(Received for publication, January 14, 1997)
From the Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada and the ¶ Department of Physiology and Institute for Human Gene Therapy, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6100
Cystic fibrosis (CF) is characterized by abnormal
regulation of epithelial ion and fluid transport due to mutations in
the CF transmembrane conductance regulator (CFTR), an apical
membrane-localized Cl channel, that usually prevent
it from exiting the endoplasmic reticulum. Defective or absent CFTR in
the epithelium is believed to disrupt fluid balance in human airways
and thereby contribute to chronic respiratory inflammation. Patch-clamp
of the plasma membrane and outer membrane of the nuclear envelope of
nuclei isolated from CFTR-expressing Chinese hamster ovary cells
revealed that CFTR is associated with a regulated ATP channel in both
membrane compartments. CFTR expression was also shown to be associated with permeability to another adenine nucleotide, adenosine 3
-phosphate 5
-phosphosulfate, the universal sulfate donor in cells. These results
may provide a link between the ion channel function of CFTR and
abnormal glycoprotein processing observed in CF.
Cystic fibrosis (CF)1 is a common
genetic disease characterized by abnormal regulation of epithelial ion
and fluid transport due to mutations in CFTR, an apical
membrane-localized cAMP-regulated Cl channel (1-8).
Hundreds of different mutations (1) can affect CFTR Cl
channel function by several mechanisms (8). However, most CF is caused
by lack of CFTR in the plasma membrane due to mutations, including the
most common one (
F508-CFTR) (8), that affect the ability of fully
translated CFTR to exit from the endoplasmic reticulum (ER), where it
is synthesized (9-12). Defective or absent CFTR in the epithelium is
believed to disrupt fluid balance in human airways and thereby
compromise mucociliary clearance of inflammatory particles, including
bacteria (13). The resulting chronic inflammation and tissue damage in
the lung are the cause of most mortality in CF (14). Nevertheless, it
remains unclear if the lack of plasma membrane Cl
channel
activity can fully account for airway pathology in CF (13, 15). Human
airways are Na+-absorbing tissues (16, 17), and the role of
CFTR in transepithelial Cl
transport is still undefined
because Cl
seems to be at electrochemical equilibrium
across the apical membrane (18), and NaCl absorption is
enhanced in CF (17, 19, 20). Furthermore, other transport
and biochemical abnormalities in the lung in CF may not be readily
explained by the lack of plasma membrane CFTR Cl
channel
activity, including elevated Na+ permeability through
amiloride-sensitive channels (13, 17, 19, 21) and abnormal regulation
by cAMP of another airway Cl
channel, the outward
rectifier (22-25). In addition, altered glycoprotein processing is
observed in respiratory cells from CF patients (26-29). CF airway
epithelial cells express higher levels of the receptor for
Pseudomonas aeruginosa, which may be a consequence of its diminished sialylation (14, 28), and secreted and cell-surface glycoconjugates are hypersulfated in the lung in vivo and in
primary cultures of airway cells from CF patients (27, 30-32). The
evidence suggesting that abnormal protein sulfation is a fundamental
biochemical defect in CF (31, 32) seems paradoxical in light of the
identification of CFTR as a plasma membrane Cl
channel.
A recent study suggested that abnormal regulation of the outward
rectifier in CF involves defective plasma membrane ATP permeability (33) and results from two studies by independent groups indicated that
CFTR may either conduct ATP or be closely associated with a separate
ATP conductance (33, 34). However, these results are currently rather
controversial because they have not been observed by all investigators
(35-37). The possibility that CFTR is associated with ATP permeability
has prompted us to consider whether other transport and
protein-processing abnormalities in CF are related to this conductance.
Specifically, in the present study we have considered whether protein
processing alterations in CF might be related to an association of CFTR
with ATP permeability in intracellular membranes. We recently
demonstrated that CFTR as well as F508 CFTR function as
Cl
channels when they are localized in the ER membrane
(38). That wild-type CFTR functions in the ER as well in plasma
membranes suggests that it also likely functions in intermediate
compartments, including the Golgi, the site of most protein processing,
including sialylation and sulfation. CF in most patients may therefore
be associated with lack of Cl
channel activity not only
in the plasma membrane but in the Golgi as well. If CFTR is associated
with nucleotide conductances, altered nucleotide permeability in
intracellular membranes may possibly contribute to cellular dysfunction
in CF.
CHO cells that stably expressed wild-type CFTR and the parental
CHO cell line (controls; no CFTR) were used (11, 39, 40). Cells were
cultured, and nuclei were isolated as described previously (38).
