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Am J Physiol Cell Physiol 292: C603-C611, 2007. First published July 5, 2006; doi:10.1152/ajpcell.00088.2006
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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Abnormal regulatory interactions of I148T-CFTR and the epithelial Na+ channel in Xenopus oocytes

Laurence Suaud,1 Wusheng Yan,1 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, Pennsylvania

Submitted 23 February 2006 ; accepted in final form 30 June 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The mechanisms underlying regulatory interactions of the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na+ channel (ENaC) in Xenopus oocytes are controversial. CFTR's first nucleotide binding domain (NBD-1) may be important in these interactions, because mutations within NBD-1 impair these functional interactions. We hypothesized that an abnormal CFTR containing a non-NBD-1 mutation and able to transport chloride would retain regulatory interactions with murine ENaC (mENaC). We tested this hypothesis for I148T-CFTR, where the mutation is located in CFTR's first intracellular loop. I148T-CFTR has been associated with a severe CF phenotype, perhaps because of defects in its regulation of bicarbonate transport, but it transports chloride similarly to wild-type CFTR in model systems (Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, Muallem S. Nature 410: 94–97, 2001). cRNAs encoding {alpha}beta{gamma}-mENaC and I148T-CFTR were injected separately or together into Xenopus oocytes. mENaC and CFTR functional expression were assessed by two-electrode voltage clamp. mENaC whole oocyte expression was determined by immunoblotting, and surface expression was quantitated by surface biotinylation. Injection of I148T-CFTR cRNA alone yielded high levels of CFTR functional expression. In coinjected oocytes, mENaC functional and surface expression was not altered by activation of I148T-CFTR with forskolin/ IBMX. Furthermore, the CFTR potentiator genistein both enhanced functional expression of I148T-CFTR and restored regulation of mENaC surface expression by activated I148T-CFTR. These data suggest that the ability to transport chloride is not a critical determinant of regulation of mENaC by activated CFTR in Xenopus oocytes and provide further evidence that I148T-CFTR is dysfunctional despite maintaining the ability to transport chloride.

cystic fibrosis transmembrane conductance regulator; genistein

CYSTIC FIBROSIS (CF) results from mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) (29). CFTR is a cAMP-activated, ATP-dependent Cl– channel that also influences the transepithelial transport of other solutes, including Na+ via the epithelial sodium channel (ENaC), Cl– via an outwardly rectifying Cl– channel, K+ via Kir1.1, HCO3–, and ATP (5, 9, 19, 31) .

Functional interactions between CFTR and ENaC are observed in both epithelial and nonepithelial cells (6, 14–16, 28, 34, 37). The activation of CFTR is generally associated with an inhibition of murine and rat ENaC (11, 14, 16, 20, 34, 37). In contrast, activation of CFTR leads to activation of ENaC in the sweat duct (28), suggesting that the regulatory interactions between these two transporters are complex and perhaps tissue specific.

Regulatory interactions of CFTR and rodent ENaC in Xenopus oocytes are similar to those of the airway. ENaC-mediated Na+ transport is decreased in the presence of CFTR (15, 16, 20, 37), and increased CFTR-mediated Cl– conductance is observed in presence of ENaC (14–16, 20, 34, 37). The molecular basis for these regulatory interactions has remained elusive and somewhat controversial. Others have suggested that CFTR's first nucleotide binding domain (NBD-1) is a critical determinant of these interactions (3, 30). These data are consistent with our observations that mutant CFTRs in which the mutation is within NBD-1, such as {Delta}F508-CFTR and G551D-CFTR, lack characteristic regulatory interactions with {alpha}beta{gamma}-mENaC when activated by forskolin/3-isobutyl-1-methylxanthine (IBMX) (33, 34). In contrast, other data support the hypothesis that chloride transport, by either CFTR or other chloride channels, is a critical determinant of modulation of ENaC function in oocytes coexpressing these transporters (2, 6, 18, 24).

We aimed to test these alternate hypotheses by examining the regulatory interactions of murine ENaC (mENaC) with I148T-CFTR. I148T is a rare mutation of CFTR where the mutation is localized in CFTR's first cytoplasmic loop (CL-1). According to the CFTR mutation database (www.genet.sickkids.on.ca/cftr), I148T can be associated with phenotypically severe, pancreatic-insufficient CF. However, this association has been questioned by others, because I148T-containing alleles from patients with CF can harbor a second mutation in cis, 3199del6, which causes deletion of I1023 and V1024, that may itself be sufficient to cause CFTR dysfunction (10). Interestingly, in heterologous cells in the absence of this second mutation in cis, I148T-CFTR appears to maintain the ability to transport chloride at levels similar to wild-type CFTR (WT-CFTR) but appears deficient in its ability to regulate HCO3– transport (9); this defective regulation of HCO3– transport is hypothesized to underlie the pancreatic dysfunction associated with this mutation in CF. Our data suggest that cAMP-activated I148T-CFTR has robust capability to conduct chloride when expressed in Xenopus oocytes but is nevertheless dysfunctional with regard to regulatory interactions with mENaC in this model system. These data argue against the hypotheses that chloride transport by activated CFTR is sufficient for regulation of mENaC in oocytes and that chloride transport by CFTR is sufficient to prevent the clinical manifestations of CF.


