yielding a final lithium dodecyl
sulfate concentration of 0.0125%. The mixture was dialyzed
(Spectrapor; molecular mass cutoff, 50 kDa) for 18 h against 2
liters of buffer A containing 1.5% sodium cholate. Dialysis was then
continued against 4 liters of buffer A for 2 days with daily changes of
buffer. For lipid bilayer studies, nystatin was introduced into the
reconstituted proteoliposomes as described previously (6). An identical
protocol was employed for the reconstitution of CFTRG551D.
ATPase activity was measured as the production
of [ CFTR
proteoliposomes and liposomes without CFTR, both in 50 mM
Tris, 50 mM NaCl at pH 7.4, were mixed with catalytic
subunit of PKA (final concentration, 200 nM), 20
µM ATP, and 5 mM MgCl2, sonicated
briefly, and incubated for 1 h at room temperature. These
conditions mimic those used for in vitro phosphorylation of
CFTR immunoprecipitated from Chinese hamster ovary cells (21). To
confirm that purified, reconstituted CFTR was phosphorylated under
these conditions, [ Purified CFTR was reconstituted into proteoliposomes
containing a phospholipid mixture PE:PS:PC:ergosterol (5:2:1:1) by
weight for bilayer studies. Nystatin (120 µg/ml) was introduced into
the proteoliposomes for bilayer studies to promote proteoliposome
fusion and to facilitate detection of these fusion events (24). Fusion
of nystatin-containing liposomes was indicated by the appearance of
transient ``nystatin spikes'' in bilayer conductance. As in our
previous experiments, a 10 mg/ml solution of phospholipid (PE:PS at a
ratio of 1:1, Avanti Polar Lipids) in n-decane was painted
over a 200-µm aperture in a bilayer chamber to form bilayers. Bilayer
formation was monitored electrically by observing the increase in
membrane capacitance. In all experiments, bilayer capacitance was
greater than 200 picofarads. Fusion of liposomes was potentiated with
the establishment of an osmotic gradient across the lipid bilayer. The
cis compartment of the bilayer chamber, defined as that
compartment to which liposomes were added, contained 300 mM
KCl; the trans compartment, connected to ground, contained
50 mM KCl. Single channel currents were monitored at a
holding potential of Open and closed time histograms were
created with a logarithmic x axis with 10 bins/decade and a
lower limit of 10 ms. The maximum likelihood method was used to fit the
data with one or two exponentials (pClamp 6.0.2 software). The goodness
of fit was assessed using the log likelihood ratio test. The mean open
and closed time constants derived from histograms generated from single
experiments (approximately 5 min in duration) were comparable to values
derived from histograms generated from four combined experiments
(i.e. four separate protein preparations). Hence, we have
shown open and closed time histograms of combined experiments. Further,
combination of data from multiple experiments permits the generation of
burst duration histograms. Burst analysis was performed using
tc = 60 ms. tc, the time that separates
short, intraburst closures from long, interburst closures, was
estimated using several approaches (25, 26). The latter approach
described by Winter et al. for CFTR in biological membranes
yielded a value for tc which led to the optimal separation
of intraburst and interburst closed times. According to Winter and
co-workers, tc can be determined by examination of the
biexponential fit of the closed time histogram for single channel
activity. tc corresponds to the nadir between the two peaks
of the relationship defining the first and second closed time
constants. A value for tc of 60 ms was determined as the
mean from four different closed time histograms corresponding to the
four different protein preparations (60.5 ± 2.1 ms) as well as
from the closed time histogram generated from the combination of the
four experiments (60.1 ± 0.5 ms).
Results are expressed as means ± S.D.
Statistical significance was assessed using the log likelihood ratio
test or Student's t test.
RESULTS The scheme for purification of recombinant CFTR from Sf9 cells and
reconstitution in phospholipid liposomes was published previously (6).
We used a similar purification and reconstitution strategy in our
present studies of CFTR ATPase activities. We confirmed the homogeneity
of the purification products used in these experiments by evaluation of
silver-stained gels overloaded with CFTR protein and by Western blot
analysis of the reconstituted protein (Fig.
