RAPID COMMUNICATION
Glutathione permeability of CFTR

Paul Linsdell and John W. Hanrahan

Department of Physiology, McGill University, Montréal, Québec, Canada H3G 1Y6

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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 (gamma -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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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
<IT>P</IT><SUB>GSH</SUB> /<IT>P</IT><SUB>Cl</SUB> = exp(−<IT>E</IT><SUB>rev</SUB> <IT>F</IT> /<IT>RT</IT> ) (1)
<IT>P</IT><SUB>GSSG</SUB> /<IT>P</IT><SUB>Cl</SUB> = [exp(−<IT>E</IT><SUB>rev</SUB>2<IT>F</IT> /<IT>RT</IT> )]/4 (2)
where F is the Faraday constant, R is the gas constant, and T is temperature.

Block of CFTR Cl- current by GSH and GSSG was analyzed using the Woodhull model of voltage-dependent block (21)
<IT>i</IT>/<IT>i</IT><SUB>0</SUB> = <IT>K</IT><SUB>d(<IT>V</IT>)</SUB>/{<IT>K</IT><SUB>d(<IT>V</IT>)</SUB> + [B]} (3)
where i is the amplitude of the current remaining in the presence of blocker, i0 is the control, unblocked current amplitude, [B] is the blocker concentration, V is the membrane potential, and Kd(V) is the voltage-dependent dissociation constant, the voltage dependence of which is given by
<IT>K</IT><SUB>d(<IT>V</IT>)</SUB> = <IT>K</IT><SUB>d(0)</SUB>exp(−<IT>z</IT>′<IT>VF</IT> /<IT>RT</IT> ) (4)
where z' is the apparent valence of the blocking ion.

Experiments were carried out at room temperature (21-23°C). Values are presented as means ± SE.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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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Fig. 1.   Asymmetric glutathione (GSH) and oxidized glutathione (GSSG) permeation in cystic fibrosis transmembrane conductance regulator (CFTR). A-D: example current-voltage (I-V) relationships recorded with either GSH or GSSG present in intracellular or extracellular solution under bi-ionic conditions, as stated on each individual panel. CFTR currents were consistently much smaller with intracellular GSSG than under any of the other ionic conditions studied; reasons for this are unknown. I-V relationships such as those shown in A-D were used to calculate mean permeabilities for GSH (E) and GSSG (F), when present in intracellular solution (In) or extracellular solution (Out) under bi-ionic conditions. Numbers in parentheses are number of patches examined. Permeability ratios shown for GSH and GSSG in extracellular solution in absence of pyrophosphate (PPi) are maximum values, since no reversal potential was observed under these conditions over voltage range studied. Actual permeabilities under these conditions are likely to be considerably lower; they may be zero (see Ref. 11).

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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Fig. 2.   Blocker sensitivity of GSH efflux. Typical examples of I-V relationships recorded under bi-ionic conditions (intracellular GSH, extracellular Cl-) before (control) and immediately after addition of 200 µM 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS; n = 4 patches) or 60 µM glibenclamide (Glib; n = 3 patches) to intracellular solution.

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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Fig. 3.   Block of CFTR Cl- currents by GSH and GSSG. Example I-V relationships recorded with symmetrical Cl--containing solutions, before (control) and immediately after addition of 5 mM GSH or 10 mM GSSG to the intracellular solution.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    ACKNOWLEDGEMENTS

We thank Jie Liao for maintaining the cell cultures and Drs. Elizabeth Cowley and David Eidelman for their comments on the manuscript.

    FOOTNOTES

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.

    REFERENCES
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Abstract
Introduction
Methods
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Discussion
References

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Am J Physiol Cell Physiol 275(1):C323-C326
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