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J. Biol. Chem., Vol. 277, Issue 11, 8928-8933, March 15, 2002

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Genistein Restores Functional Interactions between F508-CFTR and ENaC in Xenopus Oocytes^*

Laurence Suaud^§¶, Jinqing Li^¶, Qinshi Jiang^¶, Ronald C. Rubenstein^**, and Thomas R. Kleyman^§§¶¶

From the  Division of Pulmonary Medicine, Children's Hospital of Philadelphia, Departments of  Medicine and ^** Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, 19104 and ^§§ Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Received for publication, December 2, 2001, and in revised form, December 19, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its Cl channel properties, has regulatory interactions with other epithelial ion channels including the epithelial Na⁺ channel (ENaC). Both the open probability and surface expression of wild type CFTR Cl channels are increased significantly when CFTR is co-expressed in Xenopus oocytes with -ENaC, and conversely, the activity of ENaC is inhibited following wild type CFTR activation. Using the Xenopus oocyte expression system, a lack of functional regulatory interactions between F508-CFTR and ENaC was observed following activation of F508-CFTR by forskolin and isobutylmethylxanthine (IBMX). Whole cell currents in oocytes expressing ENaC alone decreased in response to genistein but increased in response to a combination of forskolin and IBMX followed by genistein. In contrast, ENaC currents in oocytes co-expressing ENaC and F508-CFTR remained stable following stimulation with forskolin/IBMX/genistein. Furthermore, co-expression of F508-CFTR with ENaC enhanced the forskolin/IBMX/genistein-mediated activation of F508-CFTR. Our data suggest that genistein restores regulatory interactions between F508-CFTR and ENaC and that combinations of protein repair agents, such as 4-phenylbutyrate and genistein, may be necessary to restore F508-CFTR function in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The cystic fibrosis phenotype is a result of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR)¹ (1). In addition to functioning as a cAMP-activated, ATP-dependent Cl channel, CFTR influences the transepithelial transport of other solutes, including Na⁺ via the epithelial sodium channel (ENaC), Cl via an outwardly rectifying Cl channel, HCO3, glutathione, and ATP (2-20).

Functional interactions between CFTR and ENaC have been observed in both epithelial and non-epithelial cells (2-8). The activation of CFTR is generally associated with an inhibition of ENaC, although activation of CFTR leads to activation of ENaC in the sweat duct (9), suggesting that the regulatory interactions between these two transporters are complex. The co-expression of CFTR and ENaC in Xenopus oocytes results in a decrease in ENaC-mediated Na⁺ transport in the presence of activated CFTR (4-7), a regulatory interaction that mimics CFTR/ENaC interactions in the airway. Furthermore, CFTR Cl conductance is increased in the presence of ENaC (5-7).

The F508-CFTR mutation is the most prevalent mutation in patients with cystic fibrosis (21). The mutant protein has impaired exit from the endoplasmic reticulum and is targeted for rapid proteasomal degradation (22-24). Selected chemical or pharmacological agents, such as glycerol or 4-phenylbutyrate, improve the functional expression of F508-CFTR and may affect the interaction of F508-CFTR with specific molecular chaperones (25-28). Genistein, an isoflavone with tyrosine kinase and topoisomerase inhibitor activities, enhances functional F508-CFTR Cl channel expression in epithelial and non-epithelial cells via increases in channel open probability (29). Previous studies have concentrated on assessing changes in mutant CFTR-mediated Cl transport in response to chemical or pharmacological agents such as 4-phenylbutyrate, glycerol, and genistein. However, assessment of mutant CFTR-mediated regulation of Na⁺ transport in response to these pharmacological agents has received limited attention. We utilized the Xenopus oocyte expression system to examine whether genistein restores F508-CFTR regulatory interactions with ENaC, as previous studies have shown that F508-CFTR is expressed at the oocyte cell surface (30). We observed that F508-CFTR does not inhibit the functional expression of ENaC, in agreement with previous data (31). Furthermore, ENaC did not enhance the functional expression of F508-CFTR. However, we observed that genistein restores functional interactions between F508-CFTR and ENaC, suggesting that combinations of pharmacologic agents may prove beneficial for the repair of mutant CFTR function.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Forskolin, IBMX, and genistein were purchased from Sigma. All other reagents were purchased from Fisher.

