(Received for publication, March 12, 1996; and in revised form, March 21, 1996)
From the
The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR). Previously, we provided definitive evidence that CFTR functions as a phosphorylation-regulated chloride channel in our planar lipid bilayer studies of the purified, reconstituted protein. Recent patch-clamp studies have lead to the suggestion that CFTR may also be capable of conducting ATP or inducing this function in neighboring channels. In the present study, we assessed the ATP channel activity of purified CFTR and found that the purified protein does not function as an ATP channel in planar bilayer studies of single channel activity nor in ATP flux measurements in proteoliposomes. Hence, CFTR does not possess intrinsic ATP channel activity and its putative role in cellular ATP transport may be indirect.
The function of CFTR ()(the cystic fibrosis
transmembrane conductance regulator) as a phosphorylation and
ATP-regulated chloride channel is thought to be critical for the
elaboration of salt and water secretion across the epithelial cell
lining of multiple organs; the airways, pancreatic ductules,
gastrointestinal tract, and reproductive tract(1) . The
chloride channel activity of CFTR was confirmed using a number of
experimental approaches. First, expression of recombinant CFTR in
heterologous cell systems conferred the appearance of cAMP-regulated
chloride channels (2, 3) . Second, mutagenesis of
amino acid residues thought to reside in membrane spanning domains
caused alteration in single-channel conductance and/or anion
selectivity of recombinant CFTR(4) . Finally, reconstitution of
purified CFTR in planar phospholipid bilayers causes the appearance of
PKA and ATP-regulated chloride channels, exhibiting biophysical
properties identical with those observed in patch clamp studies of
epithelial cell membranes(5, 6, 7) .
It is not yet clear whether defects in the chloride channel activity of CFTR can account for the diverse symptoms of cystic fibrosis. In vivo nasal potential difference measurements in CF patients revealed an altered sodium conductance as well as chloride conductance (8) . It was recently shown in in vitro studies that the activity of the epithelial sodium channel ENaC was inhibited by coexpression with CFTR(9) . Further, it has been suggested that CFTR may interact with another type of chloride channel found in the apical membrane of epithelial cells, the outwardly rectifying chloride channel (ORCC). Activation of CFTR channel function by phosphorylation can stimulate activity of the ORCC(10, 11) . Hence, CFTR may act to promote epithelial cell secretion not only through its intrinsic chloride channel activity but also through regulatory interactions with other ion channels located on the apical membrane.
Conceivably, there are several mechanisms through which CFTR may
interact with other ion channels localized at the apical membrane of
epithelial cells. Functional interaction between CFTR and other
channels may be due to the generation of permissive electrochemical
gradients, convergence of signal transduction systems, or through
direct protein-protein interactions. Recently, it has been suggested
that CFTR may affect the activity of ORCC through an
``autocrine'' mechanism. According to Guggino's
research group(12) , CFTR may activate the ORCC by mediating
the efflux of ATP which then acts to stimulate ORCC through interaction
with neighboring purinergic (P) receptors. There are
several observations which support this hypothesis. First,
extracellular ATP has been shown to stimulate the ORCC channel by
interacting with P
receptors(13) . Second, as
previously mentioned, CFTR activation leads to the stimulation of the
ORCC(10, 11) . Third, several patch clamp studies have
reported that CFTR, or an intimately associated ion channel, may be
capable of conducting ATP(12, 14) . These latter
studies have generated considerable controversy as high,
unphysiological, concentrations of ATP (100 mM) were used to
assay currents. Furthermore, some research groups have recently
reported conflicting patch clamp data which suggests that ATP can not
be conducted through CFTR(15) .
In the present investigation, we sought to assess the intrinsic ATP channel activity of CFTR by examining the conductance properties of purified, reconstituted CFTR.
In order to directly assess the capacity of CFTR to conduct
ATP, we first compared the single channel activity of purified CFTR
reconstituted in planar lipid bilayers in the presence of symmetrical
chloride (140 mM KCl) solutions with activity detected in the
presence of symmetrical K-ATP (140 mM) solutions.
In all of these experiments, CFTR was phosphorylated by the addition of
purified catalytic subunit of PKA plus MgATP and separated from these
reagents by gel filtration (see ``Materials and Methods'')
prior to reconstitution in lipid bilayers. Following the fusion of
proteoliposomes with lipid bilayers, 1 mM MgATP was added to
both the cis and trans compartments of the lipid
bilayer chamber in order to activate channel function.
In the upper panel of Fig. 1A), we show activity of
two CFTR channels in the presence of symmetrical concentrations of
chloride ion (100 mM KCl). The current-voltage relationship of
the channel is nonrectifying and its unitary conductance is 10 pS,
consistent with previous measurements of native and recombinant
CFTR(2, 3, 4, 5, 6, 7) .