1A). In Fig. 1B we show that
reconstituted, purified CFTR protein does possess intrinsic ATPase
activity. The production of radiolabeled dinucleotide
[
As in the case of CFTR chloride channel activity, CFTR ATPase activity
is dependent upon phosphorylation (Fig. 2). Pretreatment
of purified CFTR with the serine/threonine protein phosphatase 2A
caused marked inhibition of ATP hydrolysis (Fig. 2A). Hence,
purified CFTR likely remains partially phosphorylated throughout the
purification procedure. Modulation of CFTR ATPase activity by
phosphorylation is shown in further studies where CFTR is fully
phosphorylated with PKA catalytic subunit. PKA pretreatment of CFTR
caused a 2-3-fold activation of the rate of ATP hydrolysis. The
substrate dependence for ATPase activity of both partially
phosphorylated (not pretreated with PKA) and fully phosphorylated
(PKA-treated) protein was determined and the data fitted by nonlinear
regression analysis using the Hill equation, v =
SmVmax/Sm
+ K
PKA modification of CFTR ATPase activity
Volume 271, Number 45,
Issue of November 8, 1996
pp. 28463-28468
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
,,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-32P]ADP from [-32P]ATP using
polyethyleneimine-cellulose chromatography for separation of the
nucleotides. Unless otherwise stated, the assay was carried out in a
15-µl reaction mixture containing 50 mM Tris, 50
mM NaCl, pH 7.5, 2 mM MgCl2, 10%
glycerol, 0.5 mM CHAPS, and 8 µCi of
[-32P]ATP. Reaction mixtures were incubated at
30 °C and were stopped by the addition of 5 µl of 10% SDS.
One-µl samples were spotted on a polyethyleneimine-cellulose plate
and developed in 1 M formic acid, 0.5 M LiCl.
The location and quantitation of the radiolabeled ATP and ADP were
determined with a Molecular Dynamics PhosphorImager. Data were analyzed
using the ImageQuant software package (Molecular Dynamics).
-32P]ATP was used in one experiment
to phosphorylate CFTR. An autoradiograph of the phosphorylated protein
run on 6% SDS-polyacrylamide gel (22) showed a single intense band
corresponding to CFTR. To remove PKA after the phosphorylation
reaction, CFTR proteoliposomes and control protein-free liposomes were
airfuged at 100,000 × g for 30 min and then washed
twice by sonication with buffer and pelleted in the Airfuge. Pellets
were resuspended in the appropriate buffer for ATPase measurements. For
bilayer studies of channel function, CFTR was phosphorylated as
described in the preceding paragraph. Subsequently, PKA was separated
from the proteoliposomes by microspin chromatography using Sephadex
G-50. To validate complete removal of PKA, we assessed the presence of
125I-labeled catalytic subunit of PKA in the proteoliposome
sample and determined that three cycles of sonication followed by
separation on a microspin column were needed for complete removal of
the labeled subunit of PKA. To dephosphorylate CFTR, proteoliposomes in
50 mM Tris, 50 mM NaCl (1 µg of purified
protein/50 µl, pH 7.4) were mixed and incubated with protein
phosphatase 2A (3.75 units/ml; Calbiochem) for 2 h at room
temperature. This concentration of protein phosphatase 2A has been
shown to dephosphorylate CFTR in biological membranes (23). Prior to
treatment, the phosphatase was dialyzed against the above Tris buffer
to partially remove glycerol in the storage medium.
40 mV applied to the cis compartment and
detected with a bilayer amplifier (custom made by M. Shen, Physics
Laboratory, University of Alabama). Data were recorded and analyzed
using pClamp 6.0.2 software (Axon Instruments, Inc). Prior to analysis
of open probability and dwell times, single channel data were filtered
digitally at 100 Hz. Ideal records were created by use of a half-height
transition protocol (pClamp 6.0.2 software).
-32P]ADP from [-32P]ATP was
determined following separation of the nucleotides on thin layer
chromatography plates, a technique commonly used for the measurement of
GTPase activity of purified G proteins (27, 29). The rate of
[-32P]ADP production by liposomes containing purified,
PKA-phosphorylated CFTR was linear over a 5-h time period (Fig.
1B). ATPase activity was conferred by purified CFTR protein
as there was no detectable ATPase activity in suspensions of
protein-free liposomes. In Fig. 1C we show that our
measurements of CFTR ATPase activity are reproducible. The mean ATPase
activity exhibited by three distinct preparations of purified CFTR is
significantly greater than that detected in preparations of protein
free liposomes (p < 0.02).
Fig. 1.
Purified, reconstituted CFTR functions as an
ATPase. Panel A. Left, silver staining after
SDS-polyacrylamide gel electrophoresis (6% acrylamide) of aliquots
containing approximately 750 ng of purified CFTR protein.
Right, immunoblot of reconstituted CFTR protein probed with
monoclonal antibody M3A7. Panel B, ATP hydrolysis exhibited
by proteoliposomes containing 75 ng of purified, PKA-phosphorylated
CFTR (
) (from preparation assessed in panel A) but not by
liposomes alone (
). The rate of hydrolysis by purified CFTR was
linear with time over 5 h. Panel C, three different
preparations of proteoliposomes containing 75 ng of phosphorylated CFTR
(+CFTR) exhibiting significantly higher ATPase activity than
that measured in paired protein-free, liposome preparations ()
(p < 0.01). ATPase activity was determined at 4 h
after initiation of the reaction in the presence of 1 mM
Mg-ATP. The mean value ± S.D. is shown.
