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Am J Physiol Cell Physiol 292: C1553-C1561, 2007. First published December 20, 2006; doi:10.1152/ajpcell.00064.2006
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Protein and Vesicle Trafficking, Cytoskeleton

Regulatory interactions of N1303K-CFTR and ENaC in Xenopus oocytes: evidence that chloride transport is not necessary for inhibition of ENaC

Laurence Suaud,1 Wusheng Yan,1 Marcelo D. Carattino,3 Amal Robay,1 Thomas R. Kleyman,3 and Ronald C. Rubenstein1,2

1Division of Pulmonary Medicine, Children's Hospital of Philadelphia and 2Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia; and 3Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania

Submitted 9 February 2006 ; accepted in final form 15 December 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Regulatory interactions of the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na+ channel (ENaC) are readily apparent in Xenopus oocytes. However, the mechanism underlying these interactions remains controversial. CFTR's first nucleotide binding fold (NBD-1) may be important in these interactions, as dysfunctional CFTRs containing mutations within NBD-1, such as {Delta}F508 and G551D, lack such functional interactions with murine ENaC (mENaC). We hypothesized that a dysfunctional CFTR containing a non-NBD-1 mutation would retain regulatory interactions with mENaC and tested this hypothesis for N1303K-CFTR, where the mutation is located in CFTR's second nucleotide binding fold (NBD-2). cRNA for {alpha}beta{gamma}-mENaC and N1303K-CFTR was injected separately or together into Xenopus oocytes. ENaC and CFTR functional expression was assessed by two-electrode voltage clamp. Injection of N1303K (class II trafficking mutation) yielded low levels of CFTR function on activation with forskolin and 3-isobutyl-1-methylxanthine (IBMX). In coinjected oocytes, N1303K did not alter mENaC functional expression or surface expression before activation of N1303K. This is similar to our prior observations with {Delta}F508. However, unlike our observations with {Delta}F508, activation of N1303K acutely decreased mENaC functional and surface expression, and N1303K currents were enhanced by coinjection of mENaC. Furthermore, genistein only mildly enhanced the functional expression of N1303K-CFTR and did not improve regulation of ENaC by N1303K-CFTR. These data suggest that a structurally and functionally intact CFTR NBD-1 in activated CFTR can regulate mENaC surface expression independent of Cl– transport in Xenopus oocytes.

cystic fibrosis transmembrane conductance regulator; epithelial Na+ channel

CYSTIC FIBROSIS (CF) results from mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) (28). CFTR is a cAMP-activated, ATP-dependent Cl– channel that also influences the transepithelial transport of other solutes, including Na+ via the epithelial Na+ channel (ENaC), Cl– via an outwardly rectifying Cl– channel, K+ via Kir1.1, HCO3–, and ATP (5, 8, 18, 30).

Functional interactions between CFTR and ENaC are observed in both epithelial and nonepithelial cells (6, 7, 14–16, 27, 32, 35). The activation of CFTR is generally associated with an inhibition of ENaC (12, 14, 16, 19, 32, 35). In contrast, activation of CFTR leads to activation of ENaC in the sweat duct (27), suggesting that the regulatory interactions between these two transporters are complex and perhaps tissue specific.

In Xenopus oocytes, regulatory interactions of CFTR and rodent ENaC appear to mimic those of the airway. There is decreased ENaC-mediated Na+ transport in the presence of CFTR (15, 16, 19, 35) and increased CFTR-mediated Cl– conductance in the presence of ENaC (7, 14–16, 19, 32, 35). However, observations of decreased ENaC-mediated Na+ transport on activation of CFTR in oocytes remain controversial (7, 23, 24).

In oocytes, the forskolin/3-isobutyl-1-methylxanthine (IBMX)-regulated inhibition of ENaC by CFTR does not require expression of the full-length CFTR protein; a structurally and functionally intact first nucleotide-binding domain (NBD-1) appears critical. Forskolin/IBMX-dependent inhibition of ENaC is observed when ENaC is coexpressed either with wild-type (WT) CFTR truncation mutants containing an intact NBD-1 (29) or with a WT CFTR peptide fragment containing NBD-1 and the regulatory (R) domain (19). A similar NBD-1/R peptide fragment containing the G551D mutation did not demonstrate forskolin/IBMX-regulated inhibition of ENaC (19). CFTRs containing NBD-1 mutations, such as {Delta}F508-CFTR and G551D-CFTR, lack regulatory interactions with {alpha}beta{gamma}-murine ENaC (mENaC) when activated by forskolin-IBMX (31, 32). These and other data (4, 29) suggest that form and/or function of NBD-1 is important in the inhibition of ENaC by activated CFTR. Interestingly, regulation of mENaC by {Delta}F508 and G551D was restored by further activation or "repair" of these mutants with genistein (31, 32), which may interact with CFTR's second nucleotide binding fold (NBD-2) (25).

N1303K-CFTR, like {Delta}F508-CFTR, has defective intracellular trafficking characteristic of a class II CFTR mutation (11). Recent data also suggest that this mutation also may be deficient in an adenylate kinase activity that may be intrinsic to NBD-2 (26). We hypothesized that the permissive temperature of the oocyte system would allow trafficking of N1303K-CFTR to the oocyte membrane and that activated N1303K would inhibit mENaC because its NBD-1 is structurally intact. We observed that N1303K-CFTR has low Cl– conductance but is still able to regulate mENaC on activation. We also observed reduced activation of N1303K by genistein and a lack of improved N1303K regulation of mENaC with genistein, which differs significantly from our previous data with {Delta}F508 and G551D (31, 32).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Forskolin, IBMX, and genistein were purchased from Sigma Chemical (St. Louis, MO). All other reagents were purchased from Fisher Chemical.

Expression of human N1303K-CFTR and mouse ENaC in Xenopus oocytes. A cDNA encoding human N1303K-CFTR was constructed by site-directed mutagenesis using a PCR-based mutagenesis technique, and its sequence was confirmed by automated analysis in The Children's Hospital of Philadelphia Nucleic Acid and Protein Core. N1303K-CFTR and mouse {alpha}beta{gamma}-ENaC were expressed in Xenopus oocytes as previously described (16, 32). Briefly, human N1303K-CFTR and murine {alpha}-, beta-, and {gamma}-ENaC cRNAs were prepared with a cRNA synthesis kit (mMESSAGE mMACHINE, Ambion, Austin, TX) according to the manufacturer's protocol. cRNA concentrations were determined spectroscopically. Oocytes obtained from adult female Xenopus laevis (NASCO, Fort Atkinson, WI) were enzymatically defolliculated and maintained at 18°C in modified Barth's saline [mM: 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.3 Ca(NO3)2, 0.41 CaCl2, 0.82 MgSO4, 15 HEPES, pH 7.6 supplemented with 10 µg/ml sodium penicillin, 10 µg/ml streptomycin sulfate, and 100 µg/ml gentamicin sulfate]. Each batch of oocytes obtained from an individual frog was injected (50 nl/oocyte) with a Nanoject II microinjector (Drummond Scientific, Broomall, PA), with {alpha}-, beta-, and {gamma}-subunits of mENaC (0.33 ng/subunit), N1303K-CFTR (10 ng), or a combination of mENaC and N1303K-CFTR cRNAs dissolved in RNase-free water.