   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 I148T-CFTR and mouse ENaC in Xenopus oocytes. Human I148T-CFTR was constructed by site-directed mutagenesis of WT-CFTR cDNA by 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. I148T-CFTR and {alpha}beta{gamma}-mENaC were expressed in Xenopus oocytes as previously described (16, 33, 34, 37). I148T-CFTR and mouse {alpha}-, beta-, and {gamma}-ENaC cRNAs were prepared using the mMESSAGE mMACHINE cRNA synthesis kit (Ambion, Austin, TX) according to the manufacturer's protocol, and cRNA concentrations were determined spectroscopically. Oocytes were obtained from adult female X. laevis by laparotomy (NASCO, Fort Atkinson, WI) according to a protocol approved by the Institutional Animal Care and Use Committee of the Children's Hospital of Philadelphia. Harvested oocytes were enzymatically defolliculated and maintained at 18°C in modified Barth's saline [MBS: 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.3 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, and 15 mM 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 using a Nanoject II microinjector (Drummond Scientific, Broomall, PA) with either {alpha}-, beta-, and {gamma}-subunits of mENaC (0.33 ng/subunit), I148T-CFTR (10 ng), or a combination of {alpha}beta{gamma}-mENaC and I148T-CFTR cRNAs dissolved in RNase-free water (50 nl/oocyte).

Electrophysiological analyses. Whole cell current measurements were performed 24–48 h after injection by using the two-electrode voltage-clamp (TEV) method as previously described (16, 33, 34, 37). Oocytes were placed in a 1-ml chamber containing modified ND96 (96 mM NaCl, 1 mM KCl, 0.2 mM CaCl2, 5.8 mM MgCl2, and 10 mM HEPES, pH 7.4) and impaled with micropipettes of 0.5- to 5-M{Omega} resistance filled with 3 M KCl. The whole cell currents were measured by voltage clamping the oocytes in 20-mV steps between –140 and +60 mV adjusted for resting transmembrane potential. Whole cell currents (I) were digitized at 200 Hz during the voltage steps, recorded directly onto a hard disk, and analyzed using pCLAMP version 8 or 8.1 software (Axon Instruments, Foster City, CA). To reduce potential error due to series resistance, we configured the voltage clamp (Axon Geneclamp 500B) to clamp the bath potential to 0 mV. This configuration allowed us to independently monitor the oocyte membrane potential during the clamp protocol. We 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 {alpha}beta{gamma}-mENaC. I148T-CFTR was activated by perfusion of the oocyte with modified ND96 buffer containing 10 µM forskolin and 100 µM IBMX for 25 min (16, 33, 34, 37). 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, chloride current carried by I148T-CFTR 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.

Whole oocyte and cell surface expression. Surface expression was examined by using a cell surface biotinylation assay as we have previously described (38). To facilitate detection of biotinylated mENaC subunits, we used a beta-mENaC subunit with a COOH-terminal V5 epitope tag (beta-V5) in these experiments, also as previously described (38). cRNAs for {alpha}beta-V5{gamma}-mENaC were either injected into Xenopus oocytes alone or coinjected with cRNA encoding I148T-CFTR. After 48 h, oocytes were mechanically stripped of their vitelline membranes in hypertonic medium (300 mM sucrose in MBS 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-V5 subunits were detected on immunoblots probed with an anti-V5 antibody (Invitrogen). Densitometry was performed using an AlphaImager 2200 system (AlphaInnotech, San Leandro, CA).

Whole oocyte expression of beta-V5-mENaC was assessed by immunoblotting of whole oocyte lysate prepared using the oocyte lysis buffer, SDS-PAGE, and immunodetection procedures described above.