[View Larger Version of this Image (13K GIF file)]
, where v and Vmax are
the initial and maximum initial rates, respectively; S is
the ATP concentration, and m is the Hill coefficient. The
constant K is related to Km by the
equation K = (Km)m. As apparent
in Fig. 2B and Table I, PKA treatment
modifies ATPase activity of CFTR by causing a reduction in apparent
Km from 1 mM to 0.3 mM,
presumably by increasing the affinity of CFTR for ATP. The shape of the
function describing ATP dependence of ATPase activity changed from
hyperbolic, for the partially phosphorylated protein, to sigmoidal for
the PKA-treated, fully phosphorylated protein. This change in shape was
reflected by a change in the Hill coefficient from 1 to 1.7. Therefore,
full phosphorylation of CFTR appears to induce positive cooperativity
between two sites of ATPase activity. The Vmax
for CFTR ATPase activity, 50 (nmol/mg of purified protein/min), was not
altered by PKA treatment. It is probable that we have underestimated
the ATPase activity of CFTR as it is likely that only a fraction of
purified protein has been reconstituted completely to its active
configuration. Previously, we estimated on the basis of reconstitution
of single channel activity, that 20-40% of total purified protein was
functional following fusion to planar lipid bilayers (6). Hence, the
ATPase activity by CFTR may be as high as 125-250 nmol/mg/min, which
corresponds to a turnover number of approximately 0.5-1 molecules of
ATP hydrolyzed/s.
Fig. 2.
Phosphorylation modulates ATPase activity of
purified CFTR. Panel A, regulation of CFTR ATPase activity
by phosphorylation. ATPase activity of PKA-treated preparations was
expressed as a percentage of the activity measured in a paired,
untreated control preparation. The bar graph shows the mean
percentage increase ± S.D. caused by PKA phosphorylation relative
to control for four different purified protein preparations. The change
in ATPase activity induced by phosphorylation was significant,
p < 0.02. Purified CFTR was dephosphorylated using
protein phosphatase 2A. This phosphatase caused marked inhibition of
CFTR ATPase activity relative to untreated control preparations in both
experiments. Panel B, untreated () or PKA-treated ()
CFTR protein was assessed for ATPase activity at varying Mg-ATP
concentrations to determine the substrate dependence of this reaction.
Each reaction mixture contained 25 ng of protein and was incubated for
4 h before quenching. One-µl aliquots of each reaction mixture
were analyzed for ADP formation. Each point shows the mean ± S.D.
of triplicate experiments. The curves were fitted by nonlinear
regression analysis using the Hill equation. The best curve fit was
assessed as the fit that corresponded to the lowest S.D. The software
program for regression analysis was Regression, version M1.11
(Blackwell Scientific Publications, Oxford, UK). The inset
in the lower right corner shows an expansion of the curve
fit for the relationship between low ATP concentrations (0.05-1.0
mM ATP) and ATPase activity for phosphorylated CFTR
().
[View Larger Version of this Image (17K GIF file)]
Kinetic parameters
Vmax
Kma
Hill
coefficientb
nmol/mg
protein/min
µM
CFTR
51.3
989.7
1
CFTR-Pc
53.8
303
1.73
a
Calculated from K =
(Km)m.
b
Obtained by nonlinear regression analysis to the Hill
equation.
c
Phosphorylated CFTR.
We have shown in previous publications that purified, reconstituted CFTR exhibits channel activity with biophysical properties identical to those observed for CFTR in cell membranes; namely, it is anion-selective, exhibits a low unitary conductance of approximately 10 picosiemens, and requires phosphorylation by the addition of exogenous PKA plus Mg-ATP for activity (6, 28, 29, 30). Addition of Mg-ATP but not its nonhydrolyzable analog, Mg-AMPPNP, caused channel activity of phosphorylated CFTR reconstituted in planar lipid bilayers (data not shown), supporting the hypothesis that ATP hydrolysis rather than binding is required to open the CFTR channel.