Electrophysiological analyses. Whole cell current measurements were performed 24–48 h after injection with a two-electrode voltage clamp (TEV) method as previously described (16, 32). Oocytes were placed in a 1-ml chamber containing modified ND96 (mM: 96 NaCl, 1 KCl, 0.2 CaCl2, 5.8 MgCl2, 10 HEPES, pH 7.4) and impaled with micropipettes of 0.5- to 5-M{Omega} resistance filled with 3 M KCl. In experiments examining whole oocyte currents in the absence of extracellular Cl–, the composition of the chamber solution was (mM) 96 Na aspartate, 1 K aspartate, 0.2 Ca(NO3)2, 5.8 Mg(aspartate)2, and 10 HEPES, pH 7.4. The whole cell currents were measured by voltage clamping the oocytes in 20-mV steps between –140 and +60 mV adjusted for baseline transmembrane potential (transmembrane potential of the oocyte under nonclamped conditions). Whole cell currents (I) were digitized at 200 Hz during the voltage steps, recorded directly onto a hard disk, and analyzed with pCLAMP 8 software (Axon Instruments, Foster City, CA). To reduce error due to series resistance, the voltage clamp (Axon Geneclamp 500B) was configured to clamp the bath potential to 0 mV. In this configuration, we independently monitored the oocyte membrane potential during our clamp protocol and routinely observed membrane potentials that were <5% depolarized from our target holding potentials.

The difference in whole cell currents measured in the absence and presence of 10 µM amiloride was used to define the amiloride-sensitive Na+ current that was carried by mENaC. N1303K-CFTR was activated by perfusion of the oocyte with buffer containing 10 µM forskolin and 100 µM IBMX for 20 or 25 min (16, 32); here (data not shown) and in our previous work (31–33, 35), maximal activation of WT and mutant CFTR-mediated currents was achieved after 10–15 min of such perfusion. In some experiments this first step was followed by incubation with 10 µM forskolin, 100 µM IBMX, and 50 µM genistein for 20 min. In all experiments, N1303K-CFTR Cl– current was defined as the difference between amiloride-insensitive current measured before and after perfusion with forskolin-IBMX (or before and after perfusion with forskolin-IBMX-genistein). Whole cell currents were recorded at –100 mV for comparisons. All measurements were performed at room temperature.

Single-channel recordings. Single-channel studies of N1303K-CFTR-mediated Cl– conductance were performed as described by Suaud et al. (31). Briefly, bath and pipette solutions were identical and contained (mM) 110 N-methyl-D-glucamine, 1.8 CaCl2, 2.5 KCl, and 10 HEPES, pH 7.4. Oocytes expressing N1303K-CFTR alone or both N1303K-CFTR and {alpha}beta{gamma}-mENaC were placed in a hypertonic solution (bath solution supplemented with 200 mM sucrose, 10 µM forskolin, 100 µM IBMX, and 50 µM genistein) for 5–10 min, and the vitelline membranes were then removed manually. Before recordings, oocytes were incubated for at least 20 min in the bath solution supplemented with 10 µM forskolin, 100 µM IBMX, and 50 µM genistein. Patch pipettes with a tip resistance of 6–20 M{Omega} were used. Single-channel recordings were performed in the cell-attached configuration with an Axopatch 200B PatchClamp Amplifier (Axon) and a DigiData 1322A interface (Axon) connected to a Pentium 4 1.5-GHz PC (Gateway). Data were acquired with pCLAMP 9 (Axon) at 4 kHz, filtered at 1,000 Hz by a four-pole low-pass Bessel filter built in the amplifier, and stored on the hard disk. Single-channel currents were further filtered at 100 Hz with a Gaussian filter for analysis purposes.

Whole oocyte and cell surface expression. Surface expression was examined by a cell surface biotinylation assay as we described previously (36). To facilitate detection of biotinylated mENaC subunits, these experiments used a beta-mENaC subunit with a COOH-terminal V5 epitope tag (beta-V5), also as previously described (36). cRNAs for {alpha}beta-V5{gamma}-mENaC were coinjected into Xenopus oocytes. After 48 h, oocytes were mechanically stripped of their vitelline membranes in hypertonic medium (300 mM sucrose in modified Barth's saline without penicillin, streptomycin, and gentamicin) and surface proteins were labeled with sulfo-NHS-Biotin (Pierce). Oocytes (10 per group) were subsequently lysed in 0.15 M NaCl, 0.01 M Tris-Cl, pH 8.0, 0.01 M EDTA, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 1.0 mM phenylmethylsulfonyl fluoride, 0.1 mM N-{alpha}-p-tosyl-L-lysine chloromethyl ketone, 0.1 mM L-1-tosylamide-2-phenylethyl-chloromethyl ketone, and 2 µg/ml aprotinin for 1 h at 4°C and centrifuged at 13,000 g for 15 min at 4°C. Biotinylated proteins were precipitated with streptavidin-agarose (Pierce) and subjected to SDS-PAGE. Biotinylated beta-subunits were detected on blots probed with an anti-V5 antibody (Invitrogen). Densitometry was performed with an AlphaImager 2200 system (AlphaInnotech, San Leandro, CA).

Whole oocyte expression of beta-V5-mENaC was assessed by immunoblot of whole oocyte lysate prepared with the lysis buffer and procedure described above.

Statistical analyses. Statistical comparisons were typically performed with the Student's t-test. A pairwise t-test was used for pre-/posttreatment in experiments using an individual oocyte. Where these data were not normally distributed, a Wilcoxon signed-rank test was used and the results are reported in the text as median (25%, 75%). A two-tailed t-test was used when comparing currents obtained from oocytes injected with a cRNA for a single transporter (i.e., ENaC or N1303K-CFTR vs. oocytes coinjected with cRNAs for both ENaC and N1303K-CFTR). ANOVA techniques were used when multiple comparisons were performed. In oocytes injected with ENaC and stimulated with or without forskolin-IBMX-genistein, because a Poisson distribution, rather than a Gaussian distribution, best described the data, the data were subject to a square root transformation before P value determination by a two-tailed t-test (37). P values <0.05 were accepted to indicate statistical significance. All statistical analyses were performed with SigmaStat version 2.03.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Functional expression of N1303K-CFTR in Xenopus oocytes. We used the Xenopus oocyte expression system and TEV technique to examine the functional expression of N1303K-CFTR. In vitro studies suggest that N1303K-CFTR is a class II CFTR trafficking mutant that does not achieve mature glycosylation and leads to a Cl– permeability defect similar to {Delta}F508-CFTR (11). We hypothesized that the permissive temperature of oocytes (18°C) would allow N1303K-CFTR delivery to the oocyte plasma membrane. We measured whole cell currents at varying clamping potentials in oocytes injected with N1303K-CFTR cRNA (Fig. 1) and generated I-voltage (V) curves from data obtained before and after 20 min of incubation with 10 µM forskolin-100 µM IBMX to activate endogenous protein kinase A (Fig. 1A). Whole cell currents, measured at a holding potential of –100 mV, increased 1.8-fold from –0.05 ± 0.01 to –0.09 ± 0.06 µA (mean ± SE; n = 25, P = 0.02) in response to forskolin-IBMX. These data suggest that expression of N1303K leads to small amounts of forskolin/IBMX-stimulated Cl– current in oocytes. The linear I-V curve obtained is characteristic of CFTR expression in oocytes (31, 32).