Expression of Human CFTR (Wild Type (WT) and F508) and Mouse ENaC in Xenopus Oocytes-- Human CFTR (WT and F508) and mouse ENaC were expressed in Xenopus oocytes essentially as described previously (7). Briefly, human WT-CFTR, human F508-CFTR, and mouse -, -, and -ENaC cRNAs were prepared using a cRNA synthesis kit (mMESSAGE mMACHINE, Ambion Inc., 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 (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 0.3 mM Ca(NO₃)₂, 0.41 mM CaCl₂, 0.82 mM MgSO₄, 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 with -, - and -subunits of ENaC (0.33 ng/subunit), WT-CFTR (10 ng), F508-CFTR (10 ng), or a combination of ENaC and CFTR (WT or F508) cRNAs dissolved in RNase-free water using a Nanoject II microinjector (Drummond Scientific, Broomall, PA).

Electrophysiological Analyses-- Whole cell current measurements were performed 24-48 h after injection using the two-electrode voltage clamp method as described previously (7). 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, 10 mM Hepes, pH 7.4) and impaled with micropipettes of 0.5-5-megaohm resistance filled with 3 M KCl. The whole cell currents were measured by voltage clamping the oocytes in 20-mV steps between 140 mV and +60 mV adjusted for base-line transmembrane potential. Whole cell currents (I) were digitized at 200 Hz during the voltage steps, recorded directly onto a hard disc, and analyzed using pClamp 8 software (Axon Instruments, Foster City, CA). Ion replacement studies were performed in an identical manner except that equimolar N-methyl-D-glucamine (NMDG) replaced Na⁺ in the ND96 solution. 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. We routinely observed membrane potentials that were <5% depolarized from our target holding potentials, despite high conductances observed in oocytes expressing ENaC and WT-CFTR.

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 ENaC. Activation of WT- or F508-CFTR was accomplished by perfusion of the oocyte with buffer supplemented with 10 µM forskolin and 100 µM IBMX for 25 min (7). In some experiments, this first step was followed by an incubation with 10 µM forskolin, 100 µM IBMX, and 50 µM genistein for 20 min. In all experiments, F508-CFTR Cl current was defined as the difference between current measured 20 min after perfusion with forskolin/IBMX (or 15 min after perfusion with forskolin/IBMX/genistein) and the current measured before forskolin/IBMX (±genistein) stimulation. Whole cell currents were recorded at 100 mV for comparisons. All measurements were performed at room temperature.

Statistical Analyses-- Statistical comparisons were performed using the Student's t test. A pairwise t test was used for pre-/post-treatment in experiments using an individual oocyte. 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 CFTR (WT or F508)) versus oocytes co-injected with a cRNA for both ENaC and CFTR (WT or F508). p values < 0.05 were accepted to indicate statistical significance.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of F508-CFTR and ENaC in Xenopus Oocytes-- The Xenopus oocyte expression system was used to examine the functional expression of F508-CFTR and its functional interaction with ENaC. Although F508-CFTR is a "trafficking-defective" mutant, F508-CFTR is delivered to the oocyte plasma membrane (30) as oocytes are maintained at 18 °C. Temperatures 27 °C are permissive for F508-CFTR trafficking (32, 33). The two-electrode voltage clamp technique was used to measure whole cell currents at varying clamping potentials in oocytes expressing F508-CFTR (Fig. 1A). I/V curves were obtained before and after 20 min of incubation with 10 µM forskolin, 100 µM IBMX to activate endogenous protein kinase A. Whole cell currents, measured at a clamp potential of 100 mV, increased 7.5-fold from 0.2 ± 0.0 to 1.5 ± 0.3 µA (n = 19) in response to forskolin/IBMX, in agreement with previous observations (30). Oocytes expressing -ENaC exhibited amiloride-sensitive whole cell currents (Fig. 1B), as reported previously (7). The amiloride-sensitive whole cell current, measured at a clamp potential of 100 mV, was 5.1 ± 1.4 µA (n = 9). In agreement with previous observations, amiloride-sensitive currents were unchanged in response to 10 µM forskolin, 100 µM IBMX (Fig. 2, I_Na = 4.3 ± 1.0 µA (forskolin/IBMX) versus 4.6 ± 1.1 µA (+forskolin/IBMX), p is not significant; Fig. 3, I_Na = 5.1 ± 1.4 µA (forskolin/IBMX) versus 5.6 ± 1.5 µA (+forskolin/IBMX), p is not significant) (31, 34).