It has been postulated that ATP is utilized by CFTR to cause gating of
its chloride channel activity. Hence, the rate of ATP hydrolysis by
CFTR should be comparable to the rate of channel gating. As we have
quantitated ATP hydrolysis by purified CFTR we can examine this
hypothesis directly in the same protein specimen. Our planar bilayer
studies of purified protein verified patch-clamp studies that showed
that CFTR exhibits a bursting pattern of gating in the presence of 1
mM Mg-ATP (Fig. 3A). As shown in
Fig. 3B, there is a single population of channel-open times
and two populations of channel-closed times, a short and a long closed
time. Only the longer of the two closed times exhibits ATP dependence,
suggesting that only the transition from the long duration closed time
to the channel-open state is substrate-dependent. For
subsequent burst analysis, we used a value of 60 ms as the burst
delimiter (tc). tc was determined according to
the method described by Winter et al. (26). Briefly,
tc was derived from the function describing the closed dwell
time histogram as the nadir between the peak values that define the
mean short closed (intraburst) times and the long closed (interburst)
times. Following analysis of the ATP dependence of burst and interburst
duration histograms (Fig. 3C), we found that both the
transition rate to the open burst (1/between burst duration) and the
transition rate to the interburst, closed state (1/burst duration) were
altered by increasing ATP concentration. The effective bursting rate
increased with increasing ATP concentration, and this relationship was
fit by nonlinear regression analysis using the Hill equation to yield a
Km of 584 µM, maximal opening rate of
10/s, and a Hill coefficient of 2. The Hill coefficient of 2 suggests
that there is positive cooperativity between two sites through which
ATP can act to promote channel bursting. The effective closing rate
from an open burst decreased slightly with increasing ATP
concentrations, and these data were fit using an exponential decay
function to derive an estimate of the ATP concentration required for
half-maximal inhibition of closing, 434 µM. Assuming that
the overall transition rate between the two conductance states of CFTR
channel is limited by the slowest rate, i.e. bursting or
closing rate, we predict that at 1 mM ATP, the slow closing
rate will limit gating of CFTR to 1-2 transitions/s. Our quantitation
of the rate of CFTR gating is comparable to the rate of ATP hydrolysis
by CFTR, namely, 0.5-1 molecules of ATP hydrolyzed/s. Hence, we argue
on the basis of our kinetic analyses that CFTR channel activity is
coupled to ATP turnover.
Fig. 3. ATP dependence of CFTR bursting channel activity. Panel A, purified, phosphorylated CFTR exhibits bursting channel activity in planar lipid bilayers at a holding potential of
40 mV. Panel B. i, CFTR
channel-open and channel-closed dwell time histograms were generated
from four experiments obtained from four different protein
preparations, comprising data from at least 20 min of recording. The
channel-open time histogram (left) was fit by a single
exponential distribution, and the closed time histogram
(right) was best fit using two exponential distributions,
indicating the presence of both short and long duration closed times.
The nadir predicted by the fit of the closed time histogram is
indicated as (tc = 60 ms). ii, in the
accompanying graph, mean open times () and mean short closed times
() and mean long closed times (
) at various Mg-ATP concentrations
are shown. Mean long closed times () show sensitivity to Mg-ATP
concentration and decrease with increasing nucleotide concentrations.
The relationship between channel long closed times and Mg-ATP
concentration can be fit using a single exponential decay function and
predicts half-maximal inhibition of long closed times at 445
µM ATP. Panel C. i, channel burst
analysis was performed as described under ``Experimental
Procedures.'' Burst and interburst time histograms (shown
left and right, respectively) are fit by single
exponential distributions. ii, the relationship between the
rate of channel opening to a burst (
) and ATP concentration was fit
by nonlinear regression analysis according to the Hill equation. The
relationship between the rate of channel closing from a burst was fit
by a single exponential decay function (). Means ± S.D. are
shown when these values exceed the size of the symbol.
[View Larger Version of this Image (30K GIF file)]
Further evidence for coupling between CFTR channel and ATPase functions
is found in our studies of the sensitivity of these two activities for
magnesium ion and sodium azide. In Fig. 4A we
show that chelation of magnesium ion with EDTA inhibits channel opening
as well as ATPase activity. Phosphorylated CFTR protein reconstituted
in planar lipid bilayers showed channel activity when exposed to
MgCl2 (10 µM) plus Na-ATP (100
µM), where the free Mg2+ concentration was
estimated at 4 µM (31). The subsequent addition of EDTA
(1 mM) reduced free Mg2+ to the concentration
of 4 nM and abolished channel gating. Similarly, ATPase
activity of phosphorylated CFTR was reduced upon chelation of
Mg2+. As further evidence for coupling between CFTR channel
and ATPase activities, we found that both functions exhibited
sensitivity to sodium azide (Fig. 4B). Sodium azide
(NaN3) (1 mM), a well known inhibitor of the
F1F0-ATPase (32), inhibited opening of the
purified CFTR chloride channel as well as CFTR ATPase activity.
