Pyridine Nucleotide Redox Potential Modulates Cystic Fibrosis Transmembrane Conductance Regulator C1- Conductance*

C1- conductance of the apical membrane of airway epi- thelial cells has properties of a passive diffusion mechanism but is decreased by inhibition of oxidative metabo- lism. Recent reports that CAMP-dependent C1- conductance also requires ATP at the intracellular do-mains of the cystic fibrosis transmembrane conductance regulator (CFTR) suggest that ATP concentration could mediate metabolic regulation of C1- conductance. How-ever, metabolic inhibitors affect processes other than ATP free energy levels, including notably the metabolic pathways that set the redox potential of pyridine nucle- otides within the cell. We have investigated the possibility that CFTR-mediated C1- conductance is affected by the ratio of oxidized to reduced intracellular pyridine nucleotides. CFTR was expressed in airway and heter- ologous cells and studied under whole cell voltage clamp conditions, which permitted the intracellular NAD(P)+/ NAD(P)H ratio to be varied independently of ATP concentration. In three cell types expressing CFTR, whole cell dialysis with reduced pyridine nucleotides inhibited activation of C1- currents by forskolin and 8-(4-chlo-ropheny1thio)-CAMP (CPT-CAMP), whereas dialysis with oxidized

C1conductance of the apical membrane of airway epithelial cells has properties of a passive diffusion mechanism but is decreased by inhibition of oxidative metabolism. Recent reports that CAMP-dependent C1conductance also requires ATP at the intracellular domains of the cystic fibrosis transmembrane conductance regulator (CFTR) suggest that ATP concentration could mediate metabolic regulation of C1conductance. However, metabolic inhibitors affect processes other than ATP free energy levels, including notably the metabolic pathways that set the redox potential of pyridine nucleotides within the cell. We have investigated the possibility that CFTR-mediated C1conductance is affected by the ratio of oxidized to reduced intracellular pyridine nucleotides. CFTR was expressed in airway and heterologous cells and studied under whole cell voltage clamp conditions, which permitted the intracellular NAD(P)+/ NAD(P)H ratio to be varied independently of ATP concentration. In three cell types expressing CFTR, whole cell dialysis with reduced pyridine nucleotides inhibited activation of C1currents by forskolin and 8-(4-chlo-ropheny1thio)-CAMP (CPT-CAMP), whereas dialysis with oxidized pyridines increased both basal and stimulated CFTR-mediated C1-conductance. In cell-attached membrane patches, the open probability of 5-6-picosiemens C1-channels that had been activated by forskolin and CPT-CAMP was further and reversibly increased by permeant oxidants. Neither swelling-induced whole cell K' currents in CFTR-expressing cells nor swelling-induced whole cell C1-currents in multidrug resistance proteinexpressing cells were affected by NADPH. Pyridine nucleotide redox potential had little effect on phosphorylation of histone by protein kinase A. We conclude that CFTR C1conductance function can be modulated by pyridine nucleotide redox potential. This effect points to the existence of a mechanism or mechanisms by which cytosolic nucleotides other than ATP can affect plasma membrane C1-conductance and may help explain how a passive ion conductance is linked to cellular energy metabolism.
In a previous study, exposure of dog bronchial epithelium to NaCN or hypoxia inhibited CAMP-dependent CI-conductance (1). Although one mechanism of this effect could be decreased protein phosphorylation caused by depletion of intracellular * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ATP, we reasoned that it was unlikely that hypoxia or NaCN could drive intracellular ATP concentrations below the K, for ATP of CAMP-dependent protein kinases of -3 ( 2 ) . Recently, an integral membrane protein called CFTR' has been identified as the C1-channel underlying CAMP-activated apical membrane C1-conductance of C1--secreting epithelia (3). The predicted amino acid sequence of CFTR placed i t as a member of the ATP-binding cassette protein superfamily (4). Other ATPbinding cassette proteins, such as multidrug resistance (MDR) protein, bacterial permeases, and sterile-6, are solute transporters that hydrolyze ATP ( 5 ) . Sequences contained within CFTR are highly homologous to the putative nucleotide binding folds of MDR protein (6) Recently, it was reported that hydrolyzable nucleotides were required to support CFTR C1-channel activity in excised membrane patches, and the K,,, of ATP for this requirement was 300 PM (7). In contrast, in human sweat ducts, CFTR-mediated C1-conductance was supported by ATP and nonhydrolyzable ATP analogs, with an apparent K, estimated at several mM (8). The results of these studies suggest that changes in the intracellular concentration of ATP could link CAMP-dependent C1-conductance to metabolism through a requirement of CFTR for intracellular ATP.