View larger version (7K): [in this window] [in a new window]   Fig. 1.   Expression of F508-CFTR and -ENaC in Xenopus oocytes. F508-CFTR was expressed in oocytes, and TEV was performed as described under "Experimental Procedures" (A). Whole cell currents were measured by voltage clamping oocytes in 20-mV steps between 140 mV and +60 mV adjusted for base-line transmembrane potential. Shown are I/V relationships in the absence (closed circles) or presence (open circles) of 10 µM forskolin and 100 µM IBMX. -ENaC was expressed in oocytes, and TEV was performed (B). I/V relationships in the absence (open diamonds) or presence (closed diamonds) of 10 µM amiloride are shown. Means ± S.E. are illustrated. Error bars contained within the symbols are not apparent.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Expression of WT-CFTR and ENaC in Xenopus oocytes. WT-CFTR and ENaC were expressed separately or together in oocytes, and TEV was performed as described under "Experimental Procedures." Changes in whole cell currents (100-mV holding potential) after stimulation with 10 µM forskolin, 100 µM IBMX that were not inhibited by 10 µM amiloride are illustrated (closed bars) (A). Amiloride-sensitive whole cell currents (100-mV holding potential) were determined in oocytes expressing ENaC or co-expressing ENaC and WT-CFTR before (dark gray bars) and following (open bars) stimulation with 10 µM forskolin, 100 µM IBMX (B). Data obtained from the same WT-CFTR/ENaC co-injected oocytes are presented in panels A and B. Means ± S.E. are illustrated. ns, not significant.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Co-expression of F508-CFTR and -ENaC in Xenopus oocytes. F508-CFTR and ENaC were expressed separately or together in oocytes, and TEV was performed as described under "Experimental Procedures." Changes in whole cell currents (100-mV holding potential) after stimulation with 10 µM forskolin, 100 µM IBMX that were not inhibited by 10 µM amiloride are illustrated (closed bars) (A). ns, not significant. Amiloride-sensitive whole cell currents (100-mV holding potential) were determined in oocytes expressing ENaC or co-expressing ENaC and F508-CFTR before (dark gray bars) and following (open bars) stimulation with 10 µM forskolin, 100 µM IBMX (B). Data obtained from the same F508-CFTR/ENaC co-injected oocytes are presented in panels A and B. Means ± S.E. are illustrated.

Co-expression of WT-CFTR and -ENaC in Xenopus Oocytes-- Several groups have previously reported that when WT-CFTR and ENaC were co-expressed in Xenopus oocytes, ENaC-mediated Na⁺ currents were inhibited in response to CFTR activation (4-7). Furthermore, ENaC enhances forskolin/IBMX-stimulated CFTR Cl currents (5-7). Fig. 2 shows whole cell currents measured at a holding potential of 100 mV in oocytes injected with cRNAs for WT-CFTR (10 ng) or -ENaC (0.33 ng/subunit) or co-injected with cRNAs for both WT-CFTR and -ENaC. Oocytes co-injected with both CFTR and ENaC expressed an amiloride-sensitive whole cell current (1.3 ± 0.3 µA at 100 mV) in the absence of forskolin/IBMX. Following CFTR activation with forskolin/IBMX, the amiloride-sensitive whole cell current was reduced to 0.7 ± 0.2 µA (Fig. 2, n = 15, p < 0.01).