Figure 1
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  		Fig. 1. Expression of N1303K-cystic fibrosis transmembrane conductance regulator (CFTR) in Xenopus oocytes. A: N1303K-CFTR was expressed in oocytes and 2-electrode voltage clamp (TEV) performed as described in EXPERIMENTAL PROCEDURES. Whole cell currents were measured by voltage clamping oocytes in 20-mV steps between –140 and +60 mV adjusted for baseline transmembrane potential. Shown are current-voltage (I-V) relationships (n = 25) in ND96 buffer before (bullet) or after ({circ}) incubation with 10 µM forskolin and 100 µM 3-isobutyl-1-methylxanthine (IBMX) for 25 min. Error bars contained within symbols are not apparent. B: N1303K-CFTR was expressed in oocytes and TEV performed. I-V relationships (n = 19) in the absence ({blacktriangledown}) or presence ({triangledown}) of 10 µM forskolin, 100 µM IBMX, and 50 µM genistein are shown. Means ± SE are illustrated. Error bars contained within symbols are not apparent. C: N1303K-CFTR was expressed in oocytes, and TEV was performed as described above with the exception that the ND96 buffer was supplemented with 5 mM tetraethylamine chloride, 5 mM BaCl2, and 200 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) to block putative endogenous channels. I-V relationships (adjusted for resting membrane potential) are shown for the same oocytes (n = 8) before stimulation (bullet), after stimulation with 10 µM forskolin and 100 µM IBMX ({circ}), and after stimulation with 10 µM forskolin, 100 µM IBMX, and 50 µM genistein ({blacktriangledown}). Means ± SE are illustrated. Error bars contained within symbols are not apparent.

 
The isoflavone genistein increases Cl– transport by activated WT and mutant CFTRs including {Delta}F508-CFTR and G551D-CFTR (13, 31, 32). We therefore assessed the effect of genistein on oocytes injected with N1303K-CFTR. Figure 1B demonstrates the I-V relationship of N1303K-expressing oocytes before and after incubation with 10 µM forskolin-100 µl IBMX for 25 min followed by incubation with 10 µM forskolin-100 µM IBMX-50 µM genistein for 15 min. Whole cell currents, measured at a clamp potential of –100 mV, increased twofold from –0.12 ± 0.01 to –0.24 ± 0.03 µA (n = 19, P = 0.001) after addition of IBMX, forskolin, and genistein. These data are consistent with IBMX/forskolin-stimulated N1303K-CFTR-mediated Cl– current being at best modestly enhanced by genistein in Xenopus oocytes. Interestingly, this stimulation of activated N1303K-CFTR by genistein is minimal compared with our previously observed stimulation of {Delta}F508-CFTR and G551D-CFTR by genistein (5.7-fold and 4-fold stimulation by genistein, respectively) (31, 32).

To ensure that these small increases in whole oocyte current were not a result of nonspecific activation of endogenous channels, we also assessed I-V relationships of N1303K-injected oocytes in the presence of 5 mM tetraethylamine chloride, 5 mM BaCl2, and 200 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in the bath solution. These data are shown in Fig. 1C and are similar to the I-V relationships in the absence of these inhibitors of putative endogenous channels (Fig. 1, A and B). Unstimulated N1303K-mediated current at –100 mV was –0.38 ± 0.09 µA (n = 8). The magnitude of this current increased to –0.55 ± 0.14 µA (P = 0.021) after stimulation with IBMX-forskolin and to –0.71 ± 0.24 (P = 0.047 compared with unstimulated oocytes) after addition of IBMX, forskolin, and genistein. These data suggest that endogenous oocyte channels do not confound our observations.

Coexpression of N1303K-CFTR and {alpha}beta{gamma}-ENaC. Several groups have reported that when WT CFTR and either murine or rat ENaC were coexpressed in Xenopus oocytes ENaC-mediated Na+ currents were inhibited in response to CFTR activation (7, 15, 16, 19, 32, 35). Furthermore, the coexpression of ENaC enhances forskolin/IBMX-stimulated CFTR Cl– currents (7, 15, 16, 32, 35). The mechanism by which this regulation occurs is not clear. Some have suggested that the decrease in ENaC-mediated currents reflects a series resistor error (23, 24). Others have suggested that this decrease in ENaC functional expression requires CFTR-mediated Cl– transport (2, 20). Such transport would presumably lead to an increase in cytosolic Cl– concentration, which can inhibit ENaC-mediated Na+ transport (10). In contrast, our group's data suggest that activated CFTR acutely decreases the amount of mENaC in the oocyte membrane (35), and others have suggested that an intact NBD-1 is required for this effect (19, 29). On the basis of these considerations, we hypothesized that activated N1303K-CFTR might retain the ability to decrease mENaC functional and surface expression because it has an intact NBD-1. Furthermore, we felt that if activated N1303K-CFTR decreased mENaC functional and surface expression, it would be inconsistent with the notion that Cl– transport was important in such regulatory interactions.

We therefore assessed the interregulation of N1303K-CFTR and {alpha}beta{gamma}-mENaC in oocytes. Figure 2 depicts whole cell currents measured at a holding potential of –100 mV in oocytes injected with N1303K-CFTR (10 ng), {alpha}beta{gamma}-mENaC (0.33 ng/subunit), or both N1303K-CFTR and {alpha}beta{gamma}-mENaC. Oocytes coinjected with N1303K-CFTR and {alpha}beta{gamma}-mENaC had 4.1-fold increased forskolin/IBMX-stimulated, amiloride-insensitive current compared with oocytes injected with N1303K-CFTR alone [Fig. 2A, –0.12 ± 0.04 µA (N1303K-CFTR) vs. –0.49 ± 0.15 µA (N1303K-CFTR/{alpha}beta{gamma}-ENaC); P = 0.02]. Thus coexpression of {alpha}beta{gamma}-mENaC in oocytes resulted in enhancement of the amiloride-insensitive component of the forskolin/IBMX-stimulated current, which likely represents N1303K-CFTR-mediated Cl– current. This enhancement of N1303K-CFTR Cl– current by coexpression of {alpha}beta{gamma}-mENaC in oocytes is similar to the previously described enhancement of WT CFTR current by coexpression of {alpha}beta{gamma}-mENaC in oocytes (7, 15, 16, 21, 32).