Beyond maintaining ATP levels, however, substrate metabolism affects other intracellular conditions that influence diverse cellular functions. For example, inhibition of oxidative metabolism by NaCN or hypoxia depletes intracellular ATP by preventing the transfer of electrons extracted from metabolic substrates to oxygen. This results in an increase in the intracellular ratio of reduced to oxidized species of electron carriers, including the pyridine nucleotides (9). This change in redox potential is transmitted throughout the cell because of the wide participation of pyridine nucleotides as cosubstrates in enzymatic processes. In particular, redox potential controls proteinprotein associations by affecting the formation of disulfide bonds (10). Ran and Benos (11) found that disulfide bond formation promoted by oxidizing conditions led to multichannel activity and greater conductance in reconstitution studies of epithelial anion channels. Modulation of ion channel activity by intracellular redox potential has been reported in a number of cells (12,13).
The C1-conductance function of CFTR has been well characterized in heterologous expression systems, in which nonpolarized cells infected with viral constructs containing recombinant human CFTR cDNA respond to elevation of intracellular CAMP with increased C1-conductance (15-18). The character-

8668
Pyridine Nucleotides Modulate CFTR istics of this CAMP-dependent C1-conductance match those of single C1-channels activated by CAMP in C1--secreting epithelia (19,20). In the present study, we measured CAMP-activated whole cell current in CFTR-expressing cells with cytosolic ATP clamped at 3 mM while varying the concentration of reduced and oxidized pyridine nucleotides. We also measured the open probability of CFTR C1-channels in cell-attached membrane patches in the presence of permeant oxidants and reductants. Our results demonstrate that activation of the C1-conductance mediated by CFTR has a step that is sensitive to the redox status of pyridine nucleotides.

MATERIALS AND METHODS
Cells-Mouse NIH 3T3 fibroblasts were cultured by standard techniques in Dulbecco's modified Eagle's medium H + 10% fetal bovine serum supplemented with penicillin and streptomycin. The origin and culture of the CFTl epithelial cell line have been reported (21). The construction and production of the murine amphotropic retrovirus vectors LCFSN and LISN have been described previously (22). LCFSN contains the normal human CFTR cDNA under transcriptional control of the promoter elements in the retrovirus vector long terminal repeat sequence and a neo' (neomycin resistance) gene under control of a n internal SV40 promoter. LISN contains the cDNA for the a-subunit of the interleukin-2 receptor and a neo' gene and was used to generate control cells. 3T3 fibroblasts and CFTl cells were infected with LCFSN or LISN. Drug-resistant clones were selected after growth in G418. Infected cells retained CFTR-associated properties beyond passage 20. For the studies reported here, cells of passages 10-16 were subcultured onto 35-mm plastic dishes. Sf9 cells were grown in Spinner flasks in Grace's medium (23). Cells were subcultured onto 35-mm plastic dishes and exposed to a plaque pure baculovirus modified by replacement of the viral polyhedron gene with the human CFTR gene or with the p-galactosidase gene. Infected Sf9 cells were killed by viral infection within 48-72 h. High levels of CFTR protein were expressed by 36 h by immunoblot analysis. Insect cells were used for whole cell voltage clamp within 24-40 h of infection. T84 cells, a human colonic carcinoma cell line that expresses CFTR (241, were obtained from ATCC and maintained in Dulbecco's modified Eagle's medium with high glucose (4.5 gfliter) supplemented with 10% fetal bovine serum, penicillin, and streptomycin.