The forskolin/IBMX-stimulated amiloride-insensitive whole cell current measured at a clamp potential of 100 mV in oocytes co-expressing WT-CFTR and ENaC (7.6 ± 2.1 µA; n = 15) was 3.5-fold greater than that obtained in oocytes expressing CFTR alone (2.2 ± 0.4 µA; n = 18; p = 0.01; Fig. 2). The larger forskolin/IBMX-stimulated amiloride-insensitive current in oocytes expressing both CFTR and ENaC, as compared with oocytes expressing CFTR alone, is in agreement with previous reports that ENaC enhances functional CFTR expression (5-7).

Co-expression of F508-CFTR and -ENaC-- Whole cell currents measured at a clamp potential of 100 mV in oocytes injected with cRNAs for either F508-CFTR (10 ng), -ENaC (0.33 ng/subunit), or both F508-CFTR and -ENaC are shown in Fig. 3. Oocytes co-injected with both F508-CFTR and ENaC expressed amiloride-sensitive whole cell currents (5.1 ± 0.7 µA) in the absence of forskolin/IBMX. Following F508-CFTR activation with forskolin/IBMX, the amiloride-sensitive whole cell current was essentially unchanged (4.9 ± 0.6 µA, n = 19, p is not significant), in agreement with previous studies (31). Furthermore, forskolin/IBMX-stimulated whole cell currents in oocytes expressing F508-CFTR alone (1.3 ± 0.3 µA) and amiloride-insensitive forskolin/IBMX-stimulated whole cell currents in oocytes expressing F508-CFTR and ENaC (1.6 ± 0.3 µA) were similar in magnitude (n = 19; p is not significant). These data are consistent with a lack of functional regulatory interactions between F508-CFTR and ENaC expressed in Xenopus oocytes.

Genistein Restores Functional Interactions between F508-CFTR and -ENaC-- Previous studies reported that the open probability of F508-CFTR is lower than the open probability of WT-CFTR (35). Furthermore, the open probabilities of both F508-CFTR and WT-CFTR have been reported to increase in response to the isoflavone genistein (29). We therefore examined whether genistein restored functional interactions between F508-CFTR and ENaC. Linear whole cell I/V relationships were observed in oocytes expressing F508-CFTR (Fig. 4A) or WT-CFTR (Fig. 4B) after 15 min of incubation with 10 µM forskolin, 100 µM IBMX, 50 µM genistein. Similarly, oocytes expressing -ENaC and stimulated with forskolin/IBMX/genistein exhibited amiloride-sensitive whole cell currents with a linear I/V relationship (Fig. 4C).

View larger version (9K): [in this window] [in a new window]   Fig. 4.   Effect of genistein on F508-CFTR, CFTR, and -ENaC. F508-CFTR (A), WT-CFTR (B), or -ENaC (C) were expressed in oocytes, and whole cell currents were measured in response to voltage clamping oocytes between 140 mV and +60 mV in 20-mV steps. In panels A and B, the I/V relationships for F508-CFTR and WT-CFTR, respectively, are shown before (closed symbols) and after (open symbols) treatment with 10 µM forskolin, 100 µM IBMX, 50 µM genistein. Panel C illustrates whole cell currents in ENaC-injected oocytes after treatment with 10 µM forskolin, 100 µM IBMX, 50 µM genistein before (open squares) and after (closed squares) the addition of 10 µM amiloride. Means ± S.E. are illustrated. Error bars contained within the symbols are not readily apparent.

Previous studies suggested that chronic treatment with genistein inhibits ENaC by reducing surface expression of Na⁺ channels in A6 renal epithelial cells (36, 37). In contrast, protein kinase A activates ENaC in part via an increase in surface expression of channels (38, 39). Amiloride-sensitive whole cell currents were measured in oocytes expressing ENaC before and after 15 min of incubation with 10 µM forskolin, 100 µM IBMX, 50 µM genistein (Fig. 5). Whole cell amiloride-sensitive currents modestly, but significantly, increased in response to forskolin/IBMX/genistein (7.1 ± 1.0 µA (forskolin/IBMX/genistein) versus 9.0 ± 0.9 µA (+forskolin/IBMX/genistein); n = 23; p = 0.004; Fig. 5). In contrast, amiloride-sensitive currents decreased in response to genistein alone (7.3 ± 1.5 µA (ENaC  genistein); 4.0 ± 0.6 µA (ENaC + genistein); n = 8, p = 0.015).