Statistical analyses. Statistical comparisons were performed using the Student's t-test. A pairwise t-test was used for pre-/posttreatment in experiments using an individual oocyte (i.e., amiloride-sensitve currents before and after forskolin/IBMX treatment). A two-tailed t-test was used when comparing currents obtained from oocytes injected with a cRNA for a single transporter (i.e., {alpha}beta{gamma}-mENaC or I148T-CFTR) versus oocytes coinjected with cRNAs for both {alpha}beta{gamma}-mENaC and I148T-CFTR. ANOVA techniques were used when multiple comparisons were performed. P values ≤0.05 were accepted to indicate statistical significance. All statistical analyses were performed using SigmaStat version 2.03 software.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Functional expression of I148T-CFTR in Xenopus oocytes. We used the Xenopus oocyte expression system and TEV technique to examine the functional expression of I148T-CFTR. Prior data suggest that I148T-CFTR exhibits intracellular maturation (as assessed by molecular weight shifts due to glycolytic processing) and functional chloride transport similar to that of WT-CFTR but is deficient in its ability to regulate HCO3– transport (4, 9, 32). We measured whole cell currents at varying clamping potentials in oocytes injected with 10 µg of I148T-CFTR cRNA and generated current/voltage (I-V) curves from data obtained before and after 20 min of incubation with 10 µM forskolin and 100 µM IBMX to activate endogenous protein kinase A (Fig. 1A). I148T-CFTR had a CFTR-characteristic linear I-V relationship, and whole cell currents, measured at a holding potential of –100 mV, increased 28-fold from –0.12 ± 0.02 to –3.42 ± 1.18 µA (mean ± SE, n = 19, P ≤ 0.001) in response to forskolin/IBMX. The magnitude of these whole oocyte currents are similar to those of our previous studies when we assessed whole oocyte current resulting from injection of 10 µg of cRNA encoding WT-CFTR (34, 37). These data suggest that expression of I148T leads to robust forskolin/IBMX-stimulated chloride currents in oocytes that are similar in magnitude to those of WT-CFTR.


Figure 1
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  		Fig. 1. Expression of I148T-CFTR in Xenopus oocytes. A: I148T-CFTR was expressed in oocytes and two-electrode voltage clamp (TEV) was 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 in modified ND96 buffer in the absence (bullet) or presence ({circ}) of 10 µM forskolin and 100 µM IBMX. B: I148T-CFTR was expressed in oocytes and TEV was performed. I-V relationships before stimulation of I148T-CFTR (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}) are shown. Values are means ± SE for n = 19 oocytes. Error bars contained within the symbols are not apparent.

 
The isoflavone and CFTR potentiator genistein increases chloride transport by activated WT and mutant CFTRs, including {Delta}F508-CFTR and G551D-CFTR (13, 33, 34). We assessed whether genistein would also enhance the function of I148T-CFTR. Figure 1B demonstrates the I-V relationship of I148T-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 an additional 20 min. Forskolin/IBMX-stimulated whole cell currents at a holding potential of –100 mV were enhanced threefold, from –0.97 ± 0.31 to –3.06 ± 0.81 µA (n = 19, P ≤ 0.001), after addition of genistein. This enhancement of activated I148T-CFTR by genistein is similar in magnitude to the effect of genistein on WT-CFTR (~2-fold; Ref. 34) but slightly smaller in magnitude compared with our previously observed enhancement of {Delta}F508-CFTR- and G551D-CFTR-mediated currents by genistein (5.7- and 4-fold, respectively) (33, 34).

The forskolin/IBMX-stimulated currents mediated by I148T-CFTR were markedly reduced in Fig. 1B vs. Fig. 1A. Because these experiments were performed at different times with multiple, different batches of oocytes and preparations of cRNA, we feel that this is most likely a result of batch-to-batch variability in preparation of oocytes for experimentation. We also have observed similar relative batch-to-batch variation in our previously published data regarding {Delta}F508-CFTR in this system when using identical protocols (34).

Regulatory interactions of I148T-CFTR and {alpha}beta{gamma}-ENaC. In Xenopus oocytes, activation of WT-CFTR with forskolin/IBMX inhibits Na+ currents carried by either murine or rat ENaC (15, 16, 20, 34, 37). In these experiments, coexpression of murine or rat ENaC also enhances forskolin/IBMX-stimulated CFTR chloride currents (15, 16, 34, 37). The mechanism by which these regulatory interactions occur is not clear. Some have suggested that this decrease in ENaC functional expression requires CFTR-mediated chloride transport (2, 21) leading to a decrease in ENaC open probability(2), whereas others suggest that the decrease in ENaC-mediated currents reflects a series resistor error (25, 26). In contrast, we observed that activated WT-CFTR acutely decreases the amount of mENaC at the oocyte surface (37) and that this interaction may not depend on the magnitude of chloride transport (34). Our data regarding the regulatory interactions of {Delta}F508- and G551D-CFTR also are consistent with the data of others suggesting that an intact NBD-1 is required for activated CFTR to inhibit mENaC-mediated conductance (3, 33, 34).