Immunoblot Analysis of CFTR Protein-CFTR protein was detected by the method of Sarkadi et al. (25) using purified polyclonal rabbit antibodies raised against synthetic human CFTR peptides as described previously. 3T3 fibroblasts and CFTl cells were grown to confluence in plastic flasks, washed with phosphate-buffered saline, and harvested by trypsinization. Sf9 cells were collected and washed. Whole cell protein was extracted by suspension of cells in 6% (w/v) trichloroacetic acid and collection of the precipitate as a pellet (5,000 x g, 5 min). The pellet was dissolved in sodium dodecyl sulfate disaggregation buffer (50 m~ Tris phosphate (pH 6.8), 2% (w/v) sodium dodecyl sulfate, 15% (w/v) glycerol, 2% (v/v) p-mercaptoethanol, 1 mM EDTA, and 0.02% (w/v) bromphenol blue). Electrophoresis of protein samples was carried out using 4 1 5 % gradient acrylamide gels (Bio-Rad). Electroblotting of the proteins to polyvinylidene difluoride membranes (Bio-Rad) was carried out as described previously (25).
Patch Clamp Techniques-Cells were studied on the stage of an inverted microscope a t 22 "C by the techniques of Hamill et al. (26). For most whole cell studies, pipettes (2-6 megohms) were filled with an intracellular buffer with the following composition, in &iter: Tridhy-droxymethy1)aminomethane gluconate, 100; Tris-Cl-, 40; MgCl,, 2; and TES, 5 (pH 7.21, and MgATP, 3. Ca2+ activity was buffered to approximately 40 nM (1.0 mM EGTA and 0.1 m~ CaCl,; calculated with Chelator Software, Th. Schoenmakers). Bath solutions for mammalian cells contained Tris-C1 or NaC1,150; MgCl,, 2; CaCl,, 1; TES, 5; sucrose, 30; and D-glucose, 10. Voltage was clamped at -40 mV and stepped to 60 mV every 10 s for 0.4 s. Whole cell current voltage plots were obtained from the currents recorded in response to a series of voltage steps from zero, covering the range -80-140 mV in 20-mV increments. The effect of Ion whole cell C1-currents was assessed after diluting the bath solution with 150 mM NaI extracellular buffer. For Sf9 cells the bath was NaCl, 132; CaCl,, 5; MgCl,, 4; TES, 10 (pH 6.7); and glucose, 10. Cell-attached recordings were carried out i n 3T3 fibroblasts expressing CFTR. In this paradigm the C1-concentration in the pipette solution was either 155 NaCl or a combination of 40 mM Tris-C1 and 100 mM Tris gluconate.
Histone Phosphorylation-The effect of redox reagents on the activity of protein kinase A was investigated by a modification of the histone labeling procedure (27). Purified catalytic subunit of protein kinase A (Promega) was used to phosphorylate comparatively type IIA histone (Sigma) with [Y-~~PIATP as the phosphate donor. The reaction products were trapped on phosphocellulose paper and y emissions collected to quantitate the transfer of 32P to histone. Control reaction buffer contained protein kinase A (150 ndliter), 3 mM [Y-~~PIATP, 1 mg/ml type IIA histone, 0.25 mg/ml bovine serum albumin, 10 m~ MgCl,, 50 m~ TES (pH 7.2). Test buffers had the same composition with the addition of 1 &iter of one of the following: NAD+, NADH, NADP+, NADPH. In the absence of protein kinase A, transfer of 32P to histone was not different from background for any reaction conditions. Statistical Analyses-Drug-induced changes in whole cell current and differences between means were assessed for statistical significance by paired t tests (28).