Standard patch-clamp techniques were applied: (i) excised inside-out
and cell-attached configurations for the plasma membrane, and (ii)
excised patches (cytoplasmic side facing into the pipette) for the
outer nuclear membrane (38). Stable (15-60 min) seals (10-100
gigohms) were obtained using heat-polished electrodes (tip, <0.5 µm
in diameter) with 10-50 megohms resistance. Ag/AgCl electrodes were
used without salt bridges. When using low (~5.2 and 0.7 mM) Cl solutions (below) in the pipette, the
electrodes were not backfilled with Cl
-containing
solutions. These low Cl
conditions did not affect our
ability to record currents, probably because the currents that the
electrodes were required to pass in these experiments were small.
Junction potentials were corrected in all experiments by an initial
offset applied by the amplifier, with one exception relating to the
third part of Fig. 4A, which has not been corrected for the
junction potential that arose during the switch from the ATP to the
Cl
solutions. The measured offset was ~6 mV, which does
not significantly affect the interpretations. All experiments were
performed at room temperature as described previously (38). Data were
digitally filtered at 300 Hz unless indicated. Channel openings and
closings were automatically detected using the Tac computer program,
and amplitudes were determined by computer-assisted manual measurements of detected events. Data analyses were limited to records with low
base-line drift. Kinetic analyses were restricted to single-channel patches. Results are expressed as mean ± S.E. The following
solutions were used: Solution A, high Cl
solution
containing 120 mM
N-methyl-D-glucamine chloride, 3 mM MgCl2, 0.1 mM CaCl2, 1.1 mM EGTA, 10 mM HEPES, and 5 mM
glucose, pH 7.3 with HCl; Solution B, high ATP solution containing 100 mM Na2ATP, 5 mM KCl, 1 mM MgSO4, 0.1 mM CaCl2,
1.1 mM EGTA, and 10 mM HEPES, pH 7.3 or 7.1 with NaOH for plasma and nuclear membrane patches, respectively;
Solution C, high PAPS solution containing 100 mM
Li4PAPS, 5 mM KCl, 1 mM
MgSO4, 0.1 mM CaCl2, 1.1 mM EGTA, and 10 mM HEPES, pH 7.3 with NaOH.
Unless otherwise specified, PKA (180 nM) and MgATP (1 mM) were added to or included in the bath or pipette
solutions for excised plasma membrane or nuclear membrane patches,
respectively. Thus, MgATP2- concentration was always ~2
mM. In the ATP solutions, the extra 99 mM ATP
is mostly ATP4- because it was added as Na+ or
Tris+ salts. Therefore, the free Mg2+
concentration was always
1 µM. Thus, neither
Mg2+ nor MgATP2- were significant variables in
our experiments. The bath was continuously perfused until PKA was added
to it. The perfusion was halted during these periods because of the
high cost of PKA and the large quantities required for perfusion.
Cell-attached patches were stimulated by a mixture designed to elevate
intracelluar levels of cAMP containing forskolin (5 µM),
3-isobutyl-1-methylxanthine (0.1 mM), and the cAMP analog
chlorophenylthio-cAMP (0.1 mM) applied directly to the bath
to achieve the desired final concentrations. DIDS was added to the
pipette or bath for plasma membrane or nuclear membrane patches,
respectively.
Stably transfected CHO cells were used to examine whether plasma
membrane-localized as well as ER-localized CFTR are conductive to ATP.
We first reconfirmed (39) the presence of CFTR Cl
channels in the plasma membrane of CFTR-expressing CHO cells. With
Cl
solutions in the bath and pipette, elevation of
intracellular cAMP levels activated CFTR Cl
channel
activities in cell-attached patches (eight of nine patches) with
familiar properties (39, 41, 42) (data not shown). In excised
inside-out patches with the catalytic subunit of PKA and MgATP in the
bath, 6.1 ± 0.7 pS linear Cl
channels were observed
(16 of 18 patches) with typical CFTR characteristics (Fig.
1A).
We used the inside-out excised patch configuration to determine whether
membranes from these cells exhibited ATP conductances. With 100 mM ATP solutions in the bath and pipette, channel
activities were observed with features similar to those of CFTR
Cl channels (Figs. 1 and 2): i) linear
current-voltage (I/V) relation with a slope conductance of 4.5 ± 0.3 pS (13 of 14 patches; Fig. 1, B and C); ii)
PKA-dependence, because in the absence of PKA, they were not observed
(0 of 5 patches; Fig. 2A); and iii) insensitivity to DIDS
(Fig. 2B), whereas 50 uM glybenclamide, an
inhibitor of CFTR Cl
channels (33, 43), reduced channel
open probability (Po) (3 of 3 patches; Fig.
