Department of Physiology, McGill University, Montréal, Québec, Canada H3G 1Y6
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ABSTRACT |
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The cystic fibrosis transmembrane conductance
regulator (CFTR) forms an ion channel that is permeable both to
Cl and to larger organic
anions. Here we show, using macroscopic current recording from excised
membrane patches, that the anionic antioxidant tripeptide glutathione
is permeant in the CFTR channel. This permeability may account for the
high concentrations of glutathione that have been measured in the
surface fluid that coats airway epithelial cells. Furthermore, loss of
this pathway for glutathione transport may contribute to the reduced
levels of glutathione observed in airway surface fluid of cystic
fibrosis patients, which has been suggested to contribute to the
oxidative stress observed in the lung in cystic fibrosis. We suggest
that release of glutathione into airway surface fluid may be a novel
function of CFTR.
cystic fibrosis; chloride channel; lung defense; airway surface fluid; multidrug resistance protein ; cystic fibrosis transmembrane conductance regulator
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INTRODUCTION |
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CYSTIC FIBROSIS (CF) is caused by mutations in a single
gene: the one that encodes the CF transmembrane conductance regulator (CFTR). CFTR, which is a member of the "ATP-binding cassette" (ABC) family of membrane transport proteins, forms a phosphorylation- and ATP-dependent Cl
channel (3, 4). It remains unclear how the reduced epithelial Cl
permeability caused by
the functional absence of CFTR leads to the complexity of symptoms seen
in CF lung disease. This, coupled with the structural similarity
between CFTR and other ABC proteins, has long hinted that CFTR may also
perform some other function.
The anionic tripeptide glutathione (-glutamyl-cysteinyl-glycine;
GSH) is the most important extracellular antioxidant in the lung (1,
20). Airway surface fluid (ASF) contains ~400 µM GSH, ~50 times
the concentration found in plasma and 100 times that found in the
extracellular fluid of many other tissues (1, 5, 17, 20). The source of
the high concentration of GSH found in ASF is unknown (20).
Interestingly, the concentration of GSH in ASF is greatly reduced in CF
(16), which may exacerbate the severe oxidative stress that results
from chronic inflammation of the CF lung (19). Indeed,
antioxidant therapy for CF has previously been suggested (2, 16, 19).
Recently we demonstrated that a broad range of large organic anions
were able to permeate through the CFTR
Cl channel from the
cytoplasmic side of the membrane, and we suggested that release of such
anions into ASF might be a novel physiological function of CFTR (11).
Here we show that both GSH and oxidized glutathione (GSSG) can permeate
through CFTR from the intracellular solution. These results suggest
that CFTR, in addition to its role as a
Cl
channel, may function as
a permeation pathway by which GSH is released into ASF. Such a function
would suggest a previously unidentified link between CFTR and lung
antioxidant defense. Loss of this GSH permeation pathway may contribute
to the pathogenesis of CF.
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METHODS |
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Macroscopic CFTR current recordings were carried out on inside-out membrane patches excised from baby hamster kidney cells stably expressing CFTR, as described in detail elsewhere (9, 11). Briefly, channels were activated by exposure of the cytoplasmic face of excised patches to 40-60 nM protein kinase A catalytic subunit (PKA) plus 1 mM MgATP. All current traces shown have had the background leak current, recorded before addition of PKA, digitally subtracted, as described previously (9, 11). Current traces were filtered at 100 Hz using an eight-pole Bessel filter, digitized at 250 Hz and analyzed using pCLAMP6 computer software (Axon Instruments, Foster City, CA).
Recording solutions contained (in mM) 150 NaCl, 2 MgCl2, 10 TES, or 154 NaGSH or
Na2GSSG plus 2 Mg(OH)2 and 10 TES. All solutions were adjusted to pH 7.4 using NaOH. Where the pipette solution did not
contain Cl, the pipette
Ag-AgCl wire was protected by an NaCl-containing agar bridge inside the
pipette. Voltages were corrected for measured liquid junction
potentials of up to 12 mV existing between dissimilar pipette and bath
solutions. All chemicals were obtained from Sigma (St. Louis, MO)
except 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS;
Pfaltz & Bauer, Waterbury, CT) and glibenclamide (glyburide; Calbiochem, La Jolla, CA).
Macroscopic current-voltage (I-V) relationships were constructed using depolarizing voltage ramp protocols as described previously (9, 11). The current reversal potential, Erev, was estimated by fitting a polynomial function to the I-V relationship and was used to estimate permeability (P) ratios according to the equations
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(1) |
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(2) |
Block of CFTR Cl current by
GSH and GSSG was analyzed using the Woodhull model of voltage-dependent
block (21)
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(3) |
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(4) |
Experiments were carried out at room temperature (21-23°C). Values are presented as means ± SE.
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RESULTS |
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A broad range of large organic anions are able to carry current through
CFTR when present in the intracellular solution (11). PKA-stimulated
I-V relationships obtained with GSH
(Fig.
1A) or GSSG (Fig. 1B) in the intracellular
solution and Cl in the
extracellular solution (bi-ionic conditions) indicated that both of
these large anions were permeant in CFTR, with mean permeabilities
(according to Eqs. 1 and 2) of 0.082 ± 0.011 (n = 8) for GSH and 0.0061 ± 0.0017 (n = 5) for GSSG. In contrast, neither GSH (Fig. 1C) nor GSSG (Fig.
1D) were measurably permeant when
present in the extracellular solution under bi-ionic conditions. This
asymmetric permeability is similar to that which we previously observed
with a number of other large organic anions (11). This asymmetry is
abolished by substances that "lock" CFTR channels in the open
state, such as pyrophosphate
(PPi) (11). As shown in Fig. 1,
C and
D, addition of 10 mM
PPi to the intracellular solution
stimulated the influx of GSH and GSSG from the extracellular solution.