Figure 2
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  		Fig. 2. Expression of N1303K-CFTR and murine epithelial Na+ channel (mENaC) in Xenopus oocytes. N1303K-CFTR and {alpha}beta{gamma}-mENaC were expressed separately or together in oocytes and TEV performed as described in EXPERIMENTAL PROCEDURES. A: stimulated CFTR currents represent changes in whole cell currents that were not inhibited by 10 µM amiloride (–100-mV holding potential) in oocytes injected with N1303K-CFTR alone or coinjected with N1303K-CFTR and {alpha}-, beta-, and {gamma}-mENaC, after stimulation with 10 µM forskolin-100 µM IBMX. Means ± SE are illustrated. B: amiloride-sensitive whole cell currents (–100-mV holding potential) were determined in oocytes expressing mENaC or coexpressing mENaC and N1303K-CFTR before (open bars) and after (gray bars) stimulation with 10 µM forskolin-100 µM IBMX. Data obtained from the same N1303K-CFTR/mENaC-coinjected oocytes are presented in A and B. Means ± SE are illustrated. ns, Not significant.

 
In oocytes expressing {alpha}beta{gamma}-mENaC, the amiloride-sensitive whole cell currents, measured at a clamp potential of –100 mV, were similar in the absence or presence of 10 µM forskolin-100 µM IBMX (Fig. 2B); these data are consistent with previous reports (22) and our group's data (32, 35). Oocytes coinjected with both N1303K-CFTR and {alpha}beta{gamma}-mENaC expressed amiloride-sensitive whole cell currents (–2.48 ± 0.70 µA, n = 26) in the absence of forskolin-IBMX that were similar to the oocytes expressing {alpha}beta{gamma}-mENaC alone (compare open bars in Fig. 2B). After N1303K-CFTR activation with forskolin-IBMX, the amiloride-sensitive whole cell current was significantly lower (–1.67 ± 0.42; n = 26, p = 0.03), which is similar to our group's previous observations of WT CFTR behavior in this system (16, 32, 35). These observations directly contrast with our previous observations with G551D-CFTR, where a similar magnitude of stimulated G551D-mediated current did not result in a decrease in amiloride-sensitive whole oocyte current (31).

Our group previously demonstrated (35) that the decrease in {alpha}beta{gamma}-mENaC functional expression on coexpression and activation with WT CFTR correlated with a change in mENaC surface expression (N) in surface biotinylation experiments. We performed similar experiments here and again used {alpha}beta{gamma}-mENaC where the beta-subunit contained a COOH-terminal V5 epitope tag to increase the sensitivity of our immunodetection. As shown in Fig. 3A, mENaC(beta-V5) expression at the oocyte surface was unaltered by exposure to forskolin-IBMX for 20 min in oocytes injected with {alpha}beta{gamma}-mENaC alone and was also unaltered by coinjection of N1303K-CFTR before forskolin-IBMX activation. In contrast, mENaC(beta-V5) surface expression decreased on activation of N1303K-CFTR with forskolin-IBMX for 20 min in coinjected oocytes. There were parallel changes in surface expression of mENaC(beta-V5) and the {alpha}beta{gamma}-mENaC-mediated current obtained by TEV (Fig. 2B). These data suggest that activated N1303K-CFTR, even with its minimal Cl– transport activity, retains the ability to regulate the surface and functional expression of {alpha}beta{gamma}-mENaC in oocytes.


Figure 3
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  		Fig. 3. Surface and whole cell expression of {alpha}-, beta-V5- and {gamma}-mENaC in oocytes with or without N1303K-CFTR. Forty-eight hours after injection, oocytes were incubated for an additional 20 min in the absence or presence of 10 µM forskolin and 100 µM IBMX. Cell surface biotinylation was performed as described in EXPERIMENTAL PROCEDURES. beta-V5-mENaC was recovered by streptavidin-agarose precipitation and quantitated by immunoblot with an anti-V5 antibody. A: a representative immunoblot (n = 3 independent experiments) is shown. Densitometric analysis of these surface biotinylation experiments was performed as described in EXPERIMENTAL PROCEDURES, and results are expressed as means + SE; n = 3 independent experiments normalized to density obtained with oocytes expressing {alpha}-, beta-V5-, and {gamma}-ENaC in the absence of forskolin-IBMX stimulation. Open bars represent oocytes without forskolin-IBMX stimulation, and gray bars represent oocytes stimulated with 10 mM forskolin and 100 mM IBMX. P values were determined by ANOVA in comparison with {alpha}-, beta-V5-, and {gamma}-mENaC. B: a representative immunoblot and densitometry of whole oocytes lysates of {alpha}-, beta-V5-, and {gamma}-ENaC-injected oocytes demonstrating similar expression of beta-V5-mENaC are also shown and are similarly representative of n = 3 independent experiments. Open bars represent oocytes without forskolin – IBMX stimulation and gray bars represent oocytes stimulated with forskolin – IBMX.

 
We also assessed whole oocyte expression of mENaC(beta-V5) (Fig. 3B). Expression of mENaC(beta-V5) was similar in oocytes injected with {alpha}beta{gamma}-mENaC alone or coinjected with N1303K-CFTR. Activation of N1303K-CFTR did not alter the whole oocyte expression of mENaC(beta-V5). These data suggest that activated N1303K-CFTR is similar to WT CFTR in its ability to acutely decrease the amount of mENaC(beta-V5) present at the oocyte surface without altering the total amount of mENaC(beta-V5) present in the oocyte.

As an additional test of whether extracellular to intracellular Cl– transport was required to observe inhibition of ENaC on N1303K-CFTR activation, we performed a series of ion substitution experiments. Oocytes were injected with cRNAs for either N1303K-CFTR (10 ng) or {alpha}beta{gamma}-mENaC (0.33 ng/subunit) alone or with both N1303K-CFTR and {alpha}beta{gamma}-mENaC cRNAs. However, in these experiments (Fig. 4), TEV was performed with a bathing solution in which Cl– was replaced by aspartate. Figure 4A shows an I/V plot for oocytes injected with N1303K-CFTR before and after 25 min of activation with forskolin-IBMX in this Cl–-free bath. These data demonstrate a lack of significant forskolin/IBMX-stimulated current at –100 mV holding potential on activation of N1303K-CFTR. Whole cell currents, measured at a holding potential of –100 mV, were –0.30 ± 0.04 and –0.45 ± 0.07 µA (mean ± SE; n = 6, P = ns) before and after forskolin-IBMX, respectively. In contrast, significant forskolin/IBMX-stimulated current was present at positive holding potentials; whole cell currents, measured at a holding potential of +40 mV, increased ~1.9-fold from 0.34 ± 0.06 to 0.64 ± 0.11 µA (mean ± SE; n = 6, P = 0.038) in response to forskolin-IBMX.