Fig. 4. Coupling of the chloride channel and ATPase activities of purified CFTR. Panel A. i, purified CFTR was phosphorylated and reconstituted into lipid bilayer as described under ``Experimental Procedures.'' Chloride channel activity was observed in the presence of MgCl2 (10 µM) and Na-ATP (100 µM) at a holding potential of
40 mV. This record shows activity of three CFTR channels
with openings indicated as
O1-O3. Mean channel-open
probability was 0.3 in this particular experiment. Addition of EDTA (1
mM) as indicated completely abolished the CFTR channel
activity, where the free Mg2+ concentration was estimated
at 4 nM. This experiment is representative of four studies.
ii, effect of magnesium on ATPase activity. In the
left lane (Mg), CFTR (75 ng) was phosphorylated
as described previously and dialyzed against a buffer containing 50
mM Tris, 50 mM NaCl, and 5 mM EDTA
for several hours prior to the ATPase assay. ATPase activity was
determined 4 h after the addition of Na-ATP, in the absence of
added Mg2+ (n = 3). In the right
lane (+Mg), ATPase activity of phosphorylated CFTR was
determined in the presence of 5 mM Mg2+
(n = 3). Bars indicate the mean ± S.D.
for ATPase measurements. Panel B. i, chloride
channel activity exhibited by purified, phosphorylated CFTR in the
presence of Mg-ATP (100 µM) was inhibited by the addition
of NaN3 (1 mM) to both cis and
trans compartments of the bilayer chamber (n
= 5). ii, the bar graph shows that ATPase
activity of phosphorylated CFTR (75 ng) was inhibited by pretreatment
with 5 mM NaN3 (n = 3). The
mean ± S.D. is shown.
[View Larger Version of this Image (25K GIF file)]
Finally, we assessed coupling between the ATPase and channel functions
of CFTR by determining the effect of a site-directed mutation on both
activities. The relatively common cystic fibrosis mutation, CFTRG551D,
leads to severe disease (33). Glycine 551 lies within a sequence in the
first nucleotide-binding fold, D-X-[G/A]-G-Q, which shows
remarkable conservation in ABC transporters and in G proteins and has
been implicated in NTP-binding and hydrolysis (34, 35, 36). In the present
studies, we found that purified CFTRG551D exhibits altered
chloride channel activity when reconstituted in lipid bilayers
(Fig. 5A). Although the unitary conductance
of CFTRG551D is comparable to that of the wild type protein,
approximately 10 picosiemens, arguing against global changes in protein
structure, the open probability of the mutant protein was much lower
than that determined in the wild type protein, 0.011
versus 0.48, respectively. Similarly, reconstituted
CFTRG551D protein exhibits altered ATPase activity (Fig.
5B). At 1 mM Mg-ATP the rate of ATP hydrolysis
by the mutant protein occurs at approximately 10% that of the wild
type (2.5 nmol/mg of protein/min versus 20 nmol/mg of
protein/min). Hence, we have shown that a mutation within a sequence
conserved among NTPases causes a reduction both in channel gating and
ATPase activity.
Fig. 5. CFTRG551D exhibits defective ATPase and channel activities. Panel A, channel activity of a single molecule of reconstituted CFTRG551D is observed following fusion of proteoliposomes with the lipid bilayer at a holding potential of
40
mV. Fusion was detected as a nystatin conductance spike. The CFTRG551D
channel opens infrequently and for a brief period of time. Panel
B, relationships between ATP concentration and ATPase activity of
reconstituted CFTR (, n = 3) and CFTRG551D (
,
n = 3) were fitted by nonlinear regression analysis
using the Hill equation. The mean ± S.D. is shown. Each sample of
CFTR and CFTRG551D contained 75 ng of purified protein.
[View Larger Version of this Image (12K GIF file)]
DISCUSSION
In these studies of purified, reconstituted CFTR protein we have provided the first direct biochemical evidence that this protein functions as an ATPase. We measured the ATPase activity of purified CFTR as 50 nmol/mg/min and estimated that this activity may be as high as 125-250 nmol/mg/min. Significantly, Ko and Pedersen (37) recently reported that a fusion protein containing the first nucleotide-binding fold of CFTR was also capable of hydrolyzing ATP, albeit at a somewhat slower rate of 30 nmol/mg/min. Our estimate of CFTR ATPase activity is less than that measured for P-glycoprotein, a closely related member of the ABC superfamily of transporters, with hydrolytic rates measured as 300 (nmol/mg/min) (38) and 1,650 (nmol/mg/min) (39) and less than rates determined for purified P-type ATPases; Na+,K+-ATPase (800 nmol/mg/min) and Ca2+-ATPase (600 nmol/mg/min) (40). On the other hand, CFTR ATPase activity is similar to that reported for the intrinsic GTPase rates of dynamin (41) and much higher than intrinsic rates reported for the low molecular weight GTPases such as ras (42). The low rate of ATP hydrolysis by CFTR may explain why it cannot be measured in native cell membranes2 nor in some preparations of fusion proteins containing the first nucleotide-binding fold of CFTR (43).