We therefore assessed the interregulation of I148T-CFTR and mENaC in oocytes. Figure 2 depicts whole cell currents measured at a holding potential of –100 mV in oocytes injected with I148T-CFTR (10 ng) or {alpha}beta{gamma}-mENaC (0.33 ng/subunit) or both I148T-CFTR and {alpha}beta{gamma}-mENaC cRNAs. Oocytes coinjected with I148T-CFTR and {alpha}beta{gamma}-mENaC had no change in forskolin/IBMX-stimulated, amiloride-insensitive (I148T-CFTR-mediated) current compared with oocytes injected with I148T-CFTR alone [ICl = –3.20 ± 1.20 (I148T-CFTR, n = 19) vs. –2.86 ± 0.78 µA (I148T-CFTR/{alpha}beta{gamma}-mENaC, n = 17), P = not significant (NS); Fig. 2A]. Thus coexpression of {alpha}beta{gamma}-mENaC in oocytes does not alter the functional expression of I148T-CFTR. These data are significantly different to our group's previous observations with WT-CFTR, where coexpression of {alpha}beta{gamma}-mENaC enhanced WT-CFTR functional expression by 3.5- to 5-fold (34, 37).


Figure 2
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  		Fig. 2. Expression of I148T-CFTR and murine epithelial sodium channel {alpha}beta{gamma}-subunits ({alpha}beta{gamma}-mENaC) in Xenopus oocytes. I148T-CFTR (10 ng of cRNA) and {alpha}beta{gamma}-mENaC (0.33 ng cRNA/subunit) were expressed separately or together in oocytes, and TEV was performed as described in EXPERIMENTAL PROCEDURES. A: changes in whole cell currents that were not inhibited by 10 µM amiloride (–100 mV holding potential) in oocytes injected with I148T-CFTR alone or in oocytes coinjected with I148T-CFTR and {alpha}beta{gamma}-mENaC after stimulation with 10 µM forskolin/100 µM IBMX are shown. 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 I148T-CFTR before (open bars) and after (shaded bars) stimulation with 10 µM forskolin/100 µM IBMX. Data obtained from the same I148T-CFTR/{alpha}beta{gamma}-mENaC coinjected oocytes are shown in A and B. Values are means ± SE.

 
In oocytes expressing {alpha}beta{gamma}-mENaC alone, the amiloride-sensitive whole cell currents were similar before and after incubation with 10 µM foskolin/100 µM IBMX for 25 min (Fig. 2B); these data are consistent with previous reports from our group and others (23, 34, 37). Oocytes coinjected with both I148T-CFTR and {alpha}beta{gamma}-mENaC expressed amiloride-sensitive whole cell currents before forskolin/IBMX stimulation (–0.32 ± 0.10 µA, n = 17) that were significantly lower than the amiloride-sensitive whole cell currents recorded under the same conditions in oocytes injected with {alpha}beta{gamma}-mENaC alone (–2.06 ± 0.62 µA, n = 18, P = 0.01). These observations are similar to our group's data regarding CFTRs with appropriate intracellular trafficking, such as WT-CFTR (34, 37) and G551D-CFTR (33). However, unlike WT-CFTR, activation of I148T-CFTR with forskolin/IBMX did not lead to a further reduction in {alpha}beta{gamma}-mENaC-mediated amiloride-sensitive whole cell currents in coinjected oocytes (–0.32 ± 0.10 µA before forskolin/IBMX vs. –0.40 ± 0.13 µA after forskolin/IBMX, n = 17, P = NS; Fig. 2B). These data are similar to our previous data for the CFTR mutants {Delta}F508 and G551D that have defective regulatory interactions with mENaC; activation of {Delta}F508 and G551D with forskolin/IBMX also does not lead to acute decreases in mENaC functional expression (33, 34). These data are therefore consistent with activated I148T-CFTR having defective regulatory interactions with {alpha}beta{gamma}-mENaC in Xenopus oocytes despite maintaining a robust ability to transport chloride.

We have previously demonstrated that the decreases in mENaC functional expression upon coexpression of WT-CFTR in oocytes correlates with changes in mENaC surface expression. We also observed that activation of WT-CFTR leads to a further acute decrease in mENaC surface expression (37). We performed similar surface biotinylation experiments in the present study to assess the correlation of functional and surface expression of mENaC. We again used {alpha}beta{gamma}-mENaC where the beta-subunit contained a COOH-terminal V5 epitope tag (beta-V5) to increase the sensitivity of our immunodetection in these experiments. As shown in Fig. 3A, mENaC(beta-V5) expression at the oocyte surface was unaltered by treatment with forskolin/IBMX in oocytes injected with {alpha}beta{gamma}-mENaC alone and decreased after coinjection of I148T-CFTR in the absence of forskolin/IBMX activation. Furthermore, mENaC surface expression did not change upon activation of I148T-CFTR with forskolin/IBMX in coinjected oocytes. These surface expression data parallel the mENaC functional expression data obtained uisng TEV (Fig. 2B) and are consistent with the observed changes in mENaC functional expression resulting at least in part from changes in the number of channels in the plasma membrane.