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Genistein restores regulatory interactions between F508-CFTR and -ENaC. F508-CFTR and ENaC were expressed separately or together in oocytes, and TEV was performed as described under "Experimental Procedures." Changes in whole cell currents (100-mV holding potential) after stimulation with 10 µM forskolin, 100 µM IBMX (closed bar) or 10 µM forskolin, 100 µM IBMX, 50 µM genistein that were not inhibited by 10 µM amiloride (light gray bars) are illustrated (A). Amiloride-sensitive whole cell currents (100-mV holding potential) were determined in oocytes expressing ENaC or co-expressing ENaC and F508-CFTR before (dark gray bars) and following (open bars) stimulation with 10 µM forskolin, 100 µM IBMX, 50 µM genistein (B). Data obtained from the same F508-CFTR/ENaC co-injected oocytes are presented in panels A and B. Means ± S.E. are illustrated. ns, not significant.

Forskolin/IBMX-stimulated whole cell currents in oocytes expressing F508-CFTR alone increased 5.7-fold following the addition of 50 µM genistein (0.3 ± 0.1 µA (genistein) versus 1.7 ± 0.4 µA (+genistein); n = 23; p < 0.001; Fig. 5). Furthermore, the amiloride-insensitive component of the forskolin/IBMX/genistein-stimulated current in oocytes expressing F508-CFTR and -ENaC was 3.6-fold greater than the forskolin/IBMX/genistein-stimulated current measured in oocytes expressing F508-CFTR alone (1.7 ± 0.4 µA (F508-CFTR, n = 23) versus 6.2 ± 0.9 µA (F508-CFTR/ENaC, n = 24); p < 0.001; Figs. 5 and 6). A 2.4 ± 0.5-fold (p < 0.05) increase in forskolin/IBMX/genistein-stimulated amiloride-insensitive current in oocytes co-expressing F508-CFTR/ENaC (n = 12), when compared with oocytes expressing F508-CFTR alone (n = 13), was also observed when bath Na⁺ was replaced with the impermeant cation NMDG⁺ (Fig. 6). These data suggest that ENaC enhances the forskolin/IBMX/genistein-mediated activation of F508-CFTR. Furthermore, this enhanced activation of F508-CFTR is not dependent on ENaC-mediated Na⁺ transport.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   F508-CFTR/ENaC regulatory interactions restored by genistein do not require Na+ transport. Whole cell currents (100-mV holding potential) were determined in oocytes expressing F508-CFTR alone (open bars) or co-expressing F508-CFTR and -ENaC (closed bars). ND96, changes in whole cell currents that were not inhibited by 10 µM amiloride (presumed to be F508-CFTR-mediated) were determined following stimulation with 10 µM forskolin, 100 µM IBMX, 50 µM genistein in standard ND96 (NaCl) buffer. NMDG-Cl96, changes in whole cell currents were determined following stimulation with 10 µM forskolin, 100 µM IBMX, 50 µM genistein in oocytes bathed in a Na+-free solution (Na+ was replaced with NMDG). Data (means ± S.E.) are expressed relative to the mean change in whole cell current of oocytes injected with F508-CFTR alone. ns, not significant.