Figure 4
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  		Fig. 4. Function and regulatory interactions of N1303K-CFTR and mENaC in Xenopus oocytes in the absence of extracellular Cl–. In these experiments, the bath solution [mM: 96 Na aspartate, 1 K aspartate, 0.2 Ca(NO3)2, 5.8 Mg(aspartate)2, 10 HEPES, pH 7.4] for TEV did not contain Cl–. A: N1303K-CFTR was expressed in oocytes and TEV performed as described in EXPERIMENTAL PROCEDURES. Whole cell currents were measured by voltage clamping oocytes in 20-mV steps between –140 and +60 mV adjusted for baseline transmembrane potential. Shown are I-V relationships before (bullet) or after ({circ}) incubation with 10 µM forskolin and 100 µM IBMX for 25 min. Error bars contained within symbols are not apparent. B: amiloride-sensitive whole cell currents (–100-mV holding potential) were determined in oocytes expressing mENaC or coexpressing mENaC and N1303K-CFTR before (open bars) and after (gray bars) stimulation with 10 µM forskolin-100 µM IBMX for 25 min. These data (means ± SE) are presented as the relative amiloride-sensitive currents before and after forskolin-IBMX stimulation, with P values determined by a pairwise t-test.

 
In oocytes expressing {alpha}beta{gamma}-mENaC alone, the relative amiloride-sensitive whole cell currents, measured at a clamp potential of –100 mV in the Cl–-free bath, were again not significantly changed by incubation with 10 µM forskolin-100 µM IBMX (Fig. 4B). Importantly, even in this Cl–-free bath, activation of N1303K-CFTR with forskolin-IBMX led to a significant reduction in the relative amiloride-sensitive whole cell current in oocytes co-injected with N1303K-CFTR and {alpha}beta{gamma}-mENac (Fig. 4B); the relative amiloride-sensitive current after N1303K activation was 0.79 ± 0.08 (n = 10, P = 0.027). The data in Fig. 4B are presented as relative amiloride-sensitive current because there was significant batch-to-batch variability among the four separate preparations of oocytes utilized; this batch-to-batch variability yielded a non-Gaussian data distribution that precluded the analysis of these raw data by parametric statistics before normalization. Importantly, even when the medians for these data were compared with nonparametric statistical techniques (Wilcoxon signed rank test), oocytes injected with mENaC alone had no significant change in amiloride-sensitive current in response to forskolin-IBMX, while oocytes coinjected with N1303K-CFTR and {alpha}beta{gamma}-mENaC again demonstrated significant reduction in amiloride-sensitive current in response to forskolin-IBMX. For oocytes injected with mENaC alone, median (25%, 75%) amiloride-sensitive currents were –0.30 (–0.40, –0.23) µA before and –0.34 (–0.62, –0.20) µA after forskolin-IBMX (n = 9, P = not significant). In contrast, oocytes coinjected with N1303K-CFTR and {alpha}beta{gamma}-mENaC had significantly decreased median amiloride-sensitive currents of –0.32 (–0.50, –0.14) µA after forskolin-IBMX compared with –0.39 (–0.54, –0.20) µA before N1303K activation (n = 10, P = 0.02). These data are therefore consistent with regulation of mENaC by activated N1303K-CFTR in the absence of inward Cl– transport and suggest that regulation of mENaC by activated CFTR can occur in the absence of Cl– transport.

Interregulation of N1303K-CFTR and {alpha}beta{gamma}-mENaC after forskolin-IBMX-genistein stimulation. Our data suggest that N1303K-CFTR has poor Cl– conductance in response to activation with forskolin-IBMX. This may indicate that N1303K has defects in other functional properties. We therefore examined whether the CFTR potentiator genistein, which activates G551D-CFTR, {Delta}F508-CFTR, and WT CFTR Cl– conductance and restores functional interactions between {alpha}beta{gamma}-mENaC and the G551D-CFTR and {Delta}F508-CFTR mutants (31, 32), is able to activate N1303K-CFTR and improve its regulation with mENaC. As shown in Fig. 5A, the amiloride-insensitive, or N1303K-mediated, component of the forskolin/IBMX/genistein-stimulated (forskolin-IBMX for 25 min followed by genistein for 15 min) whole cell current in oocytes coexpressing N1303K-CFTR and {alpha}beta{gamma}-mENaC was 9.3-fold greater than the forskolin/IBMX/genistein-stimulated current measured in oocytes expressing N1303K-CFTR alone [N1303K-CFTR –0.15 ± 0.02 µA (n = 19) vs. N1303K-CFTR/mENaC –1.4 ± 0.52 µA (n = 19); P = 0.006].


Figure 5
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  		Fig. 5. Influence of genistein on the regulatory interactions between N1303K-CFTR and {alpha}beta{gamma}-mENaC. N1303K-CFTR and {alpha}beta{gamma}-mENaC were expressed separately or together in oocytes and TEV performed as described in EXPERIMENTAL PROCEDURES. A: changes in whole cell currents (–100-mV holding potential) before (–0.15 ± 0.02 µA) and after stimulation with 10 µM forskolin-100 µM IBMX-50 µM genistein that were not inhibited by 10 µM amiloride are illustrated. B: amiloride-sensitive whole cell currents (–100-mV holding potential) were determined in oocytes expressing {alpha}beta{gamma}-mENaC or coexpressing {alpha}beta{gamma}-mENaC and N1303K-CFTR before (open bars) and after (dark gray bars) stimulation with 10 µM forskolin-100 µM IBMX-50 µM genistein. Data obtained from the same N1303K-CFTR/{alpha}beta{gamma}-mENaC-coinjected oocytes are presented in A and B. Means ± SE are illustrated.

 
Coinjection of {alpha}beta{gamma}-mENaC increases WT CFTR open probability (Po) and surface expression in oocytes (16). In contrast, coinjection of {alpha}beta{gamma}-mENaC increases forskolin/IBMX/genistein-stimulated G551D-CFTR-mediated current in oocytes but does not alter G551D's Po (31). We attempted to determine whether this significant increase in forskolin/IBMX/genistein-stimulated, N1303K-mediated whole oocyte current with coinjection of {alpha}beta{gamma}-mENaC reflected a change in N1303K-CFTR's Po. Unfortunately, we were unable to observe single-channel CFTR currents in 50 patches obtained from oocytes expressing either N1303K-CFTR alone or N1303K-CFTR with {alpha}beta{gamma}-mENaC. However, we were able to record mENaC-mediated outward Na+ single-channel currents in oocytes coexpressing N1303K-CFTR and {alpha}beta{gamma}-mENaC (data not shown). This suggests that these oocytes were viable and able to express channels, but that the number of N1303K-CFTR channels at the oocyte surface was insufficient to detect single channel events. Alternatively, forskolin-IBMX-genistein was inefficiently stimulating channel activity under our conditions. These results agree with the low level of currents observed on whole cell recordings, and with previous data suggesting that this mutation primarily affects channel processing (3, 11).