The results presented in this paper support the hypothesis that CFTR ATPase and channel activities are coupled. Both CFTR ATPase activity and channel gating are regulated by PKA phosphorylation. Our kinetic analyses suggest that PKA phosphorylation enhances CFTR ATPase activity by increasing affinity for ATP, possibly through exposure of a second catalytic site. Although this hypothesis must be examined directly in future studies, we speculate that the phosphorylated R domain may function to coordinate the activities of the two nucleotide-binding folds of CFTR. It is well known that CFTR channel gating requires phosphorylation, and a role of the R domain in the coordination of ATP-dependent channel gating has also been implicated (12). Further, we determined that the rates of ATP turnover and channel gating by phosphorylated, purified CFTR are similar, approximately 1/s, attesting to the proposal that these activities are coupled.
The requirement for CFTR ATPase activity in channel gating was assessed by inhibition of ATPase activity by site-directed mutagenesis. Glycine 551 of CFTR lies within a motif that is conserved in nucleotidases, and mutation of this residue to aspartic acid caused a marked reduction in both ATPase activity and channel gating. Hence, the mutation CFTRG551D likely causes altered channel activity (44) and human disease (33) because it possesses defective ATPase activity. Further, chemical modulation of CFTR ATPase activity caused comparable inhibition of the rate of channel gating. In contrast to some electrophysiological studies of CFTR in biological membranes (17, 18), we could observe a clear dependence of channel activation on magnesium ion. This difference may relate to an improved ability to regulate free magnesium ion concentrations in bilayer studies of purified protein.
To understand how CFTR functions in cells it is important to determine
which of its putative activities are intrinsic or dependent upon the
context of other membrane proteins. This issue is of particular
relevance to CFTR and other members of the ABC superfamily of membrane
proteins as it has been hypothesized that they may mediate cellular
transport through direct protein-protein interactions within the
membrane (19). Studies of purified, reconstituted CFTR can be used to
verify or to argue against putative activities of CFTR (6, 30). For
example, the chloride channel activity of CFTR was originally verified
in planar lipid bilayer studies of purified CFTR (6). Conversely, the
putative ATP
channel function of CFTR could not be
confirmed in our recent studies using purified protein (30), leading us
to speculate that CFTR-mediated cellular efflux of ATP reported in some
studies (45) may be due to an effect of CFTR on associated membrane
proteins.
Finally, the results in the present paper clearly show that CFTR possesses intrinsic ATPase activity and support the hypothesis that CFTR utilizes ATP to gate its channel function. Hence, the ATP dependence of channel activity is unlikely to be mediated by neighboring membrane proteins. These results pave the way for more detailed investigations of the mechanism through which ATP is utilized to drive CFTR channel gating. Furthermore, other putative activities for this protein, such as its possible function as a pump for organic substrates, can now be tested directly (3).
FOOTNOTES
The first two authors contributed equally to this work.
¶ Medical Research Council Scientist. To whom correspondence should be addressed: Division of Cell Biology, Research Institute, Hospital for Sick Children, 555 University Ave., Toronto, Canada M5G 1X8. Tel.: 416-813-5981; Fax: 416-813-5028; E-mail: bear{at}sickkids.on.ca.
1 The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; ABC, ATP-binding cassette; PKA, protein kinase A; PKC, protein kinase C; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PE, phosphatidylethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine; tc, time that separates short, intraburst closures from long, interburst closures; Mg-AMPPNP, Mg-5
-adenylyl imidodiphosphate.
2 M. Ramjeesingh, W. Wang, and C. E. Bear, unpublished observations.
Acknowledgments
CFTR monoclonal antibody was generously provided by Dr. N. Kartner (Department of Pharmacology, University of Toronto). We thank Dr. Chris Miller, Dr. M. Buchwald, and Dr. S. Grinstein for critical reading of the manuscript and for many helpful suggestions.