Whole cell amiloride-sensitive currents observed after stimulation by forskolin/IBMX/genistein in oocytes expressing ENaC and F508-CFTR (5.6 ± 0.6 µA, n = 24) were significantly lower than the amiloride-sensitive currents obtained after stimulation by forskolin/IBMX/genistein in oocytes expressing ENaC alone (9.0 ± 0.9 µA, n = 23, p = 0.003). Although amiloride-sensitive whole cell currents significantly increased in oocyte expressing ENaC alone following stimulation by forskolin/IBMX/genistein (Fig. 5), the amiloride-sensitive currents recorded in oocytes co-expressing ENaC and F508-CFTR remained stable following stimulation with forskolin/IBMX/genistein (5.2 ± 0.7 (forskolin/IBMX/genistein) versus 5.6 ± 0.6 µA (+forskolin/IBMX/genistein); n = 24; p is not significant). These data suggest that activation of F508-CFTR by forskolin/IBMX in the presence of genistein partially restores CFTR-mediated inhibition of ENaC activity.

Functional interactions between WT-CFTR and ENaC were examined in oocytes treated with genistein (Fig. 7). Whole cell currents measured in oocytes co-expressing WT-CFTR and ENaC in response to forskolin/IBMX/genistein were 13.0 ± 1.9 µA (n = 12; Fig. 7), 5.2-fold greater than forskolin/IBMX/genistein-stimulated currents in oocytes expressing CFTR alone (2.5 ± 0.5 µA, n = 13, p < 0.001). Furthermore, oocytes co-expressing WT-CFTR and ENaC responded to forskolin/IBMX/genistein with a modest but statistically insignificant reduction in amiloride-sensitive whole cell currents (Fig. 7; 3.2 ± 0.6 µA (forskolin/IBMX/genistein); 2.6 ± 0.5 µA (+forskolin/IBMX/genistein); n = 12; p is not significant).

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Regulatory interactions between WT-CFTR and -ENaC in the presence of genistein. WT-CFTR and ENaC were expressed separately or together in oocytes, and TEV was performed as described under "Experimental Procedures." Changes in whole cell currents (100-mV holding potential) after stimulation with 10 µM forskolin, 100 µM IBMX (closed bar) or 10 µM forskolin, 100 µM IBMX, 50 µM genistein that were not inhibited by 10 µM amiloride (light gray bars) are illustrated (A). Amiloride-sensitive whole cell currents (100-mV holding potential) were determined in oocytes co-expressing ENaC and WT-CFTR before (dark gray bars) and following (open bars) stimulation with 10 µM forskolin, 100 µM IBMX, 50 µM genistein (B). Data obtained from the same WT-CFTR/ENaC co-injected oocytes are presented in panels A and B. Means ± S.E. are illustrated. ns, not significant.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CFTR is a Cl-selective channel as well as a regulator of other epithelial channels and transport proteins. In cystic fibrosis, the absence of CFTR in the airway epithelia leads to hyperactive ENaC, which is hypothesized to lead to enhanced reabsorption of airway fluids and impaired mucociliary clearance (40). ENaC hyperactivity results in hyperpolarization of airway epithelia, as is evidenced by increases in the change in the transepithelial potential of the nasal airway in response to amiloride (41). In contrast, activation of CFTR in sweat ducts leads to an enhancement in ENaC activity, suggesting that regulatory interactions between CFTR and ENaC may be tissue-specific (9).

Previous studies have demonstrated that regulatory interactions between CFTR and ENaC in airway epithelia are replicated in Xenopus oocytes. Functional expression of WT-CFTR, but not F508-CFTR, is associated with an inhibition of functional ENaC expression in oocytes (31). Such CFTR-mediated inhibition of ENaC is associated with a decrease in Na⁺ channel open probability, not with changes in levels of ENaC mRNA or protein expression (8, 42, 43). The forskolin/IBMX-regulated inhibition of ENaC by CFTR does not require expression of the full-length CFTR protein. Inhibition of ENaC was observed when ENaC is co-expressed either with CFTR truncation mutants containing an intact first nucleotide binding domain (NBD-1) (44) or with a CFTR peptide fragment containing NBD-1 and the regulatory (R) domain (45). A similar NBD-1/R peptide fragment containing the G551D mutation did not demonstrate forskolin/IBMX-regulated inhibition of ENaC (45). Furthermore, Ji et al. (6) demonstrated that regulatory interactions between CFTR and ENaC were dependent on the presence of the carboxyl terminus of the -subunit and the amino terminus of the -subunit of ENaC.