Figure 3
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  		Fig. 3. Influence of I148T-CFTR on surface and whole oocyte expression of mENaC. {alpha}beta{gamma}-mENaC (0.33 ng cRNA/subunit) where the beta-subunit contained a COOH-terminal V5 epitope was expressed in oocytes without or with I148T-CFTR (10 ng of cRNA). After 48 h, 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. mENaC beta-V5 was recovered by stepavidin-agarose precipitation and quantitated by immunoblotting 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. These data are expressed as mean density (±SE) normalized to the density observed with oocytes expressing {alpha}-, beta-V5-, and {gamma}-mENaC in the absence of forskolin/IBMX stimulation. These data are derived from n = 3 independent experiments. Shaded bars represent ooctyes without forskolin/IBMX stimulation' open bars represent oocytes stimulated with 10 µM forskolin and 100 µM IBMX. P values were determined by ANOVA compared with {alpha}beta-V5{gamma}-mENaC oocytes that were not treated with forskolin/IBMX. B: a representative immunoblot and densitometric analysis of the immunoblots of the whole oocytes lysates of the experiment in A are shown. These data are similarly representative of n = 3 independent experiments.

 
We also assessed whole oocyte expression of mENaC(beta-V5) (Fig. 3B). The whole oocyte content of mENaC(beta-V5) was not altered by acute treatment with forskolin/IBMX in oocytes injected with {alpha}beta{gamma}-mENaC alone, was decreased by coinjection with I148T-CFTR cRNA, and was not further decreased upon activation of I148T-CFTR by forskolin/IBMX. These data are similar to our previous observations with WT-CFTR and significantly differ from our observations with {Delta}F508-CFTR, where coinjection of the same amount of cRNA (10 ng) did not alter whole oocyte expression of mENaC(beta-V5) (37). Thus competition for oocyte translational machinery is likely not confounding these experiments. These data are therefore consistent with I148T-CFTR, in the absence of activation, maintaining the ability to decrease the whole oocyte expression of mENaC. This decrease in whole oocyte expression in coinjected oocytes parallels the decrease in surface expression and may result from I148T-CFTR decreasing the synthesis of mENaC and its trafficking to the oocyte membrane or increasing the rate at which mENaC is degraded either before or after reaching the plasma membrane.

Interregulation of I148T-CFTR and {alpha}beta{gamma} mENaC after IBMX/forskolin/genistein stimulation. We examined whether the CFTR potentiator genistein, which improves functional interactions between {alpha}beta{gamma}-mENaC and the G551D-CFTR and {Delta}F508-CFTR mutants (33, 34), is similarly able to restore I148T-CFTR's regulatory interactions with mENaC. As shown in Fig. 4A, the amiloride-insensitive, or I148T-CFTR-mediated, component of the forskolin/IBMX/genistein-stimulated whole cell current in oocytes coexpressing I148T-CFTR and {alpha}beta{gamma}-mENaC was 1.7-fold greater than the forskolin/IBMX/gensitein-stimulated current measured in oocytes expressing I148T-CFTR alone (I148T-CFTR: –3.06 ± 0.81 µA, n = 21 vs. I148T-CFTR/{alpha}beta{gamma}-mENaC: –5.29 ± 0.81 µA, n = 23, P = 0.005).


Figure 4
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  		Fig. 4. Influence of genistein on the regulatory interactions between I148T-CFTR and {alpha}beta{gamma}-mENaC. I148T-CFTR (10 ng of cRNA) and {alpha}beta{gamma}-mENaC (0.33 ng cRNA/subunit) were expressed separately or together in oocytes, and TEV was performed as described in EXPERIMENTAL PROCEDURES. A: changes in whole cell currents (–100 mV holding potential) after stimulation with 10 µM forskolin/100 µM IBMX/50 µM genistein that were not inhibited by 10 µM amiloride. 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 I148T-CFTR before (open bars) and after (shaded bars) stimulation with 10 µM forskolin/100 µM IBMX/50 µM genistein. Data obtained from the same I148T-CFTR/{alpha}beta{gamma}-mENaC coinjected oocytes are presented in A and B. Values are means ± SE.