Replacement of KCl with symmetrical K-gluconate (100 mM)
solutions eliminated the appearance of channel steps, confirming that
CFTR is anion-selective and cannot pass gluconate. The subsequent
substitution of K-gluconate with K-ATP failed to evoke the
appearance of channel behavior, suggesting that ATP
(ATP
or ATP
) is not
permeant through CFTR. Finally, we confirmed that the CFTR protein had
remained in the lipid bilayer during bath changes, as the replacement
of K
-ATP with KCl restored the appearance of channel
function. The unitary conductance for chloride ion was not different
from control following re-establishment of symmetrical chloride
solutions, 10.1 versus 10.0 pS, respectively. However, we did
detect a change in channel open probability at high ATP concentration,
decreasing from 0.78 to 0.24, suggesting that exposure to 100 mM K
-ATP may inhibit gating of CFTR.
Figure 1:
Single channel ATP currents are not
detected in planar bilayer studies of purified CFTR. A,
activity of two prephosphorylated CFTR channels, opening to level 1 or
2, was detected at the holding potential of -40 mV in the
presence of symmetrical 100 mM KCl solutions and 1 mM MgATP in both compartments. Complete exchange of the KCl solutions
with K-gluconate in both cis and trans compartments
eliminated channel activity, confirming that gluconate was not permeant
through the CFTR channel. The subsequent replacement of K-gluconate
with K-ATP failed to restore channel activity, indicating
that ATP was impermeant. On the other hand, channel activity was
restored with the replacement of K
-ATP with KCl. A constant
concentration of MgATP (1 mM) was maintained in both
compartments during all solution exchanges. The channel records
obtained in the presence of symmetrical KCl, prior to and following
exposure to 100 mM K
-ATP, have been replotted
using a faster time scale to show that the open probability, not
unitary conductance, was altered by exposure to the high ATP
concentration. Similar results were obtained in 3 experiments. B, in this figure we show that in the presence of symmetrical
KCl solutions, single channel steps of equal amplitude are evoked with
driving forces of equal but opposite polarity. On the other hand, in
the presence of asymmetrical salt solutions, K
-ATP (100
mM) in the cis compartment and KCl in the trans compartments, single channel steps can only be observed when the
polarity of the potential drives chloride conductance, not when the
polarity would drive ATP conductance. The open probability of the
chloride channel activity of CFTR is reduced when the cis compartment contained K
-ATP. These results are
representative of 3 studies.
In a second set
of studies, we examined the capacity of CFTR to conduct chloride while
exposed to asymmetrical Cl/ATP
concentrations. In the upper two panels of Fig. 1B), we show that single channel steps of similar
amplitude result from the application of equal but opposite electrical
driving forces in the presence of symmetrical KCl solutions. This
behavior is consistent with previous reports of the nonrectifying I
- V relationship of CFTR chloride channel activity (2, 3, 4, 5, 6, 7) .
On the other hand, in the presence of asymmetrical anion solutions
((KCl) versus (K
-ATP)), single channel currents
were only evident when the applied electrical potential (+40 mV)
drove chloride current from the trans to the cis compartment. Single channel currents were not evident upon
application of an electrical potential (-40 mV) which should
drive ATP
current from the cis to the trans compartments. These results support the data presented
in Fig. 1and suggest that CFTR cannot conduct
ATP
. Further, it is clear from the single channel
chloride current record obtained at +40 mV that the presence of
K
-ATP in the contralateral bilayer compartment resulted in
a low a channel open probability of 0.26 relative to control open
probability of 0.68 measured in the presence of symmetrical KCl
solutions. As in the preceding bilayer study shown in Fig. 1A), high ATP concentrations (100 mM)
appear to inhibit CFTR channel function. On the other hand, there was
no change in the amplitude of the chloride current step which occurs
when CFTR opens, suggesting that ATP did not modify the conductive
pathway for chloride ion.
It is conceivable that ATP may be conducted through CFTR but at a rate which is too slow for
detection in planar lipid bilayer studies of single channel activity,
we utilized the ``Garty'' method for assay of electrogenic
anion flux anion in liposomes containing purified chloride channel
protein(15, 16) . Briefly, this assay, as modified by
Miller et al.(18) , involves the creation of an
outward concentration gradient for chloride movement across
proteoliposomes containing CFTR. The outward movement of chloride
creates a positive potential inside the vesicle, thereby generating an
inward driving force for chloride conductance, quantifiable by
Cl
addition (Fig. 2A).
We show in Fig. 2B, that as predicted on the basis of
existing single-channel data, liposomes containing PKA-phosphorylated
CFTR exhibit significantly greater concentrative uptake of
Cl
than liposomes containing
nonphosphorylated CFTR (p < 0.001).