2C). A 10-fold ATP gradient shifted the reversal potential
from 0 mV to approximately
12 mV, indicating permeability to ATP
(Fig. 1C). Of note, this reversal potential is close to that
expected for permeation by a quadrivalent anion, i.e.
ATP4
(RT/4F ln Co/Ci = ~15 mV),
the overwhelmingly dominant ionic species in these experiments.
Cl
was eliminated as the current carrier because similar
channels (4.6 ± 0.2 pS) were present (10 of 11 patches; Fig.
3A) in Tris2ATP and
Na2ATP solutions in which the Cl
concentration was reduced from the normal 5.2 mM (Figs.
1B and 2) to only 0.7 mM (Fig. 3).
Na+ was also excluded as the current carrier because in
inside-out patches under symmetrical conditions, 4.6 ± 0.1 pS
channels were present when Na2ATP was replaced with
Tris2ATP (eight of nine patches; Fig. 3B), and
no channels were observed when Na2ATP was replaced with
sodium gluconate (zero of four patches; Fig. 3C). Importantly, no ATP channels were observed in control cells (0 of 10 patches; Fig. 2D).
In inside-out plasma membrane patches with Cl and ATP in
the pipette and bath solutions, respectively, currents at +70 mV had
slope conductances of 6-8 pS, whereas at
70 mV they were 4-5 pS (6 of 6 patches; Fig. 4), consistent with the currents being carried by Cl
and ATP, respectively. Replacement of
bath ATP by perfusion with 120 mM NaCl solution restored
6-8 pS activities at
70 mV (Fig. 4A). Under reversed
bi-ionic conditions in inside-out patches (i.e. pipette,
ATP; bath, Cl
), 6-8 pS Cl
currents were
observed at
70 mV, whereas 4-5 pS ATP currents were present at +70
mV (8 of 9 patches; data not shown).
We previously demonstrated by patch-clamp of the outer membrane of
nuclei isolated from CFTR-expressing CHO cells that ER-localized CFTR
was functional as a Cl channel, with properties similar
to those exhibited when it resided in the plasma membrane (38). Using a
similar approach, we evaluated the ATP conductance of the ER, using
nuclei isolated from the same CHO cells. With PKA and MgATP in the
pipette (because the cytosolic face of the membrane patch faces into
the pipette in this configuration), PKA-dependent channels
with linear I/V relation and 4.6 ± 0.5 pS conductance were
recorded in symmetrical ATP solutions (33 of 35 patches; Fig.
5). Other properties of the ATP channels in the
nuclear/ER membrane were also similar to those observed for wild-type
CFTR in the plasma membrane (Fig. 5).
Our studies suggest that CFTR is associated with a regulated ATP
channel in the plasma membrane and ER, and by extension in membranes in
intermediate compartments including Golgi. The single ATP channel
conductance, I/V relation, PKA sensitivity, pharmacological sensitivities, and dependence on expression of CFTR we have observed in
the present study are similar to those reported previously (33, 34) in
different cell types, suggesting that CFTR is closely associated with
or is in fact an ATP conductance. We do not think that the
Cl conduction pathway in CFTR is the ATP conduction
pathway, although the resemblance of the biophysical, kinetic, and
pharamacological properties of the ATP conductance to those of the CFTR
Cl
conductance is striking and suggests that gating of
the ATP permeability is strongly tied to CFTR gating. The reasons why
some investigators have observed nucleotide conductances associated
with CFTR (33, 34) although others have not (35-37) are not addressed
by our study, and our results provide no insights into this issue. We would point out, however, that we have also observed CFTR
Cl
channels that were not associated with ATP
conductances in a different CHO cell
clone,2 suggesting that coupling of the two
permeabilities may be cell-type dependent. The clear channel activities
we observe will enable us in future studies to define variables that
might regulate the manifestation of the ATP conductances and therefore
resolve these conflicting observations.
The presence of a CFTR-associated ATP conductance in intracellular
membranes raises the possibility that altered ATP permeability in
intracellular compartments in CF may contribute to disease pathogenesis. ATP-translocase activity has been detected in ER and
Golgi membranes (44), and ATP binding to ER proteins, ATP-dependence of
the structure or activities of ER-resident proteins, and lumenal ATP
requirement for protein translocation in the ER (45-47) suggest that
intralumenal ATP content is physiologically regulated. ATP binding by
ER-localized molecular chaperones, including BiP and calnexin, is
necessary for ATPase activity, structural changes, and oligomerization
required for chaperone functions (45-47). These observations may have
significance for CF because CFTR and F508-CFTR both associate with
calnexin in the ER membrane (40), and the trafficking defect of
F508-CFTR is likely a chaperone-assisted process (12). If ATP
permeability through ER- or Golgi-localized CFTR affected intralumenal
ATP concentrations, mutant CFTRs could conceivably modify lumenal ATP
concentrations and consequently affect protein processing. However,
measurements of lumenal ATP concentrations will be required to test
this hypothesis.