All of these properties of GSH and GSSG permeation are similar to those
we described previously for a number of different large organic anions
(11). As discussed in more detail elsewhere (11), the reasons for the
disruption of asymmetric permeability caused by
PPi are not currently known but
may involve inhibition of ATP hydrolysis-dependent transitions between
different channel open states. The mean relative permeability of GSH
and GSSG under different conditions, calculated according to
Eqs. 1 and 2 (see METHODS), are summarized in Fig. 1,
E and
F. Note that the permeability of the
divalent anion GSSG is very low compared with GSH and other large
monovalent anions (Fig. 1, E and
F) (11).
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The suggestion that intracellular large organic anions were permeant in
CFTR and not simply transported by some CFTR-regulated transport
process was supported by the finding that currents carried by such
large anions were inhibited by the CFTR open-channel blockers DNDS and
glibenclamide (11). Both DNDS and glibenclamide also blocked the
current carried by intracellular GSH under bi-ionic conditions (Fig.
2). This suggests that GSH and
Cl share a common
permeation pathway through CFTR, as previously suggested for other
large anions (11).
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If GSH and GSSG are able to permeate through the CFTR
Cl channel pore, then they
might also be expected to act as open channel blockers of CFTR
Cl
current. Indeed, the
large organic anions gluconate and glutamate, both of which are
permeant in CFTR when present in the intracellular solution (11), are
also low-affinity blockers of
Cl
permeation through the
channel (10). As shown in Fig. 3, addition of GSH (5 mM) or GSSG (10 mM) to the intracellular solution reduced the
macroscopic CFTR Cl
current
recorded with 150 mM NaCl present on both sides of the membrane. Block
by intracellular GSH and GSSG was immediate and readily reversible,
consistent with an open channel block mechanism. Analysis of the
blocking effects of GSH and GSSG using the Woodhull model (21; see
METHODS) suggested
Kd(0) = 15.8 ± 3.7 mM and z' =
0.11 ± 0.02 (n = 5) for GSH
and Kd(0) = 23.7 ± 2.9 mM and z' =
0.02 ± 0.03 (n = 4) for
GSSG. Thus, although the affinity of channel block by these two anions
is relatively low, it is at least 20 times higher than that estimated
for intracellular gluconate or glutamate ions at a membrane potential
of 0 mV (~600 and 1,100 mM, respectively; Ref. 10). The ability of
intracellular GSH and GSSG to act as blockers of CFTR
Cl
current is consistent
with their being able to enter the pore from the intracellular end.
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DISCUSSION |
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Despite early skepticism due to its structure, CFTR has been
conclusively proven to be a
Cl channel. Reduced
transepithelial Cl
transport is thought to be the primary defect in CF (15); however, the
link between this reduced
Cl
permeability and the
pathogenesis of CF lung disease remains unclear. Previously, we
suggested that CFTR may fulfill another direct transport role, allowing
efflux of a broad range of large organic anions from the cytoplasm
(11). Based on our present results, we propose that a potential
physiological substrate for this transport pathway is the important
antioxidant molecule GSH. The ability of GSH to permeate through CFTR
suggests a direct link between CFTR and antioxidant defense in the
lung.
GSH is synthesized in the cytoplasm, where levels of 1-10 mM are typical (17). High concentrations of GSH (~400 µM) are also found in ASF of healthy individuals (1). We propose that GSH may be released into ASF via CFTR, further emphasizing the role of CFTR in determining ASF composition (18). Because cytoplasmic concentrations of GSH are so high, a passive permeation pathway would presumably be sufficient to mediate release of such amounts of GSH into ASF. Loss of this GSH transport pathway may contribute to CF lung disease. The levels of GSH are greatly reduced in ASF of CF patients (16), potentially exacerbating the oxidant stress that results from neutrophil-dominated inflammation of the CF lung (16, 19). GSH levels in ASF are also reduced in idiopathic pulmonary fibrosis and adult respiratory distress syndrome and are increased in cigarette smokers with no clinical evidence of lung disease (16).
GSH and GSSG transport by CFTR suggests a functional similarity between CFTR and the structurally related ABC proteins, multidrug resistance protein (MRP) and canalicular multispecific organic anion transporter (cMOAT; also known as MRP2). Both MRP and cMOAT mediate ATP-dependent export of large intracellular organic anions, in particular glutathione-S-conjugates (6, 14). GSSG is a substrate for both MRP- and cMOAT-mediated transport (8, 14). Unconjugated GSH may be transported by cMOAT (14) but is thought not to be transported by MRP (8); however it may act as a cosubstrate for MRP-mediated transport of unconjugated compounds (12, 13). Potential functional overlap between CFTR and other ABC proteins is of particular interest, since it has been suggested that MRP may be able to functionally substitute for CFTR in the lung and alleviate CF symptoms (7). Our results suggest that such functional substitution may be due to restoration of GSH or GSH-conjugate transport.
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ACKNOWLEDGEMENTS |
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We thank Jie Liao for maintaining the cell cultures and Drs. Elizabeth Cowley and David Eidelman for their comments on the manuscript.
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FOOTNOTES |
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This work was supported by the Canadian Cystic Fibrosis Foundation (CCFF), Canadian Medical Research Council (MRC), and National Institute of Diabetes and Digestive and Kidney Diseases. P. Linsdell is a CCFF postdoctoral fellow. J. W. Hanrahan is an MRC scientist.
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. §1734 solely to indicate this fact.
Address for reprint requests: P. Linsdell, Dept. of Physiology, McGill Univ., 3655 Drummond St., Montréal, Québec, Canada H3G 1Y6.
Received 6 March 1998; accepted in final form 14 April 1998.
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