Figure 2:
Electrogenic Cl
uptake mediated by CFTR-containing
liposomes. A, the Garty
Cl
flux
assay. This diagram shows the principle underlying the
``Garty'' flux assay of chloride and ATP conductance through
purified CFTR reconstituted in liposomes. Proteoliposomes are loaded
with KCl (150 mM), and external Cl
was
replaced with glutamate by rapid buffer exchange on a Sephadex G-50
``spin-column'' to establish a large outward Cl
gradient. Chloride-permeable liposomes will become polarized
(positive inside) and
Cl
added to the
external medium will be taken up by the liposomes. B, time
course of electrogenic
Cl uptake by liposomes containing
CFTR. The
Cl
uptake at by liposomes
containing phosphorylated CFTR (7 studies) (
) was significantly
greater than uptake by liposomes containing unphosphorylated CFTR at
5-120 min after creation of an outwardly chloride gradient
(
). Means ± S.D. have been shown for each time point (6
studies). In these studies, reconstituted CFTR was phosphorylated by
addition of catalytic subunit of PKA (200 nM) plus MgATP (1
mM). These data were fit using logarithmic functions by
Kaleidagraph (Abelbeck Software).
We then compared the
concentrative uptake of Cl
and
[
H]ATP by proteoliposomes possessing an
inwardly-directed gradient for anion conductance. Concentrative uptake
was determined at 30 min after the establishment of the outward
chloride gradient, an interval close to the time determined for
half-maximal uptake of
Cl
by
phosphorylated CFTR.
Cl
uptake by
CFTR-free liposomes and by liposomes containing unphosphorylated CFTR
showed no significant difference (p = 0.327),
confirming previous studies showing that unphosphorylated CFTR is not
functional as a chloride channel (2, 3, 4, 5, 6, 7) .
Cl
uptake by PKA-phosphorylated CFTR was
approximately 5-fold greater than that measured in liposomes with
unphosphorylated CFTR. On the other hand,
[
H]ATP
taken up by the same
liposomes showed no dependence on CFTR protein. At 30 min,
[
H]ATP
uptake by CFTR-free
lipsosomes and liposomes containing phosphorylated CFTR was not
significantly different (Fig. 3). These results suggest that
CFTR does not mediate the conductance of [
H]ATP.
Figure 3:
Electrogenic [H]ATP
uptake is not mediated by liposomes containing CFTR. Three liposome
preparations were compared; liposomes with no CFTR (Control) (n = 4), liposomes containing unphosphorylated CFTR (n = 4), and liposomes containing phosphorylated CFTR (n = 4) (see ``Materials and Methods''). One
microgram of CFTR protein was present in each 100-µl sample of
liposomes. Electrogenic uptake of
Cl
and
[
H]ATP was determined simultaneously for each
liposome preparation (100 µl), 30 min after exposure of liposomes
to an outwardly directed chloride concentration gradient. Liposomes
containing phosphorylated CFTR exhibited a significant increase in
concentrative uptake of
Cl
(
),
relative to uptake by liposomes without added CFTR protein (control) (p < 0.001) and liposomes containing unphosphorylated CFTR (p < 0.001). Liposomes containing phosphorylated CFTR
showed no significant uptake of [
H]ATP
(&cjs2113;) relative to liposomes with no added CFTR (p = 0.115) or unphosphorylated CFTR (p =
0.421).
In summary, our results show that purified, reconstituted CFTR
cannot conduct ATP. While purified CFTR clearly exhibits PKA- and
M-ATP-dependent chloride channel activity as described
previously(2, 3, 4, 5, 6, 7) ,
ATP conductance through CFTR could not be detected in
either planar lipid bilayer studies of single channel activity or as
potential-driven uptake by proteoliposomes.
As described, some
previous patch clamp studies reported that CFTR expression is
associated with ATP channel activity. This
observation formed the basis for the ``autocrine'' model for
CFTR regulation of the outwardly rectifying chloride
channel(13) . According to this model, ORCC channels are
stimulated via G protein coupled to purinergic receptors
(P
) which are activated by ATP conductance through CFTR.
The present studies show that purified CFTR cannot conduct
ATP
, and we suggest that certain features of the
autocrine model should be modified. For example, while we have shown
that CFTR is not an ATP channel, we cannot exclude the possibility that
CFTR may mediate ATP flux through a nonelectrogenic transport
mechanism. This putative mechanism would explain existing data
generated in cell lines wherein nonepithelial cells transfected with
wild-type CFTR exhibited enhanced radiolabeled ATP efflux relative to
cells which didn't express CFTR or variant forms of CFTR known to
cause disease in humans(12) . However, patch clamp data showing
ATP
currents associated with CFTR expression could
not be reconciled with such a mechanism. Alternatively, it is
conceivable that ATP conductance may be mediated through a channel
which is functionally coupled to activated CFTR. The presence or
absence of such functional interactions may account for some of the
variability which exists between different patch clamp studies of the
putative ATP channel activity of
CFTR(12, 14, 15) .
Finally, it is clear that the mechanism through which CFTR participates in cellular ATP transport and the role of this transport process in epithelial cell fluid secretion cannot be entirely defined in studies of purified, reconstituted CFTR protein alone. However, we have shown the utility of this reconstitution system in identifying those functions which are intrinsic to CFTR and those activities which are not intrinsic and may require interactions with neighboring proteins.