RESULTS
Expression of CFTR protein in Sf9 (insect), NIH 3T3 (mouse fibroblast), and CFTl (human cystic fibrosis airway epithelia) cells was verified by Western blot analysis (Fig. lA). CFTR expressed in Sf9 cells infected with the baculovirus vector containing the human CFTR cDNA was detected as a 140-kDa band by Western blot analysis, consistent with previous reports that CFTR is not glycosylated in these cells (25). Protein extracted from 3T3 and CFTl cells infected with the LCFSN vector contained a broad immunoreactive band in the 155-175-kDa region, a staining pattern expected of variably glycosylated CFTR protein (291, and similar to the staining of protein extracted from T84 cells. Whole cell C1-currents stimulated by a combination of forskolin (25 J~M ) and CPT-CAMP (0.5 mM) were measured as a functional assay of CFTR expression. Experiments that were carried out in Sf9 cells are illustrated in Fig.  1B. Forskolin and CPT-CAMP caused no significant change in basal currents in control Sf9 cells, including uninfected cells and cells infected with a recombinant baculovirus containing the cDNA for P-galactosidase. Forskolin and CPT-CAMP stimulated outward currents at 60 mV in cells that had been exposed to recombinant CFTR baculovirus. Stimulated currents in CFTR-expressing cells were insensitive to DIDS (14) but were inhibited by external I- (Fig. 1B). The latter phenomenon was originally interpreted as an apparent greater selectivity for C1over I-by CFTR (14) but more recently as a block of anion permeation through CFTR C1-channels by concentrations of Iless than about 150 m M (30, 31). Summary current-voltage relationships of basal and stimulated currents carried out in CFTR-expressing and sham-infected Sf9 cells, 3T3 fibroblasts, and CFTl cells are shown in Fig. 1C. C1-was the major permeant anion in our solutions, and the cells were dialyzed with solution that contained no permeant cations. In each set of data the stimulated currents reversed near the reversal potential calculated from the C1-concentrations of pipette and bath SOlution and showed little voltage dependence. The data in Fig. 1 demonstrate expression of CFTR protein and C1-conductance function in three different expression systems.
Inhibition of oxidative metabolism, as employed in a previous study (I), is expected to decrease intracellular ATP but is also predicted to increase the ratio of reduced to oxidized pyridine nucleotides (9). We took advantage of whole cell dialysis to clamp intracellular ATP at 3 mM. Protocols to activate and characterize CFTR-mediated C1-currents (similar to that in Fig. 1B C. Clamp voltage was held a t -40 mV and pulsed to 60 mV every 10 s for 0.4 s. The broad pulses were recorded a t higher chart speed to allow better resolution. Current-voltage relationships were obtained after each high resolution pulse recording. Forskolin (25 p~) , CPT-cAMP (0.5 mM), DIDS (0.5 mM), and I-(65 mM) were present beginning at the times indicated by arrows. Panel C, current-voltage relationships before and during CAMP exposure. Control cells, basal (circles), and forskolin-exposed (souares) cells are compared with CFTR-expressing cells, basal (right side-up tri&gles) and forskolin exposed (upside-down triangles) bells. and stimulation of current by forskolin and CPT-CAMP was decreased compared with CFTR-expressing cells studied with no pyridine nucleotide in the pipette solution (Fig. 2). In contrast, both the resting and stimulated currents in CFTR-expressing cells dialyzed with NADP+ were greater than under control conditions. These effects of reduced and oxidized NADP on the CAMP-dependent C1-conductance associated with CFTR expression were found in Sf9 cells, in 3T3 fibroblasts, and in CFTl airway epithelial cells. NADP+/NADPH dialysis had no effect on currents recorded from uninfected cells (data not shown).
We verified that the elevated basal currents and large responses to CAMP in cells dialyzed with NADP' were mediated by CFTR. In the experiment illustrated in Fig. 3A, basal outward (CI-) current stabilized a t 3 nA. Addition of forskolin and CPT-CAMP further increased current to nearly 5 nA. The current-voltage relationship for the large basal and stimulated currents were linear (Fig. 3, B and C ) and reversed near Ecl (Fig. 3 0 . Exchanging 120 mM bath solution NaCl with NaI sharply inhibited CY current, to a value well below the nonstimulated resting current, and decreased whole cell conductance. The current-voltage relationship in the presence of I-was depolarized (Fig. 3C), characteristic of the effect of external Ion CFTR-mediated anion conductance. In a series of experiments, the inhibition of stimulated whole cell conductance by 75 mM I-was larger in cells dialyzed with 0.3 mM NADP' than in cells dialyzed with control pipette solution (Fig. 30), consistent with the larger conductance in the presence of oxidized NADP being mediated by CFTR.