It has been suggested that the ability of CFTR mutants to transport Cl correlates with their ability to inhibit ENaC (4, 46). Recent data have correlated levels of Cl transport in oocytes expressing WT-CFTR with the degree of ENaC inhibition; higher levels of Cl transport due to increased WT-CFTR expression are associated with increases in the extent of inhibition of ENaC (46). In contrast, our data with both WT-CFTR (Figs. 2 and 7) and F508-CFTR (Figs. 3 and 5) suggest that levels of Cl transport do not correlate with the degree of ENaC inhibition. For example, although we observed high levels of CFTR-mediated Cl currents in oocytes co-injected with CFTR and ENaC following treatment with forskolin/IBMX/genistein, we did not observe significant inhibition of ENaC in oocytes expressing ENaC and CFTR following activation of CFTR (Fig. 5). Furthermore, the enhanced activation of F508-CFTR by ENaC is not dependent on ENaC-mediated Na⁺ transport (Fig. 6). These data argue that Cl and Na⁺ conductances are not the sole determinants of CFTR/ENaC interactions.

Nagel and co-workers (47) suggested that the apparent CFTR-mediated inhibition of ENaC reflected a series resistor error. Our voltage clamp was configured to clamp the bath potential to 0 mV to diminish the likelihood of series resistor errors. Furthermore, in this configuration, we independently monitored oocyte membrane potential during our clamp protocol, and even at high conductances, we routinely observed membrane potentials that were <5% depolarized from our target holding potentials. In addition, the cAMP-regulated conductance in oocytes co-expressing CFTR and ENaC was significantly greater than that observed in oocytes expressing CFTR alone (Fig. 5), in agreement with recent observations (5-7). This apparent stimulation of CFTR by the presence of ENaC (Figs. 2 and 7) and the restoration of the stimulation of F508-CFTR by ENaC in the presence of genistein cannot be explained by series resistance errors (47). These data therefore provide further support for the presence of regulatory interactions between these proteins.

Our data are potentially important in devising strategies for effecting chemical or pharmacological repair of mutant CFTR function. F508-CFTR retains chloride transport properties but is mistrafficked and targeted for rapid intracellular degradation (22-24). It was hypothesized that the repair of trafficking would sufficiently restore CFTR function to ameliorate the CFTR deficiency in cystic fibrosis (48). In fact, F508-CFTR-mediated chloride transport can be restored by a number of physical or pharmacological maneuvers in vitro (49), and to a small extent in vivo, using the protein repair agent sodium 4-phenylbutyrate (50). However, in this clinical trial, there was no change in nasal epithelial amiloride-sensitive potential in vivo with 4-phenylbutyrate (50). This observation and the present data suggest that combinations of protein repair agents, such as 4-phenylbutyrate and genistein, may be necessary to restore F508-CFTR function in vivo.

	ACKNOWLEDGEMENT

We thank Dr. Shaohu Sheng for help with TEV experiments.

	FOOTNOTES

^* This work was supported by Grants DK56305 and DK58046 from the National Institutes of Health (to T. R. K. and to R. C. R., respectively).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.

^§ Recipient of a postdoctoral fellowship award from the Pennsylvania/Delaware Chapter of the American Heart Association.

^¶ Recipient of a postdoctoral fellowship award from the Cystic Fibrosis Foundation.

Recipient of a Cystic Fibrosis Foundation Leroy Matthews Individual Physician Scientist Award.

^¶¶ To whom correspondence should be addressed: Renal-Electrolyte Division, A919 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-647-3121; Fax: 412-648-9166; E-mail: kleyman@pitt.edu.

Published, JBC Papers in Press, December 28, 2001, DOI 10.1074/jbc.M111482200

	ABBREVIATIONS

The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; ENaC, epithelial sodium channel; IBMX, 3-isobutyl-1-methylxanthine; NMDG, N-methyl-D-glucamine; WT, wild type; TEV, two-electrode voltage clamp.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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