Consistent with our previous results (32), whole cell amiloride-sensitive currents modestly but significantly increased in response to 100 µM IBMX-10 µM forskolin-50 µM genistein [–1.4 ± 0.38 µA (–forskolin-IBMX-genistein) vs. –2.4 ± 0.56 µA (+forskolin-IBMX-genistein); n = 15, P = 0.001] in oocytes injected with {alpha}beta{gamma}-mENaC alone (Fig. 5B). Our group's recent data (33) suggest that this increase in mENaC functional expression is not associated with an increase in mENaC surface expression, and therefore is due to increases in either mENaC Po or unitary conductance or both. In oocytes coexpressing {alpha}beta{gamma}-mENaC and N1303K-CFTR, the amiloride-sensitive currents also modestly but significantly increased after stimulation with forskolin-IBMX-genistein [–1.9 ± 0.4 µA (–forskolin-IBMX-genistein) vs. –3.0 ± 0.5 (+forskolin-IBMX-genistein); n = 19, P < 0.001]. These data contrast with our previous observations with WT, {Delta}F508-, and G551D-CFTR, where such an increase in {alpha}beta{gamma}-mENaC-mediated current after forskolin-IBMX-genistein in CFTR/mENaC-coinjected oocytes was not present (31, 32). These data are consistent with genistein not further improving regulation of {alpha}beta{gamma}-mENaC by N1303K-CFTR and therefore being a less effective potentiator of N1303K-CFTR function than it is for {Delta}F508- and G551D-CFTR.

Synergistic activation of N1303K-CFTR by genistein and ENaC. As discussed above, forskolin/IBMX-stimulated, N1303K-CFTR-mediated currents were increased ~1.3-fold with the addition of genistein alone (Fig. 1, A and B). Oocytes coinjected with N1303K-CFTR and {alpha}beta{gamma}-mENaC had approximately fourfold greater amiloride-insensitive, forskolin/IBMX-stimulated N1303K-mediated currents compared with oocytes injected with N1303K-CFTR alone (Fig. 2A). Interestingly, oocytes coinjected with N1303K-CFTR and {alpha}beta{gamma}-mENaC had an ~10-fold greater N1303K-mediated current in the presence of genistein compared with oocytes expressing N1303K-CFTR alone and stimulated with forskolin-IBMX (Fig. 5A). These data demonstrate synergistic enhancement of N1303K-CFTR functional expression by genistein and {alpha}beta{gamma}-mENaC in the presence of forskolin-IBMX, and are similar to our previous data suggesting synergistic enhancement of G551D-CFTR (31) and I148T-CFTR (33) function by genistein and {alpha}beta{gamma}-mENaC. These data are therefore consistent with genistein and {alpha}beta{gamma}-mENaC enhancing N1303K-CFTR function in oocytes by different and complementary mechanisms. We attempted to probe the mechanisms underlying this synergy by performing single-channel recordings of N1303K-CFTR, but, as detailed above, these experiments were not successful. Furthermore, the quite low NPo for N1303K-CFTR suggested by these experiments, and our group's previous experience with biochemical detection of surface CFTR expression by surface biotinylation in oocytes, suggests that the surface expression of N1303K-CFTR in oocytes is likely below the limit of experimental detection.


   DISCUSSION
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One hypothesis of the pathophysiology of the CF airway is that the absence of CFTR leads to increased ENaC activity in the airway epithelia. This results in increased solute and liquid absorption, decreased airway surface liquid volume, and decreased mucociliary clearance (17). The regulatory interactions of CFTR and ENaC are thus likely to influence the CF airway phenotype. The interactions of CFTR and ENaC have been extensively studied in the Xenopus oocyte system, although the mechanism of these functional interactions in oocytes remains the subject of debate. Our data here add additional structural and functional information regarding features of CFTR that are necessary for these interactions.

Methodological issues. Our methods here and in our previous publications (31–33, 35) slightly differ from those of other groups who have studied the interactions of CFTR and ENaC in the oocyte system and obtained contrasting results (7, 23, 24). Our TEV protocol used a 20-mV step from –140 to +60 mV. Oocytes were allowed to "rest" between applications of the clamp protocol. In contrast, both Chabot et al. (7) and Nagel et al. (23) used a ramp protocol and observed that activation of CFTR did not result in a decrease in ENaC functional expression. Nagel et al. (23) also presented continuous recordings of current obtained with an oocyte clamped at –60 mV. Here, and in our previous publications, we have used murine ENaC, whereas Chabot et al. (7) used rat ENaC, and Nagel et al. (23) used both rat and human ENaC. The amounts of injected cRNA are also somewhat different. We do not know how these differences in experimental design and protocol may influence these data. However, we have published data comparing the interactions of human and murine ENaC with WT CFTR (35); our data demonstrate differential effects of activated WT CFTR on human and murine ENaC. Furthermore, our data for human ENaC are not inconsistent with those of Nagel et al. (23), suggesting that the species of origin of ENaC may be critical in experiments such as these. The other major difference between our data here and in previous publications (33, 35) and the observations of others is our correlation of changes in functional ENaC expression by TEV with changes in ENaC surface expression detected by biochemical assay.

Regulation of mENaC by activated CFTR does not require significant Cl– transport. Recent data have suggested that the degree of decreased functional expression of ENaC is correlated with the degree of Cl– transport by activated CFTR (2). However, our data here are inconsistent with the hypothesis that Cl– transport by activated CFTR is required for the decreased functional expression of mENaC. The forskolin/IBMX-stimulated current of N1303K-CFTR is very low (Figs. 1 and 2), yet activation of N1303K leads to decreased {alpha}beta{gamma}-mENaC functional and surface expression in a fashion similar to WT CFTR (35). Furthermore, the functional expression of mENaC also decreases on activation of N1303K-CFTR when inward Cl– transport is prevented by removing Cl– from the bath solution (Fig. 4). Thus these data suggest that high amounts of Cl– transport are not necessary for regulation of {alpha}beta{gamma}-mENaC by activated CFTR. Because high amounts of Cl– transport leading to significantly decreased oocyte membrane resistance are necessary to generate a series resistor error in TEV experiments, these data are therefore also inconsistent with the hypothesis that the decrease in {alpha}beta{gamma}-mENaC functional expression on CFTR activation results from a series resistor error (24).

These data are not contradictory to the reports that elevations of cytosolic Cl– concentrations lead to inhibition of mENaC-mediated Na+ transport (10). Rather, these data suggest that there are potentially two nonexclusive mechanisms by which activation of CFTR may inhibit mENaC. Inhibition of mENaC may result from significant CFTR-mediated inward Cl– transport such that the cytosolic Cl– concentration substantially increases. Alternatively, as described here and in our previous work (35), activation of CFTR decreases mENaC surface expression. Our data here and in previous publications support the latter mechanism being more important in oocytes under our experimental conditions. Here, Cl– transport by activated N1303K-CFTR was not required to observed inhibition of mENaC. In contrast, we recently demonstrated that activation of I148T-CFTR, which maintains the ability to transport Cl– at levels similar to WT CFTR (9, 33), did not lead to inhibition of mENaC in oocytes despite supporting significantly higher Cl– transport (33) than N1303K-CFTR did in the present experiments.