To explore the mechanism of inhibition of CFTR-mediated C1-conductance by NADPH, we compared the effects of NADPH dialysis on forskolidCPT-CAMP-stimulated C1-conductance to the effects of NADH dialysis. As demonstrated above, CFTR-expressing cells dialyzed with NADPH typically responded to forskolin and CPT-CAMP with no changes in outward current (Fig. 4A) and whole cell conductance (Fig. 4B ). In cells dialyzed with 1.2 mM NADH, forskolin and CPT-CAMP consistently induced increases in outward current (Fig. 4A) and conductance (Fig. 4B), but the forskolidCPT-CAMP-induced conductance (3.0 z 0.6 nanosiemens, n = 7) was significantly ( p < 0.03) smaller than the response in control experiments (8.01 z 0.18 nanosiemens, n = 8). We reasoned that if the inhibition of CAMP-stimulated C1-conductance was caused by the reduced status of the pyridine nucleotide, then a maneuver to shift the redox ratio to a more oxidized state should allow current to increase. To test this notion we added the cell-permeant electron carrier PMS to the bath (2.5 mM). TBOH was included (2.5 mM) to assure that PMS was fully oxidized. We expected oxidized PMS to enter the cell and oxidize a portion of cytosolic NADPH or NADH. Strikingly, the block of CAMP activation of current in CFTR-expressing cells that were dialyzed by 1.2 mM NADPH was overcome by this maneuver (Fig. 4, A  and B ) . The newly stimulated conductance had identifying characteristics of CAMP-dependent CFTR-mediated conductance, including reversal of the current-voltage plot near the predicted reversal potential of a pure C1-current (-25 uersus -33 mV, not shown), resistance to 0.5 mM DIDS, and inhibition by 75 mM I- (Fig. 4). In the same protocol, the inhibition of CAMP-dependent outward current (Fig. 4A) and whole cell conductance (Fig. 4B) by 1.2 mM NADH was not reversed by the PMSRBOH oxidant cocktail. PMS and TBOH had no effect on whole cell currents of cells not expressing CFTR (not shown). We extended the study of how pyridine nucleotides affect CFTR function by measuring the current and conductance in stimulated cells that were dialyzed with regular pipette solution or with 0.3 mM NADP+ or NAD'. We also tested whether a cell-permeant reducing agent (NaMBS) (32) would affect current stimulated in the presence of oxidized pyridine nucleotides in the pipette. The experiment in Fig. 5A is representative. Outward C1-current of a CFTR-expressing cell dialyzed with pipette solution that contained 0.3 mM NAD+ was stimulated with forskolin and CPT-CAMP. NaMBS (3 m) reversed this stimulation. The inhibition induced by NaMBS was not altered by the electron carrier PMS, which would be highly reduced in the presence of metabisulfite, but was overcome by introduction of the oxidant TBOH. The current manipulated by inter- actions between cytosolic pyridine nucleotides and permeant reductants and oxidants in this experiment was inhibited by 75 mM external I-. Data from similar experiments comparing NaMBS inhibition of forskolidCPT-CAMP-stimulated currents in cells dialyzed with normal pipette solution or pipette solution containing NADP+ or NAD+ are summarized as whole cell conductance in Fig. 5B. The larger whole cell conductance recorded with oxidized pyridine nucleotides in the pipette was more inhibited by the permeant reducing agent, suggesting that the oxidized state of the pyridine nucleotides was important for CFTR-mediated C1-conductance.
