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J Biol Chem, Vol. 275, Issue 18, 13266-13274, May 5, 2000

Epithelial Sodium Channels Regulate Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels in Xenopus Oocytes^*

Qinshi Jiang^§, Jinqing Li^§, Rachael Dubroff, Yoon J. Ahn^§, J. Kevin Foskett^¶, John Engelhardt, and Thomas R. Kleyman^¶**

From the Departments of  Medicine and ^¶ Physiology, University of Pennsylvania and Veterans Affairs Medical Center, Philadelphia, Pennsylvania 19104-6144 and the  Department of Anatomy, University of Iowa, Iowa City, Iowa 52242

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl channel properties, regulates other ion channels. CFTR inhibits epithelial Na⁺ channel (ENaC) currents in many epithelial and nonepithelial cells. Because modulation of net NaCl reabsorption has important implications in extracellular fluid volume homeostasis and airway fluid volume and composition, we investigated whether this regulation was reciprocal by examining whether ENaC regulates CFTR. Co-expression of human (h) CFTR and mouse (m) ENaC in Xenopus oocytes resulted in a significant, 3.7-fold increase in whole-cell hCFTR Cl conductance compared with oocytes expressing hCFTR alone. The forskolin/3-isobutyl-1-methylxanthine-stimulated whole-cell conductance in hCFTR-mENaC co-injected oocytes was amiloride-insensitive, indicating an inhibition of mENaC following hCFTR activation, and it was blocked by DPC (diphenylamine-2-carboxylic acid) and was DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid)-insensitive. Enhanced hCFTR Cl conductance was also observed when either the - or -subunit of mENaC was co-expressed with hCFTR, but this was not seen when CFTR was co-expressed with the -subunit of mENaC. Single Cl channel analyses showed that both CFTR Cl channel open probability and the number of CFTR Cl channels detected per patch increased when hCFTR was co-expressed with mENaC. We conclude that in addition to acting as a regulator of ENaC, CFTR activity is regulated by ENaC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Epithelial cell layers are organized with discrete apical and basolateral plasma membrane domains that differ in protein composition. This polarized distribution of proteins at the cell surface allows for the transport of solutes across an epithelial cell layer in a directed, or vectorial, manner (1, 2). The transport of Na⁺ and Cl across an epithelial cell layer may result in the reabsorption or the secretion of solute. Epithelial Na⁺ channels (ENaCs)¹ are expressed on the apical membrane of selected epithelia that transport NaCl in an absorptive manner (3, 4). Activation of ENaC will result in increased rates of transepithelial NaCl reabsorption. Cl is transported across absorptive epithelia via paracellular or transcellular pathways. ENaCs are found in epithelial cells lining the distal nephron (5-7), airway, and alveolar epithelia in the lung (8-10), and distal colon (7, 10) and have a key role in the regulation of urinary Na⁺ reabsorption, extracellular fluid volume homeostasis, and control of blood pressure.

Several distinct epithelial Cl channels are expressed in apical plasma membranes of secretory epithelia. These channels facilitate active Cl secretion (11). Na⁺ is transported across secretory epithelia via paracellular pathways. Mutations in an epithelial, cAMP-regulated Cl channel, the cystic fibrosis transmembrane conductance regulator (CFTR), are responsible for the cystic fibrosis (CF) phenotype (12-14). As expected with mutations that lead to a loss of function of secretory Cl channels, reduced epithelial secretions of NaCl in the airway, gastrointestinal tract, and certain exocrine organs results in changes in the viscosity of fluids which are a hallmark of cystic fibrosis (15).

Certain hormones that activate CFTR through a protein kinase A-mediated pathway have also been reported to activate ENaCs (16-18). Whereas activation of CFTR in secretory epithelia will result in net NaCl secretion, activation of ENaC in absorptive epithelia will result in NaCl reabsorption. Selected epithelial cells, including those found in the airway, distal nephron, and sweat ducts, express both apical membrane ENaCs as well as CFTR. Activation of CFTR in these cells appears to be associated with Cl reabsorption (19-22). In this setting, activation of both CFTR and ENaC might result in dramatic increases in NaCl entry into cells and associated cell swelling. Under these circumstances, mechanisms to modulate NaCl entry are required to avoid large shifts in cell volume. Several groups, including ours, have demonstrated in selected epithelial as well as nonepithelial cell types that activation of CFTR is associated with an inhibition of ENaCs (23-29). This interaction provides a means by which epithelial cells that express both CFTR and ENaC can modulate net NaCl entry. CFTR inhibits Na⁺ channel open probability (16, 24, 30), although the mechanism(s) underlying this inhibition have not been clearly defined. In contrast, CFTR expression in sweat duct is required for activation of ENaC by cAMP-dependent protein kinase-mediated mechanisms (31). Given the importance of the coordinate regulation of apical plasma membrane Na⁺ and Cl channels, we examined whether ENaCs regulate CFTR. Our results demonstrate that forskolin and 3-isobutyl-1-methylxanthine (IBMX)-stimulated CFTR Cl currents are increased significantly in the presence of ENaC, suggesting that ENaCs regulate CFTR.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Reagents were purchased from vendors listed below or from Sigma Chemical Co.