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A. A Aleksandrov, X.-b. Chang, L. Aleksandrov, and J. R Riordan The non-hydrolytic pathway of cystic fibrosis transmembrane conductance regulator ion channel gating J. Physiol., October 15, 2000; 528(2): 259 - 265. [Abstract] [Full Text] [PDF]
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 M. BIENENGRAEBER, A. E. ALEKSEEV, M. R. ABRAHAM, A. J. CARRASCO, C. MOREAU, M. VIVAUDOU, P. P. DZEJA, and A. TERZIC ATPase activity of the sulfonylurea receptor: a catalytic function for the KATP channel complex FASEB J, October 1, 2000; 14(13): 1943 - 1952. [Abstract] [Full Text]
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J. Luo, T. Zhu, A. Evagelidis, M. D. Pato, and J. W. Hanrahan Role of protein phosphatases in the activation of CFTR (ABCC7) by genistein and bromotetramisole Am J Physiol Cell Physiol, July 1, 2000; 279(1): C108 - C119. [Abstract] [Full Text] [PDF]
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M. Ikuma and M. J. Welsh Regulation of CFTR Cl- channel gating by ATP binding and hydrolysis PNAS, June 30, 2000; (2000) 140220597. [Abstract] [Full Text]
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M. H. Akabas Cystic Fibrosis Transmembrane Conductance Regulator. STRUCTURE AND FUNCTION OF AN EPITHELIAL CHLORIDE CHANNEL J. Biol. Chem., February 11, 2000; 275(6): 3729 - 3732. [Full Text] [PDF]
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M. Matsuo, N. Kioka, T. Amachi, and K. Ueda ATP Binding Properties of the Nucleotide-binding Folds of SUR1 J. Biol. Chem., December 24, 1999; 274(52): 37479 - 37482. [Abstract] [Full Text] [PDF]
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M. A. Harrington, K. L. Gunderson, and R. R. Kopito Redox Reagents and Divalent Cations Alter the Kinetics of Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating J. Biol. Chem., September 24, 1999; 274(39): 27536 - 27544. [Abstract] [Full Text] [PDF]
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P. M. van Endert Role of Nucleotides and Peptide Substrate for Stability and Functional State of the Human ABC Family Transporters Associated with Antigen Processing J. Biol. Chem., May 21, 1999; 274(21): 14632 - 14638. [Abstract] [Full Text] [PDF]
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K. Szabo, G. Szakacs, T. Hegedus, and B. Sarkadi Nucleotide Occlusion in the Human Cystic Fibrosis Transmembrane Conductance Regulator. DIFFERENT PATTERNS IN THE TWO NUCLEOTIDE BINDING DOMAINS J. Biol. Chem., April 30, 1999; 274(18): 12209 - 12212. [Abstract] [Full Text] [PDF]
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K. de MEER, V. A. M. GULMANS, and J. van der LAAG Peripheral Muscle Weakness and Exercise Capacity in Children with Cystic Fibrosis Am. J. Respir. Crit. Care Med., March 1, 1999; 159(3): 748 - 754. [Abstract] [Full Text]
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J. F. Cotten and M. J. Welsh Cystic Fibrosis-associated Mutations at Arginine 347 Alter the Pore Architecture of CFTR. EVIDENCE FOR DISRUPTION OF A SALT BRIDGE J. Biol. Chem., February 26, 1999; 274(9): 5429 - 5435. [Abstract] [Full Text] [PDF]
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S. Kawano, A. Kuruma, Y. Hirayama, and M. Hiraoka Anion Permeability and Conduction of Adenine Nucleotides Through a Chloride Channel in Cardiac Sarcoplasmic Reticulum J. Biol. Chem., January 22, 1999; 274(4): 2085 - 2092. [Abstract] [Full Text] [PDF]
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D. N. SHEPPARD and M. J. WELSH Structure and Function of the CFTR Chloride Channel Physiol Rev, January 1, 1999; 79(1): 23 - 45. [Abstract] [Full Text] [PDF]
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D. C. GADSBY and A. C. NAIRN Control of CFTR Channel Gating by Phosphorylation and Nucleotide Hydrolysis Physiol Rev, January 1, 1999; 79(1): 77 - 107. [Abstract] [Full Text] [PDF]
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E. A. Pasyk, X. K. Morin, P. Zeman, E. Garami, K. Galley, L. J. Huan, Y. Wang, and C. E. Bear A Conserved Region of the R Domain of Cystic Fibrosis Transmembrane Conductance Regulator Is Important in Processing and Function J. Biol. Chem., November 27, 1998; 273(48): 31759 - 31764. [Abstract] [Full Text] [PDF]
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J. F. Cotten and M. J. Welsh Covalent Modification of the Nucleotide Binding Domains of Cystic Fibrosis Transmembrane Conductance Regulator J. Biol. Chem., November 27, 1998; 273(48): 31873 - 31879. [Abstract] [Full Text] [PDF]
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K A Lansdell, J F Kidd, S J Delaney, B J Wainwright, and D N Sheppard Regulation of murine cystic fibrosis transmembrane conductance regulator Cl- channels expressed in Chinese hamster ovary cells J. Physiol., November 1, 1998; 512(3): 751 - 764. [Abstract] [Full Text] [PDF]
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A. Decottignies, A. M. Grant, J. W. Nichols, H. de Wet, D. B. McIntosh, and A. Goffeau ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p J. Biol. Chem., May 15, 1998; 273(20): 12612 - 12622. [Abstract] [Full Text] [PDF]