The single channel basis of CFTR-mediated C1-conductance has been reported to be a 4-8 picosiemens, slowly gating anion channel (15)(16)(17)(18). We reasoned that modulation of CFTR-mediated whole cell conductance by permeant oxidants and reductants should be paralleled by effects on single CFTR C1-channels. In five of seven attempts in cell-attached membrane patches of 3T3 fibroblasts expressing CFTR, forskolin and CPT-CAMP induced the appearance of small, slowly gating channels, similar to what has been reported for CFTR C1-channels in other studies (Fig. 6 A ) (15)(16)(17)(18). No channels were activated by forskolin and CPT-CAMP in fibroblasts not expressing CFTR (n = 3, not shown). The mean current-voltage relationships for CAMP-dependent channels from cell-attached recordings in stimulated cells revealed no marked voltage dependence and a single channel conductance of approximately 6 picosiemens in the range of -Vpipette from 10 to 60 mV (Fig. 6B). The zero current voltage was 22 mV with 45 mM C1-in the pipette and 0 mV with 155 C1-in the pipette, indicating C1-selectivity (Fig.  6B). As reported previously, endogenous C1-channels were present in both infected (Fig. 6C, all traces)  (not shown and (23)), but had a larger conductance, more rapid kinetics, and did not respond to CPT-CAMP (Fig. 6C). Introduction of permeant oxidants (PMSRBOH) into the bath promptly increased the activity of CFTR C1-channels but did not affect the activity of an endogenous C1-channel present in the same patch (Fig. 6C, third and fourth traces from top). The effect on CFTR C1-channels induced by PMSRBOH was an increased open probability compared with forskolin and CPT-CAMP alone ( p < 0.05) (Fig. 6D). The addition of NaMBS partially reversed the increase in CFTR C1-channel open probability seen with PMSRBOH ( p < 0.05) (Fig. 6C, bottom trace, and Fig. 6D). These single channel studies generally parallel the effects of redox agents on CAMP-dependent whole cell currents in CFTRexpressing cells and thus support the interpretation that intracellular redox potential modulates the CAMP-dependent C1conductance mediated by CFTR.
We explored whether pyridine nucleotide redox status affected currents other than CAMP-dependent C1-current by measuring currents induced in fibroblasts by hypotonic swelling. A clone of CFTR-infected 3T3 cells which expressed small quantities of CFTR protein and no CAMP-dependent C1-conductance (45) was used to test the effect of NADPH on non-CFTR-mediated conductances in fibroblasts. The presence of 1.2 m M NADPH in the pipette solution did not affect the whole cell current stimulated by hypotonic (0.5 isotonic) shock (compare Fig. 7, A and B). MDR protein, an ATP-binding cassette protein with a structure analogous to CFTR, was reported to mediate hypotonically induced C1-conductance (5,6). In 3T3 fibroblasts infected with a viral construct containing the cDNA for MDR protein (5,33) we found that stimulation of whole cell currents by hypotonic shock was not affected by NADPH in the pipette (Fig. 7 B ) . Thus, modulation of CAMP-dependent C1current by NADP redox status is a characteristic of CFTRmediated C1-conductance not shared by other conductances in fibroblasts.