Expression of hCFTR and mENaC in Xenopus Oocytes-- Human CFTR and mouse -, -, and ENaC cRNAs (32) were prepared using a cRNA synthesis kit (m-MESSAGE mMACHINE, Ambion Inc., Austin, TX). cRNA concentrations were measured spectroscopically, and the amounts of cRNA injected into oocytes are indicated under "Results" or in figure legends. Adult female Xenopus laevis were obtained from NASCO (Fort Atkinson, WI) and Xenopus-1 (Ann Arbor, MI). Oocytes were isolated and defolliculated by collagenase treatment. Oocytes were then placed in SOS (100 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM Hepes, pH 7.6) supplemented with 2.5 mM sodium pyruvate and 50 µg/ml gentamycin at 16 °C. Whole-cell current measurements were made 2 days after cRNA injection using the two-electrode voltage clamp method. Single 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 7.2, by NaOH), connected to a reference bath electrode by a 3 M KCl-agar bridge. Conventional two-electrode voltage clamp was performed at room temperature using an OC-725C oocyte clamp amplifier (Warner Instrument Corp., Hamden, CT) connected to a PowerMac 7100 via an ITC-16 interface (Instrutech Corp., Great Neck, NY). Pulse+PulseFit software (HEKA elektronik, Lambrecht, Germany) was used to ramp the applied transmembrane potential (V_m) at 10-s intervals from 60 to 60 mV (with reference to the bath) at a rate of 16 mV/s. V_m was clamped at the prestimulation reversal potential between the voltage ramps. Transmembrane current (I) and V_m were digitized at 200 Hz during the voltage ramps and recorded directly onto a hard disk. To reduce signal noise, a fifth-order polynomial was fitted to the raw I-V_m curve from each voltage ramp. Ion replacement studies were performed by replacing NaCl in the ND96 solution either with N-methyl-D-glucamine (NMDG) Cl to produce a Na⁺-free extracellular environment or with Na⁺ methanesulfonate to produce a Cl-free extracellular environment. The differences in the whole-cell conductance measured in the presence and in the absence of 100 µM amiloride were used to define the amiloride-sensitive Na⁺ conductance that is carried by ENaC. Activation of CFTR was accomplished by perfusion of the oocyte with buffers supplemented with 100 µM IBMX and 10 µM forskolin for 30 min. In all experiments, CFTR Cl conductance was defined as the difference in conductance measured 30 min after perfusion with 10 µM forskolin and 100 µM IBMX (forskolin/IBMX) and the conductance measured prior to forskolin/IBMX stimulation. When indicated, 50 µM DPC or 200 µM DIDS was added to the perfusion buffer. The whole-cell conductance and reversal potential (E_rev) were evaluated from the slope and x intercept, respectively, of the background-corrected I-V_m curve using Igor Pro software (WaveMetrics, Lake Oswego, OR). Whole-cell currents were measured at 50 mV. Single channel recordings were performed in the cell-attached configuration. Pipette and bath solutions were Na⁺-free and contained (in mM): 96 NMDG Cl, 1 KCl, 0.2 CaCl₂, 5.8 MgCl₂, 10 Hepes, pH adjusted to 7.2 with NMDG. Electrical signals were amplified using a Dagan PC-one amplifier (Dagan, Minneapolis, MN), digitized by DigiData 1200 (Axon Instruments, Foster City, CA), and stored on disk. Pclamp7 (Axon Instruments) was used for data acquisition and analysis. All data were collected at room temperature and were filtered at 300 Hz. Closed states of less than 20 ms and open states of less than 50 ms were ignored in analyses of open probability. Continuous recording of greater than 6-min duration with a minimum of 50 opening events was used to determine open probability and the number of channels within a patch. Recordings were performed between 30 min and 3 h after stimulation with 10 µM forskolin and 100 µM IBMX.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of hCFTR and mENaC in Xenopus Oocytes-- The Xenopus oocyte expression system was used to examine the functional expression of hCFTR and mENaC because this system readily allows for the expression and detection of a variety of exogenous ion channels at the oocyte plasma membrane. Oocytes injected with 10 ng of hCFTR cRNA were bathed in a solution containing 10 µM forskolin and 100 µM IBMX to activate endogenous protein kinase A and hCFTR. Whole-cell conductance was monitored by two-electrode voltage clamp (Fig. 1A). An 11-fold increase in whole-cell conductance, from 6.5 ± 1.5 µS to 73 ± 8 µS was observed in response to the addition of forskolin and IBMX (Fig. 1B, n = 38, p < 0.01). No forskolin/IBMX-activated conductance was observed in water-injected oocytes (data not shown).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Functional expression of hCFTR in Xenopus oocytes. Whole-cell conductances were determined in oocytes injected with 10 ng of hCFTR cRNA prior to (Fsk/IBMX; control) and after (+Fsk/IBMX) maximum stimulation with 10 µM forskolin and 100 µM IBMX. Panel A shows a representative response of whole-cell conductance to activation of hCFTR with forskolin and IBMX. In panel B are representative current tracings obtained from the voltage ramp protocol which were used to determine whole-cell conductances. Broken line, control; solid line, +Fsk/IBMX. No change in whole-cell conductance was observed in response to forskolin and IBMX in water-injected oocytes (not shown). The average change in whole-cell conductance in hCFTR cRNA-injected oocytes 30 min after stimulation with forskolin/IBMX is shown in panel C. Data are expressed as the mean ± S.E. (n = 38). An 11-fold increase in whole-cell conductance, from 6.5 ± 1.5 µS (Fsk/IBMX) to 73 ± 8 µS (+Fsk/IBMX) was observed in response to forskolin and IBMX.