NBD-1 is a critical element for regulation of ENaC by activated CFTR. Interestingly, previous studies have suggested that the forskolin/IBMX-regulated inhibition of ENaC by CFTR does not require expression of the full-length CFTR protein. Forskolin/IBMX-dependent inhibition of ENaC can be observed when ENaC is coexpressed either with WT CFTR truncation mutants containing an intact NBD-1 (29) or with a WT CFTR peptide fragment containing NBD-1 and the R domain (19). A similar NBD-1/R peptide fragment containing the G551D mutation did not demonstrate forskolin/IBMX-regulated inhibition of ENaC (19). These data suggest that a structurally and functionally intact NBD-1 of CFTR is required for this regulatory interaction. Furthermore, the data demonstrating forskolin/IBMX-regulated inhibition of ENaC by the NBD-1/R peptide are also consistent with the notion that Cl– transport is not required for this interaction.

We previously showed (31, 32) that CFTRs with NBD-1 mutations (G551D-CFTR and {Delta}F508-CFTR) lack the ability to regulate {alpha}beta{gamma}-mENaC when activated by forskolin-IBMX. Our data here with N1303K are also consistent with a structurally and functionally intact NBD-1 being a critical element in this regulatory interaction. Like {Delta}F508-CFTR, N1303K-CFTR is a class II CFTR trafficking mutation that, according to our data here, is able to achieve a low level of functional expression at the permissive temperature of 18°C in oocytes. Yet, unlike {Delta}F508-CFTR, N1303K-CFTR has an intact NBD-1 and a mutant NBD-2 and interestingly retains the ability to regulate {alpha}beta{gamma}-mENaC functional and surface expression in oocytes on activation. These data with N1303K-CFTR suggest that a mutant NBD-2, which lacks intrinsic adenylate kinase activity (26), still allows for regulation of {alpha}beta{gamma}-mENaC by activated CFTR. These data are also consistent with functional nonequivalence of CFTR class II trafficking mutants and may suggest the need for further refinement of mutation-directed correction strategies.

Further evidence that genistein influence CFTR via NBD-2. Genistein activates CFTR (WT, {Delta}F508, and G551D) Cl– transport by increasing channel open time and decreasing channel closing, thereby increasing channel Po (1, 34). While the molecular details of this action remain elusive, others have suggested that genistein interacts directly with CFTR via the second nucleotide binding fold, NBD-2 (25). Our data are consistent with this suggestion. Genistein only modestly (1.3-fold) enhanced N1303K-CFTR mediated current in these experiments. These data contrast with our previous observations that genistein enhanced Cl– transport by WT CFTR and the NBD-1 mutants {Delta}F508- and G551D-CFTR by four- to sixfold (31, 32). Furthermore, while genistein improves the regulation of {alpha}beta{gamma}-mENaC by activated {Delta}F508- and G551D-CFTR (31, 32), it did not enhance regulation of {alpha}beta{gamma}-mENaC by activated N1303K. These results are consistent with CFTR's NBD-2 being a critical determinant in the genistein action as a mutant CFTR potentiator.

Conclusions. These data suggest that N1303K-CFTR retains regulatory interactions with {alpha}beta{gamma}-mENaC in oocytes. Given the low level of N1303K-CFTR functional expression, and our observations that regulation of mENaC occurred in the absence of inward Cl– transport, these data provide additional evidence that CFTR-mediated Cl– transport is not required for such interactions to occur. Rather, these data provide additional support for the notion that NBD-1 is a critical element for these interactions and that the class II CFTR mutants {Delta}F508 and N1303K are functionally distinct. These data also provide further evidence that genistein may act through CFTR's NBD-2. These considerations suggest a potential added level of complexity in considering pharmacological repair of mutant CFTR function.


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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-054354 (T. R. Kleyman and R. C. Rubenstein) and DK-58046 (R. C. Rubenstein). R. C. Rubenstein was an Established Investigator of the American Heart Association.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. C. Rubenstein, Div. of Pulmonary Medicine, Abramson 410C, Children's Hospital of Philadelphia, 34th St. and Civic Center Blvd., Philadelphia, PA 19104 (e-mail: rrubenst{at}mail.med.upenn.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Al Nakkash L, Hu S, Li M, Hwang TC. A common mechanism for cystic fibrosis transmembrane conductance regulator protein activation by genistein and benzimidazolone analogs. J Pharmacol Exp Ther 296: 464–472, 2001.[Abstract/Free Full Text]

2. Bachhuber T, Konig J, Voelcker T, Murle B, Schreiber R, Kunzelmann K. Cl– interference with the epithelial Na+ channel ENaC. J Biol Chem 280: 31587–31594, 2005.[Abstract/Free Full Text]

3. Berger AL, Ikuma M, Hunt JF, Thomas PJ, Welsh MJ. Mutations that change the position of the putative {gamma}-phosphate linker in the nucleotide binding domains of CFTR alter channel gating. J Biol Chem 277: 2125–2131, 2002.[Abstract/Free Full Text]

4. Boucherot A, Schreiber R, Kunzelmann K. Role of CFTR's PDZ1-binding domain, NBF1 and Cl– conductance in inhibition of epithelial Na+ channels in Xenopus oocytes. Biochim Biophys Acta 1515: 64–71, 2001.[Medline]

5. Braunstein GM, Roman RM, Clancy JP, Kudlow BA, Taylor AL, Shylonsky VG, Jovov B, Peter K, Jilling T, Ismailov II, Benos DJ, Schwiebert LM, Fitz JG, Schwiebert EM. Cystic fibrosis transmembrane conductance regulator facilitates ATP release by stimulating a separate ATP release channel for autocrine control of cell volume regulation. J Biol Chem 276: 6621–6630, 2001.[Abstract/Free Full Text]

6. Briel M, Greger R, Kunzelmann K. Cl– transport by cystic fibrosis transmembrane conductance regulator (CFTR) contributes to the inhibition of epithelial Na+ channels (ENaCs) in Xenopus oocytes co-expressing CFTR and ENaC. J Physiol 508: 825–836, 1998.[Abstract/Free Full Text]

7. Chabot H, Vives MF, Dagenais A, Grygorczyk C, Berthiaume Y, Grygorczyk R. Downregulation of epithelial sodium channel (ENaC) by CFTR co-expressed in Xenopus oocytes is independent of Cl– conductance. J Membr Biol 169: 175–188, 1999. [Corrigenda. J Membr Biol 186: 2002, p. 185.][CrossRef][ISI][Medline]

8. Choi JY, Lee MG, Ko S, Muallem S. Cl–-dependent HCO3– transport by cystic fibrosis transmembrane conductance regulator. JOP 2: 243–246, 2001.[Medline]

9. Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, Muallem S. Aberrant CFTR-dependent HCO3– transport in mutations associated with cystic fibrosis. Nature 410: 94–97, 2001.[CrossRef][Medline]

10. Dinudom A, Young JA, Cook DI. Na+ and Cl– conductances are controlled by cytosolic Cl– concentration in the intralobular duct cells of mouse mandibular glands. J Membr Biol 135: 289–295, 1993.[ISI][Medline]