 
As described above, I148T-CFTR-mediated currents were increased approximately threefold with the addition of genistein alone (Fig. 1B), and coinjected oocytes had no greater amiloride-insensitive forskolin/IBMX-stimulated currents than oocytes injected with I148T-CFTR alone (Fig. 2A). In contrast, I148T-CFTR/mENaC coinjected oocytes had ~5.5-fold greater current with addition of genistein (–5.29 ± 0.81 µA, n = 23) than in forskolin/IBMX-stimulated oocytes injected with I148T-CFTR alone (–0.97 ± 0.31 µA, n = 19). These data suggest synergistic enhancement of I148T-CFTR functional expression by genistein and {alpha}beta{gamma}-mENaC in the presence forskolin/IBMX, and are qualitatively similar to our previous observations of synergistic activation of G551D-CFTR by genistein and {alpha}beta{gamma}-mENaC (33). These data are therefore consistent with genistein improving the regulation of I148T-CFTR by {alpha}beta{gamma}-mENaC in oocytes.

Consistent with our previous results (33, 34), whole cell amiloride-sensitive currents modestly but significantly increased in response to 10 µM foskolin/100 µM IBMX/50 µM genistein [–3.61 ± 0.87 µA (–forskolin/IBMX/genistein) vs. –4.44 ± 1.12 µA (+forskolin/IBMX/genistein) n = 17, P = 0.048] in oocytes injected with {alpha}beta{gamma}-mENaC alone (Fig. 4B). In oocytes coexpressing {alpha}beta{gamma}-mENaC and I148T-CFTR, the amiloride-sensitive currents did not change following stimulation with forskolin/IBMX/gensitein [–0.82 ± 0.17 µA (–forskolin/IBMX/gensitein) vs. –0.84 ± 0.18 (+forskolin/IBMX/genistein), n = 23, P = NS]. These data are similar to our previous observations with WT-, {Delta}F508-, and G551D-CFTR, where this forskolin/IBMX/genistein stimulation in mENaC-mediated current observed in oocytes injected with {alpha}beta{gamma}-mENaC alone was not present in CFTR/{alpha}beta{gamma}-mENaC coinjected oocytes; such data are consistent with improved regulation of {alpha}beta{gamma}-mENaC by the forskolin/IBMX-activated mutant CFTRs in the presence of genistein (33, 34).

We performed surface biotinylation and whole oocyte expression experiments to probe the mechanism by which genistein acts on mENaC and influences mENaC regulation by activated I148T-CFTR (Fig. 5). Data shown in Fig. 5A suggest that mENaC(beta-V5) expression at the oocyte surface was unaltered by treatment with forskolin/IBMX/genistein in oocytes injected with {alpha}beta{gamma}-mENaC alone, was decreased with coinjection of I148T-CFTR in the absence of forskolin/IBMX/genistein activation, and was further decreased upon activation of I148T-CFTR with forskolin/IBMX/genistein in coinjected oocytes. Interestingly, these surface expression data do not parallel the mENaC functional expression data obtained using TEV and shown in Fig. 4B. Instead, these data suggest that the increase in mENaC functional expression with forskolin/IBMX/genistein results from an increase in mENaC open probability or unitary conductance, rather than a change in surface expression. Furthermore, in coinjected oocytes, mENaC(beta-V5) surface expression acutely decreased upon activation of I148T-CFTR with forskolin/IBMX/genistein. This decreased surface expression likely balanced the suggested forskolin/IBMX/genistein-stimulated increase in mENaC open probability or unitary conductance and yielded the similar mENaC-mediated currents in coinjected oocytes before and after forskolin/IBMX/genistein stimulation (Fig. 4B). These data are therefore consistent with genistein restoring the regulation of mENaC surface expression by activated I148T-CFTR.


Figure 5
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  		Fig. 5. Influence of genistein on surface and whole oocyte expression of mENaC. {alpha}beta{gamma}-mENaC (0.33 ng cRNA/subunit) where the beta-subunit contained a COOH-terminal V5 epitope was expressed in oocytes without or with I148T-CFTR (10 ng of cRNA). After 48 h, oocytes were incubated for an additional 20 min in the absence or presence of 10 µM forskolin and 100 µM IBMX, followed by an additional 15 min in 50 µM genistein. Cell surface biotinylation was performed as described in EXPERIMENTAL PROCEDURES. mENaC beta-V5 was recovered by stepavidin-agarose precipitation and quantitated by immunoblotting with an anti-V5 antibody. A: a representative immunoblot and densitometric analysis of these surface biotinylation experiments. These data are expressed as mean density (±SE) normalized to the density observed with oocytes expressing {alpha}-, beta-V5-, and {gamma}-mENaC in the absence of forskolin/IBMX/genistein stimulation. These data are derived from n = 3 independent experiments. Solid bars represent ooctyes without forskolin/ IBMX/genistein stimulation; open bars represent oocytes stimulated with 10 µM forskolin, 100 µM IBMX, and 50 µM genistein. P values were determined by ANOVA. B: a representative immunoblot and densitometric analysis of the immunoblots of the whole oocytes lysates of the experiment in A are shown. These data are similarly representative of n = 3 independent experiments, with P values determined by ANOVA.