We previously isolated and characterized cDNAs encoding the -, -, and -subunits of the mouse ENaC (32). Oocytes injected with mENaC cRNAs (3.3 ng/subunit) expressed an amiloride-sensitive whole-cell conductance of 75 ± 12 µS (Fig. 2). An amiloride-sensitive whole-cell conductance was not observed in water-injected oocytes (data not shown).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Functional expression of mENaCs in Xenopus oocytes. Oocytes were injected with ENaC cRNAs (3.3 ng/subunit) and whole-cell conductances were measured in the absence (amiloride) or presence of 100 µM amiloride. Panel A shows a representative response of whole-cell conductance and the response to amiloride. Panel B shows representative current tracings obtained from the voltage ramp protocol which were used to determine whole-cell conductances. Solid line, control; broken line, presence of amiloride. An amiloride-sensitive whole-cell conductance of 75 ± 12 µS was observed (panel C). Data are expressed as the mean ± S.E. (n = 12).

Co-expression of ENaC and CFTR in Xenopus Oocytes-- It was reported previously that co-expression of CFTR and ENaC in Xenopus oocytes inhibited ENaC-mediated Na⁺ conductance (29, 33, 34). We used the oocyte expression system to examine whether ENaCs regulate CFTR. Oocytes were co-injected with human CFTR (10 ng) and mouse ENaC (3.3 ng/subunit) cRNAs, and whole-cell conductances were determined by two-electrode voltage clamp (Fig. 3A). As shown in Fig. 3B, an amiloride-sensitive conductance of 81 ± 11 µS was observed in the absence of forskolin/IBMX (n = 38). However, after CFTR had been fully activated by forskolin/IBMX, no amiloride-sensitive whole-cell conductance was observed (268 ± 29 µS pre-amiloride; 274 ± 29 µS, post-amiloride, (n = 38); Fig. 3C). In addition, replacement of Na⁺ with the impermeant cation NMDG in the bath solution did not significantly affect the forskolin/IBMX-stimulated whole-cell conductance (241 ± 29 µS, Na⁺ bath; 261 ± 32 µS, NMDG bath (n = 9); Fig. 4), confirming the functional inhibition of ENaC expression after CFTR activation, in agreement with previous observations (29, 33, 34).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Co-expression of hCFTR and mENaC in Xenopus oocytes. Oocytes were injected with hCFTR (10 ng) and ENaC (3.3 ng/subunit) cRNAs, and whole-cell conductances were measured in response to 100 µM amiloride and to 10 µM forskolin with 100 µM IBMX. Panel A is a representative response of the whole-cell conductance to amiloride and to forskolin/IBMX. The additions of amiloride and of forskolin/IBMX to the bath solution are indicated. Amiloride was washed out of the bath solution when oocytes were initially exposed to IBMX and forskolin (+Fsk/IBMX). Amiloride was added again to the bath solution at a later time (+Fsk/IBMX/amiloride). Panel B shows representative current tracings obtained from the voltage ramp protocol which were used to determine whole-cell conductances. Solid line, control; short-dashed line, +amiloride; dash-dotted line, +Fsk/IBMX; long-dashed line, +Fsk/IBMX/amiloride. Before activation of CFTR with forskolin/IBMX, the average change in whole-cell conductance in response to amiloride was 81 ± 11 µS (panel C). After stimulation with forskolin/IBMX (30 min), no inhibition of whole-cell conductance was observed in response to amiloride (panel D). The average forskolin/IBMX-stimulated whole-cell conductance was 268 ± 29 µS before amiloride and rose slightly to 274 ± 29 µS after the addition of 100 µM amiloride to the bath solution. Data are expressed as the mean ± S.E. (n = 38).