11. Gregory RJ, Rich DP, Cheng SH, Souza DW, Paul S, Manavalan P, Anderson MP, Welsh MJ, Smith AE. Maturation and function of cystic fibrosis transmembrane conductance regulator variants bearing mutations in putative nucleotide-binding domains 1 and 2. Mol Cell Biol 11: 3886–3893, 1991.[Abstract/Free Full Text]

12. Hopf A, Schreiber R, Mall M, Greger R, Kunzelmann K. Cystic fibrosis transmembrane conductance regulator inhibits epithelial Na+ channels carrying Liddle's syndrome mutations. J Biol Chem 274: 13894–13899, 1999.[Abstract/Free Full Text]

13. Illek B, Zhang L, Lewis NC, Moss RB, Dong JY, Fischer H. Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Am J Physiol Cell Physiol 277: C833–C839, 1999.[Abstract/Free Full Text]

14. Ismailov II, Awayda MS, Jovov B, Berdiev BK, Fuller CM, Dedman JR, Kaetzel M, Benos DJ. Regulation of epithelial sodium channels by the cystic fibrosis transmembrane conductance regulator. J Biol Chem 271: 4725–4732, 1996.[Abstract/Free Full Text]

15. Ji HL, Chalfant ML, Jovov B, Lockhart JP, Parker SB, Fuller CM, Stanton BA, Benos DJ. The cytosolic termini of the beta- and gamma-ENaC subunits are involved in the functional interactions between cystic fibrosis transmembrane conductance regulator and epithelial sodium channel. J Biol Chem 275: 27947–27956, 2000.[Abstract/Free Full Text]

16. Jiang Q, Li J, Dubroff R, Ahn YJ, Foskett JK, Engelhardt J, Kleyman TR. Epithelial sodium channels regulate cystic fibrosis transmembrane conductance regulator chloride channels in Xenopus oocytes. J Biol Chem 275: 13266–13274, 2000.[Abstract/Free Full Text]

17. Knowles MR, Boucher RC. Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 109: 571–577, 2002.[CrossRef][ISI][Medline]

18. Konstas AA, Koch JP, Tucker SJ, Korbmacher C. CFTR-dependent upregulation of Kir1.1 (ROMK) renal K+ channels by the epithelial sodium channel (ENaC). J Biol Chem 277: 25377–25384, 2002.[Abstract/Free Full Text]

19. Kunzelmann K, Kiser GL, Schreiber R, Riordan JR. Inhibition of epithelial Na+ currents by intracellular domains of the cystic fibrosis transmembrane conductance regulator. FEBS Lett 400: 341–344, 1997.[CrossRef][ISI][Medline]

20. Kunzelmann K, Schreiber R, Boucherot A. Mechanisms of the inhibition of epithelial Na+ channels by CFTR and purinergic stimulation. Kidney Int 60: 455–461, 2001.[CrossRef][ISI][Medline]

21. Ling BN, Zuckerman JB, Lin C, Harte BJ, McNulty KA, Smith PR, Gomez LM, Worrell RT, Eaton DC, Kleyman TR. Expression of the cystic fibrosis phenotype in a renal amphibian epithelial cell line. J Biol Chem 272: 594–600, 1997.[Abstract/Free Full Text]

22. Mall M, Hipper A, Greger R, Kunzelmann K. Wild type but not {Delta}F508 CFTR inhibits Na+ conductance when coexpressed in Xenopus oocytes. FEBS Lett 381: 47–52, 1996.[CrossRef][ISI][Medline]

23. Nagel G, Barbry P, Chabot H, Brochiero E, Hartung K, Grygorczyk R. CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytes. J Physiol 564: 671–682, 2005.[Abstract/Free Full Text]

24. Nagel G, Szellas T, Riordan JR, Friedrich T, Hartung K. Non-specific activation of the epithelial sodium channel by the CFTR chloride channel. EMBO Rep 2: 249–254, 2001.[CrossRef][ISI][Medline]

25. Randak C, Auerswald EA, Assfalg-Machleidt I, Reenstra WW, Machleidt W. Inhibition of ATPase, GTPase and adenylate kinase activities of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator by genistein. Biochem J 340: 227–235, 1999.[CrossRef][ISI][Medline]

26. Randak CO, Welsh MJ. ADP inhibits function of the ABC transporter cystic fibrosis transmembrane conductance regulator via its adenylate kinase activity. Proc Natl Acad Sci USA 102: 2216–2220, 2005.[Abstract/Free Full Text]

27. Reddy MM, Light MJ, Quinton PM. Activation of the epithelial Na+ channel (ENaC) requires CFTR Cl– channel function. Nature 402: 301–304, 1999.[CrossRef][Medline]

28. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, Drumm ML, Iannuzzi MC, Collins FS, Tsui LC. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066–1073, 1989.[Abstract/Free Full Text]

29. Schreiber R, Hopf A, Mall M, Greger R, Kunzelmann K. The first-nucleotide binding domain of the cystic-fibrosis transmembrane conductance regulator is important for inhibition of the epithelial Na+ channel. Proc Natl Acad Sci USA 96: 5310–5315, 1999.[Abstract/Free Full Text]

30. Schwiebert EM, Egan ME, Hwang TH, Fulmer SB, Allen SS, Cutting GR, Guggino WB. CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP. Cell 81: 1063–1073, 1995.[CrossRef][ISI][Medline]

31. Suaud L, Carattino M, Kleyman TR, Rubenstein RC. Genistein improves regulatory interactions between G551D-cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel in Xenopus oocytes. J Biol Chem 277: 50341–50347, 2002.[Abstract/Free Full Text]

32. Suaud L, Li J, Jiang Q, Rubenstein RC, Kleyman TR. Genistein restores functional interactions between {Delta}F508-CFTR and ENaC in Xenopus oocytes. J Biol Chem 277: 8928–8933, 2002.[Abstract/Free Full Text]

33. Suaud L, Yan W, Rubenstein RC. Abnormal regulatory interactions Of I148T-CFTR and the epithelial Na+ channel in Xenopus oocytes. Am J Physiol Cell Physiol 292: C603–C611, 2007.[Abstract/Free Full Text]

34. Weinreich F, Wood PG, Riordan JR, Nagel G. Direct action of genistein on CFTR. Pflügers Arch 434: 484–491, 1997.[CrossRef][ISI][Medline]

35. Yan W, Samaha FF, Ramkumar M, Kleyman TR, Rubenstein RC. Cystic fibrosis transmembrane conductance regulator differentially regulates human and mouse epithelial sodium channels in Xenopus oocytes. J Biol Chem 279: 23183–23192, 2004.[Abstract/Free Full Text]

36. Yan W, Suaud L, Kleyman TR, Rubenstein RC. Differential modulation of a polymorphism in the COOH terminus of the {alpha}-subunit of the human epithelial sodium channel by protein kinase C{delta}. Am J Physiol Renal Physiol 290: F279–F288, 2006.[Abstract/Free Full Text]

37. Zar JH. Biostatistical Analysis. Englewood Cliffs, NJ: Prentice-Hall, 1984.




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