 
We also assessed the influence of forskolin/IBMX/genistein on the whole oocyte expression of mENaC(beta-V5) in these experiments (Fig. 5B). Similar to Fig. 3B, the whole oocyte content of mENaC(beta-V5) was not altered by acute treatment with forskolin/IBMX/genistein in oocytes injected with {alpha}beta{gamma}-mENaC alone, was decreased by coinjection with I148T-CFTR cRNA, and was not further decreased upon activation of I148T-CFTR by forskolin/IBMX/genistein. These data are consistent with genistein not acutely altering whole oocyte expression of mENaC in oocytes injected with either {alpha}beta{gamma}-mENaC alone or upon activation of I148T-CFTR in coinjected oocytes.


   DISCUSSION
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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The absence of functional CFTR leads to increased ENaC activity in the airway epithelia, which is hypothesized to result in increased solute and liquid absorption, decreased airway surface liquid volume, and decreased mucociliary clearance (17). The regulatory interactions of CFTR and ENaC are therefore likely to influence the CF airway phenotype. The interactions of these proteins have been extensively studied in the Xenopus oocyte system, because the oocyte system, at a first approximation, reproduces the reciprocal relationship between increased CFTR and decreased ENaC function apparent in the airway. Less is known about the mechanism of potential tissue specificity of these interactions, as is suggested by CFTR's facilitation of ENaC function in the sweat duct epithelia (28). Thus, despite these extensive studies, the mechanism of CFTR/ENaC functional interaction, especially in oocytes, remains the subject of debate. In the present study, we assessed the functional interactions of I148T-CFTR and mENaC to gain further insight into the mechanism underlying these interactions, as well as to examine in more detail potential functional defects in I148T-CFTR. These findings are discussed in the context of our group's previous observations regarding the interactions of mENaC with WT-, {Delta}F508-, and G551D-CFTR, which are summarized in Table 1.


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  		Table 1. Interactions between wild-type and mutant CFTRs and mENaC in oocytes

 
I148T-CFTR/mENaC functional interactions. Previous observations that I148T-CFTR maintains robust chloride transport function but may be clinically associated with a CF phenotype, perhaps because of an inability to regulate bicarbonate transport (9), prompted us to test the hypothesis that I148T-CFTR may also have defective interactions with ENaC. Of note, the I148T-CFTR construct used in this work, and in the work of Choi et al. (9), did not harbor a second mutation in cis, 3199del6, which causes deletion of I1023 and V1024, as has been described in patients with CF and an I148T allele. Such mutations in cis may cause CFTR dysfunction regardless of whether residue 148 is an I or a T (10). Thus any functional defects in I148T-CFTR noted in this or the previous studies regarding bicarbonate transport (9) are solely due to the I148T mutation and are not potentially confounded by a second mutation in cis.

Our data confirm the observations of others (9) that I148T-CFTR maintains the ability to transport chloride at levels similar to WT-CFTR. However, despite this robust chloride transport, we found that I148T-CFTR had functional and regulatory interactions with mENaC that differed significantly from those of WT-CFTR previously observed by our group (34, 37). These data provide additional molecular support for the hypothesis that I148T-CFTR may be associated with a clinically apparent CF phenotype due to aberrant functional interactions with the transporters of other ions that result in defective regulation of transport of these other ions, such as Na+ and bicarbonate, in epithelia.

Ability of CFTR to mediate chloride transport is not sufficient for regulation of mENaC. The mechanism by which activated CFTR decreases mENaC functional expression in oocytes remains controversial. Some have suggested that CFTR-mediated chloride transport is required for this effect with observations that the degree of decreased functional expression of ENaC parallels the magnitude of chloride transport by CFTR (6), perhaps because increases in cytosolic Cl– may cause a decrease in ENaC open probability (2). Others have suggested the hypothesis that the decrease in mENaC functional expression upon CFTR activation results from a series resistor error and have suggested that such effects also occur with non-CFTR-mediated chloride transport (25, 26). In contrast, our group's previous data (37) suggest that the decrease in functional mENaC expression upon activation of WT-CFTR parallels an acute change in mENaC expression at the oocyte surface. Furthermore, our present data suggest that forskolin/IBMX-stimulated I148T-CFTR does not acutely alter mENaC functional and surface expression despite the presence of robust ability to transport chloride. These data are therefore inconsistent with the hypothesis that chloride transport by activated CFTR is required and sufficient for the decreased functional expression of mENaC regardless of whether this is a result of the amount of chloride transport mediated by CFTR or a series resistor error.