It was proposed that CFTR may normally provide a Cl
conductance in Golgi membranes to facilitate lumenal acidification by a
proton pump and that lack of such activity in CF, by compromising Golgi
pH, might account for altered glycoprotein processing (26). Nevertheless, a direct test of this model failed to provide support for
it (48). Nor can altered protein sulfation in CF airway epithelial
cells be accounted for by changes in plasma membrane sulfate transport
or cell content of inorganic sulfate (31, 49). Intracellular sulfation
reactions utilize sulfate that has been "activated" by its
incorporation into PAPS (50). PAPS is synthesized in the cytoplasm by a
reaction involving ATP and sulfate, catalyzed by a bifunctional
ATP-sulfurylase-adenosine 5
-phosphosulfate kinase, and transported
into the Golgi lumen by a Golgi-specific saturable process that is
temperature-dependent, independent of pH gradients or ATP
hydrolysis, and inhibitable by stilbenes (51, 52). Once transported
into the Golgi lumen, PAPS donates sulfate to sulfotransferases for
protein sulfation. PAPS is an intermediate for synthesis of all
naturally occurring sulfated compounds, including sulfated
glycoproteins, fibronectins, sulfatides, and glycosaminoglycans (51,
52). We have considered whether PAPS, as an adenine nucleotide, might
also be permeable through CFTR.
With high PAPS on both sides of excised plasma membrane patches from
CFTR-expressing CHO cells, channels were observed with essentially
identical biophysical and pharmacological properties as the ATP
channels observed in the plasma membrane and ER (Fig. 6). The channels had a linear I/V relation, had a
4.7 ± 0.1-pS conductance (Fig. 6C), and were
DIDS-insensitive (eight of eight patches; Fig.
7B). They were observed in the presence of
PKA (26 of 28 patches; Fig. 7A) but were not observed in its
absence (0 of 7 patches; Fig. 7A). The channels were
PAPS-selective because a 10-fold PAPS gradient (100 mM
PAPSext/10 mM PAPScyt) shifted the
reversal potential from 0 mV to approximately 15 mV (6 of 7 patches;
Fig. 6, B and C) (the expected Nernstean
potential is ~
15 mV because PAPS, like the ATP in these
experiments, has a valence of
4). Li1+ was excluded as a
current carrier because no channel activities were observed when
currents were recorded in inside-out patches in symmetrical
Li2SO4 solution (Solution C with 150 mM Li2SO4 replacing
Li4PAPS) (0 of 13 patches; data not shown). In control cells, no activities were observed in inside-out patches in Solution C
in the presence or absence of PKA (zero of seven patches; data not
shown).
By demonstrating that PAPS permeability is associated with CFTR, our results raise the possibility that the concentration of PAPS in the Golgi lumen may be regulated in part by CFTR. In a simple model, activity of CFTR in this intracellular compartment would constitute a PAPS "leak" in parallel with the PAPS pump and would therefore tend to lower the PAPS concentration in the Golgi lumen. Lack of CFTR in this compartment in CF would shift the balance of these activities in favor of the pump, resulting in a higher Golgi lumenal PAPS concentration. Because the PAPS concentration in the Golgi lumen is likely rate-limiting for sulfation reactions (50, 53, 54), this model predicts hypersulfation of proteins in CF, consistent with observations. The physiological consequences of hypersulfation may include altered visco-elastic properties of airway secretions and diminished glycoprotein sialylation because sialylation and sulfation reactions are competitive on some glycoprotein residues (55, 56). Importantly, because decreased sialylation has been implicated in the adherence of Pseudomonas to airway epithelial cells (14, 28, 57), the observed PAPS permeability of CFTR may therefore contribute to the heretofore unexplained propensity of Pseudomonas to colonize the lungs of CF patients (57). This PAPS hypothesis therefore links CFTR to glycoprotein processing and possibly to mucus viscosity and bacterial adherence. Because bacterial infection accounts for up to 90% of morbidity and mortality in patients with CF (58), detailed investigations of the PAPS hypothesis are now warranted to determine its relevance for understanding pathophysiology observed in CF.
We thank J. Riordan and X.-B. Chang for providing transfected CHO cells, D. Mak and I. Brockhausen for helpful discussions, D. Wong for technical assistance, and D. Mak, P. Ross, and J. Engelhardt for comments on the manuscript.