Under the conditions of our experiments, i.e. high levels of CFTR expression, constant 3 mM ATP, and permeant CAMP, the only protein elements known to be required for CAMP-dependent activation of CFTR-mediated whole cell C1-currents are CFTR and endogenous protein kinase A. To determine if redox potential could affect the function of protein kinase A, we examined the protein kinase A-mediated incorporation of 32P into histone in reaction mixtures that contained oxidized or reduced nucleotides. As summarized in Fig. 8, neither reduced nor oxidized pyridine nucleotides had significant effects on protein kinase A function. DISCUSSION In sweat ductal and airway epithelia, apical membrane C1conductance has been reported to diminish under conditions of metabolic inhibition. Apical membrane C1-conductance is traditionally modeled as a process with the characteristics of passive ion diffusion, so the apparent metabolic dependence of C1conductance was unpredicted. CFTR has been identified as the epithelial CAMP-dependent C1-conductance. CFTR has consensus nucleotide binding sites which have been proposed to bind or hydrolyze ATP, and ATP concentration at the cytosolic membrane surface has been implicated in the regulation of CFTR C1-conductance in sweat ductal epithelium and gating of CFTR channels in excised membrane patches (7,8). Intracellular ATP concentration has been proposed to couple apical membrane C1-conductance to metabolic state. In the present study, however, we clamped intracellular ATP a t a physiologic concentration and found that variation of pyridine nucleotide redox potential affected CFTR C1-conductance. When three diverse cell types that expressed CFTR were dialyzed with reduced pyridine nucleotides, CAMP-dependent C1-current was diminished. Conversely, during dialysis of CFTR-expressing cells with oxidized pyridine nucleotides, basal currents were larger, and the responses of CFTR-mediated C1-currents to CAMP were enhanced. Because ATP concentration is not expected to have changed during these experiments, the results show that redox potential chemically clamped by NAD(P)H and NAD(P)+ modulates CFTR C1-conductance.
Several observations confirmed that the C1-currents affected by pyridine nucleotides in CFTR-expressing cells were indeed mediated by CFTR. First, the currents of uninfected cells of epithelial, nonepithelial, and insect origin, which demonstrated no CAMP-dependent C1-current, were unaffected by dialysis with oxidized or reduced nucleotides or by exposure to permeant oxidants and reductants. Second, the C1-currents activated by CAMP in CFTR-expressing Sf9, CFT1, and 3T3 cells exposed t o intracellular oxidized pyridine nucleotides were DIDS-insensitive and were inhibited by external I-, two important characteristics of CAMP-dependent C1-currents mediated by CFTR. Most definitively, we recorded single CFTR C1-channels in cell-attached membrane patches of 3T3 cells expressing CFTR and found that the activity of these channels was modulated by permeant oxidants and reductants in a way that was consistent with the modulation of whole cell C1-conductance.
The mechanism by which the redox status of intracellular pyridine nucleotides affects CFTR C1-conductance is unknown. Under the conditions of our experiments, CFTR and protein kinase A are the only proteins with well established roles in the activation of CAMP-dependent C1-conductance (3). Required soluble reactants, i.e. ATP and CAMP, were provided exogenously, and we determined that redox potential clamped by pyridine nucleotides had no effect on the ability of protein kinase A to phosphorylate histone. Other regulatory proteins known to interact with CFTR, such as protein kinase C (35), cGMP-dependent kinase (361, and protein phosphatases (161, do not require pyridine nucleotides as cofactors. Thus, modulation of CAMP-dependent C1-currents by cytosolic NADP)/ NAD(P)H could be explained by reversible regulation of the CFTR protein itself. Perhaps more plausible, however, is the possibility that CFTR may interact with proteins not yet identified but nonetheless important for CAMP-activated c1-conductance, such as cytoskeletal elements. Either the function of such putative proteins or their possible interactions with CFTR could be influenced by pyridine nucleotide redox potential.
The differential effects of NAD and NADP on CFTR C1conductance function may allow some speculation as to the nature of the mechanism(s) by which pyridine nucleotides affect CFTR. Although NADH, like NADPH, inhibited activation of CFTR C1-conductance by CAMP, inhibition by NADH was not as complete and was not reversed by permeant oxidants. Moreover, whereas cells dialyzed with NAD+ very consistently responded to forskolin and CPT-CAMP with characteristic CFTR C1-conductance, the largest responses occurred in cells dialyzed with NADP'. It is not uncommon for proteins to exhibit different relative affinities for reduced and oxidized forms of NADP and NAD. For example, the order of affinities for pyridine nucleotide binding to catalase is NADPH > NADH > NADP' > NAD' (44). Thus, tight binding of NADPH to CFTR, or a CFTR-associated protein, can be speculated to favor a CFTR conformation that is not activated by cAMP-dependent phosphorylation, whereas NADH may have a weaker binding affinity for the same site. Dialysis of CFTR-expressing cells with high concentrations of reduced nucleotides would then account for the inhibition of CFTR which we observed. Under the oxidized conditions imposed by dialysis with NADP+, or by exposure of NADPH dialyzed cells to permeant oxidants, NADPH bound to CFTR would be displaced by or converted to NADP', relieving NADPH-mediated inhibition of C1-conductance. NADH dialysis may mediate inhibition of CFTR which cannot be reversed by exposure to permeant oxidants because this maneuver would convert a portion of NADH to NAD', speculated to be the least avidly bound nucleotide and perhaps unable to compete with NADH for binding.