Our data in the present and in previous work do not specifically address whether increases in cytosolic Cl– concentration may also influence ENaC functional expression in these experiments via decreasing ENaC open probability(2). We have not directly measured changes in intracellular Cl– because of expression of WT- vs. I148T-CFTR in these experiments. However, it seems somewhat unlikely to us that intracellular Cl– would increase significantly more with activation of WT- vs. I148T-CFTR given the similar levels of whole oocyte CFTR-mediated current measured by TEV in the present study for I148T-CFTR and in our previous studies for WT-CFTR (34, 37).

Is NBD-1 a critical element for regulation of mENaC by activated CFTR? WT-CFTR truncation mutants containing an intact NBD-1 (30), or with a WT-CFTR peptide fragment containing NBD-1 and the regulatory (R) domain (20), are also able to inhibit ENaC functional expression in a forskolin/IBMX-dependent manner. Although these data are consistent with the notion that chloride transport is not required for this regulation, these data also implicate NBD-1 as a critical element in such regulation. That a similar NBD-1/R peptide fragment containing the G551D mutation (20) and the CFTR mutants G551D (33) and {Delta}F508 (34) do not manifest forskolin/IBMX-regulated inhibition of ENaC also suggests that a structurally and functionally intact NBD-1 is required for this regulatory interaction.

At first glance, I148T-CFTR should have a structurally and functionally intact NBD-1, because this mutation is located within CFTR's first cytoplasmic loop. One would therefore have predicted that I148T-CFTR should demonstrate forskolin/IBMX-dependent inhibition of mENaC functional and surface expression. Since I148T-CFTR does not acutely decrease mENaC functional activity and surface expression upon stimulation, these data suggest either that domains of CFTR other than NBD-1 may be important in the regulatory interactions of CFTR and mENaC or that changes in CFTR's first cytoplasmic loop may influence NBD-1 form and/or function. The latter of these possibilities is supported by the strongly hydrophilic nature of CFTR's cytoplasmic loops, which have the potential for intramolecular interactions with other charged portions of the CFTR molecule such as NBD-1 (35). This possibility is also supported by the crystal structures of the potentially CFTR homologous bacterial ABC transporters MsbA (7, 8) and BtuCD (22), where the first cytolplasmic loop of the transmembrane domain interacts directly with the nucleotide binding domain.

Potentiation of mutant CFTR function with genistein improves regulatory interactions. Genistein potentiates CFTR (WT, {Delta}F508, and G551D) chloride transport by increasing channel open probability (1, 36). Genistein may interact directly with CFTR via CFTR's second nucleotide binding domain, NBD-2 (27), although the complete molecular detail of this interaction remains elusive. In the present study, we have demonstrated that genistein is able to potentiate chloride transport by I148T-CFTR, to enable mENaC to potentiate chloride transport by I148T-CFTR in a synergistic fashion, and to restore the ability of activated I148T-CFTR to acutely decrease the surface expression of mENaC in coinjected oocytes. These latter two observations suggest that genistein improves the regulatory interactions of I148T-CFTR with mENaC. These effects are similar to genistein's actions to potentiate {Delta}F508- and G551D-CFTR-mediated chloride transport and improve the regulatory interactions of these mutants with mENaC (33, 34).

Genistein also potentiates cAMP-stimulated HCO3– and Cl– currents in permeabilized Calu-3 monolayers that express CFTR (12). Our data suggest that genistein slightly increases mENaC functional expression without altering surface expression in the absence of CFTR but is also able to "repair" the regulatory interactions between I148T-CFTR and mENaC. If there is a common mechanism by which activated CFTR regulates HCO3– transport and ENaC trafficking/surface expression, we can speculate that genistein may also repair regulation of HCO3– transport by I148T-CFTR.

In conclusion, we have demonstrated that I148T-CFTR has abnormal regulatory interactions with mENaC in Xenopus oocytes despite maintaining robust levels of chloride transport. The CFTR potentiator genistein can improve such regulatory interactions. These data provide further molecular and physiological evidence that the I148T mutation is associated with significant CFTR regulatory dysfunction and are consistent with I148T being a CF disease-causing mutation. These data also provide further evidence that CFTR potentiators such as genistein may be useful as therapeutic agents.


   GRANTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-58046 (to R. C. Rubenstein). R. C. Rubenstein is an Established Investigator of the American Heart Association.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. C. Rubenstein, Division of Pulmonary Medicine, Children's Hospital of Philadelphia, Abramson 410C, 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.


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 DISCUSSION
 GRANTS
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L. Suaud, W. Yan, M. D. Carattino, A. Robay, T. R. Kleyman, and R. C. Rubenstein
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