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J. Luo, M. D. Pato, J. R. Riordan, and J. W. Hanrahan Differential regulation of single CFTR channels by PP2C, PP2A, and other phosphatases Am J Physiol Cell Physiol, May 1, 1998; 274(5): C1397 - C1410. [Abstract] [Full Text] [PDF]
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C. J Mathews, J. A Tabcharani, X.-B. Chang, T. J Jensen, J. R Riordan, and J. W Hanrahan Dibasic protein kinase A sites regulate bursting rate and nucleotide sensitivity of the cystic fibrosis transmembrane conductance regulator chloride channel J. Physiol., April 15, 1998; 508(2): 365 - 377. [Abstract] [Full Text] [PDF]
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K A Lansdell, S J Delaney, D P Lunn, S A Thomson, D N Sheppard, and B J Wainwright Comparison of the gating behaviour of human and murine cystic fibrosis transmembrane conductance regulator Cl- channels expressed in mammalian cells J. Physiol., April 15, 1998; 508(2): 379 - 392. [Abstract] [Full Text] [PDF]
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 X. Zhong and P. C. Tai When an ATPase Is Not an ATPase: at Low Temperatures the C-Terminal Domain of the ABC Transporter CvaB Is a GTPase J. Bacteriol., March 15, 1998; 180(6): 1347 - 1353. [Abstract] [Full Text]
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 H. A. Berger, S. M. Travis, and M. J. Welsh Fluoride stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity Am J Physiol Lung Cell Mol Physiol, March 1, 1998; 274(3): L305 - L312. [Abstract] [Full Text] [PDF]
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S. M. Travis, H. A. Berger, and M. J. Welsh Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator PNAS, September 30, 1997; 94(20): 11055 - 11060. [Abstract] [Full Text] [PDF]
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B.-H. Qu, E. H. Strickland, and P. J. Thomas Localization and Suppression of a Kinetic Defect in Cystic Fibrosis Transmembrane Conductance Regulator Folding J. Biol. Chem., June 20, 1997; 272(25): 15739 - 15744. [Abstract] [Full Text] [PDF]
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J. Ahn, J. T. Wong, and R. S. Molday The Effect of Lipid Environment and Retinoids on the ATPase Activity of ABCR, the Photoreceptor ABC Transporter Responsible for Stargardt Macular Dystrophy J. Biol. Chem., June 30, 2000; 275(27): 20399 - 20405. [Abstract] [Full Text] [PDF]
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Y.-x. Hou, L. Cui, J. R. Riordan, and X.-b. Chang Allosteric Interactions between the Two Non-equivalent Nucleotide Binding Domains of Multidrug Resistance Protein MRP1 J. Biol. Chem., June 30, 2000; 275(27): 20280 - 20287. [Abstract] [Full Text] [PDF]
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Q. Mao, R. G. Deeley, and S. P. C. Cole Functional Reconstitution of Substrate Transport by Purified Multidrug Resistance Protein MRP1 (ABCC1) in Phospholipid Vesicles J. Biol. Chem., October 27, 2000; 275(44): 34166 - 34172. [Abstract] [Full Text] [PDF]
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M. Matsuo, K. Tanabe, N. Kioka, T. Amachi, and K. Ueda Different Binding Properties and Affinities for ATP and ADP among Sulfonylurea Receptor Subtypes, SUR1, SUR2A, and SUR2B J. Biol. Chem., September 8, 2000; 275(37): 28757 - 28763. [Abstract] [Full Text] [PDF]
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I. Kogan, M. Ramjeesingh, L.-J. Huan, Y. Wang, and C. E. Bear Perturbation of the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Inhibits Its ATPase Activity J. Biol. Chem., April 6, 2001; 276(15): 11575 - 11581. [Abstract] [Full Text] [PDF]
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L. Aleksandrov, A. Mengos, X.-b. Chang, A. Aleksandrov, and J. R. Riordan Differential Interactions of Nucleotides at the Two Nucleotide Binding Domains of the Cystic Fibrosis Transmembrane Conductance Regulator J. Biol. Chem., April 13, 2001; 276(16): 12918 - 12923. [Abstract] [Full Text] [PDF]
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C. J. Ketchum, W. K. Schmidt, G. V. Rajendrakumar, S. Michaelis, and P. C. Maloney The Yeast a-factor Transporter Ste6p, a Member of the ABC Superfamily, Couples ATP Hydrolysis to Pheromone Export J. Biol. Chem., July 27, 2001; 276(31): 29007 - 29011. [Abstract] [Full Text] [PDF]
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D. J. Hennager, M. Ikuma, T. Hoshi, and M. J. Welsh A conditional probability analysis of cystic fibrosis transmembrane conductance regulator gating indicates that ATP has multiple effects during the gating cycle PNAS, March 13, 2001; 98(6): 3594 - 3599. [Abstract] [Full Text] [PDF]
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M. Ikuma and M. J. Welsh Regulation of CFTR Cl- channel gating by ATP binding and hydrolysis PNAS, July 18, 2000; 97(15): 8675 - 8680. [Abstract] [Full Text] [PDF]
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