Alternatively, the greater effectiveness of the NADPW NADP+ couple may indicate that redox modulation of CFTR C1conductance is mediated through the activity of enzymes that prefer NADPH over NADH. This specificity is a characteristic of enzymes, such as glutathione S-transferase, glutathione reductase, and protein disulfide isomerase, which utilize the typically highly reduced status of NADPH to maintain protein cysteines and free intracellular glutathione in a reduced state (10). Although CFTR has 17 cysteines, 12 of which are highly conserved among CFTRs from different species (human, rat, mouse, dogfish) (37-39), biochemical analyses of CFTR have not identified high molecular weight species that could be explained by intermolecular disulfide bond formation between CFTR molecules. Nonetheless, functional data routinely show that CFTR C1-channels appear in clusters (17) or exhibit cooperative gating patterns referred to as "wavelike" behavior (40, 41). Such observations strongly suggest that CFTR associates with itself and/or with cytoskeletal or other components in functioning as a C1-channel. Similar functional interactions of other ion channel proteins have been shown to be sensitive to redox potential, even when not due to formation of disulfide bonds (42).
The concentration of free pyridine nucleotides to which endogenous CFTR is normally exposed in intact, secretory epithelia is not well established. In a variety of rat tissues the total concentration of NADPH + NADP' is in a range of 200 p~, and the ratio NADPWNADP' is more reduced than oxidized, whereas the tissue concentration of NADH + NAD' is approximately 500 p~, and NADWNAD' is more oxidized than reduced (43). Values for NADP within this range were found in red blood cells (44) and in cultured airway epithelial c e k 2 The actual concentrations of reduced and oxidized pyridine nucleotides free in the cytosol are lower still but are difficult to predict because of tight protein binding and uneven distribution among cellular compartments (34). We selected concentrations of NAD(P)H (1.2 mM) and NAD(P)+ (0.3 mM) which exceed likely physiologic concentrations to make a clear comparison between the effects of reduced and oxidized pyridine nucleotides on CFTR C1-conductance function. The metabolic rate and the ratio of ATP to ADP within cells are related to pyridine nucleotide redox status, such that a low ATP/ADP ratio caused by inhibition of oxidative metabolism is coincident with more reduced ratios of cytosolic free pyridine nucleotides (9). Con-H. N. Kirkman, personal communication.
versely, more oxidized ratios of cytosolic free pyridine nucleotides arise from a high metabolic rate and high ATP/ADP ratio. The observation that CFTR-mediated, CAMP-dependent C1conductance was modulated by NAD(P) redox status suggests that the reduced cytosolic redox potential in airway epithelia exposed to anoxia or NaCN might contribute to the inhibition of CAMP-dependent C1-conductance (1).
Metabolic regulation of CFTR-mediated C1-conductance has been proposed as an important mechanism by which active fluid secretion is coupled to the metabolic status of epithelial tissues. Quinton and Reddy (8) found recently that CFTR-mediated C1-conductance is regulated by intracellular ATP concentration. Our results suggest that variations in NAD(P) redox status expected to occur with different metabolic states could contribute to metabolic regulation of CFTR C1-conductance. Understanding the mechanism(s) by which intracellular pyridine nucleotides regulate CFTR-mediated CAMP-dependent C1-conductance could provide new insights into the functions of CFTR and new ideas for pharmacological therapeutic approaches to the treatment of cystic fibrosis or other diseases involving CFTR.