View larger version (9K): [in this window] [in a new window]   Fig. 4.   Replacement of Na+ with the impermeant cation NMDG in the bath solution did not alter forskolin/IBMX-activated whole-cell conductance. Forskolin/IBMX-activated whole-cell conductances were measured in oocytes injected with 10ng of CFTR cRNA or co-injected with CFTR and ENaC (3.3 ng/subunit). Replacement of NaCl (white bars) in the bath solution with NMDG Cl (black bars) did not significantly alter forskolin/IBMX-stimulated whole-cell conductances (p > 0.2). Data are expressed as the mean ± S.E. (n = 9).

Surprisingly, the forskolin/IBMX-stimulated whole-cell conductance in oocytes co-injected with hCFTR and mENaC cRNAs (268 ± 29 µS) was 3.7 times as large as that observed in oocytes injected with hCFTR cRNA alone (73 ± 8 µS; n = 38; p < 0.01; Fig. 5). Additional experiments were performed to determine whether the forskolin/IBMX-stimulated whole-cell conductance in oocytes co-injected with hCFTR and mENaC cRNAs was mediated primarily by hCFTR. Replacement of Cl with the impermeant anion methanesulfonate (Fig. 6 (n = 10)) in the bath solution resulted in a significant transient increase in the inward whole-cell current when the voltage was clamped at a hyperpolarizing potential (50 mV). The maximum increase of the inward current was achieved within 30 s after ion replacement (data not shown). Furthermore, the forskolin/IBMX-stimulated whole-cell conductance in oocytes co-injected with hCFTR and mENaC cRNAs was blocked by 50 µM DPC (Fig. 7A (n = 10)) but was insensitive to 200 µM DIDS (Fig. 7B (n = 10)). Taken together, these results indicate that the forskolin/IBMX-stimulated conductance in oocytes co-injected with hCFTR and mENaC cRNAs was largely mediated by hCFTR (35-37).

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Forskolin/IBMX-activated conductances in oocytes expressing hCFTR or co-expressing hCFTR and mENaC cRNAs. Oocytes were injected with 10 ng of hCFTR cRNA or were co-injected with 10 ng of hCFTR cRNA and 10 ng of mENaC (3.3 ng/subunit) cRNAs. Whole-cell conductances were measured 30 min after the addition of 10 µM forskolin and 100 µM IBMX to the bath solution. The forskolin/IBMX-stimulated whole-cell conductance in oocytes co-injected with hCFTR and mENaC cRNAs (268 ± 29 µS) was 3.7 times as large as that observed in oocytes injected with hCFTR cRNA alone (73 ± 8 µS). Data are expressed as the mean ± S.E. (n = 38), p < 0.01.

View larger version (9K): [in this window] [in a new window]   Fig. 6.   Effects of anion substitution on forskolin/IBMX-activated whole-cell currents. Oocytes were injected with 10 ng of hCFTR or co-injected with 10 ng of hCFTR and 10 ng of mENaC (3.3 ng/subunit). Whole-cell currents were measured with the voltage clamped at a hyperpolarizing potential (50 mV) 30 min after the addition of 10 µM forskolin and 100 µM IBMX to the bath solution. Currents were measured in a NaCl bath solution (white bars) and approximately 30 s after perfusion of a solution in which NaCl was replaced with Na+ methanesulfonate (black bars). Significant increases in inward currents were observed in oocytes expressing hCFTR alone when extracellular NaCl was replaced Na+ methanesulfonate (4.3 ± 0.5 µA (NaCl), 7.8 ± 0.9 µA (Na+ methanesulfonate), n = 10, p < 0.005). Significant increases in inward currents were also observed in oocytes co-expressing hCFTR and mENaC, when extracellular NaCl was replaced Na+ methanesulfonate (11.0 ± 1.1 µA (NaCl), 20.4 ± 1.8 µA (Na+ methanesulfonate), n = 10, p < 0.001). Data are expressed as the mean ± S.E.

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Effects of anion channel inhibitors DPC or DIDS on forskolin/IBMX-stimulated whole-cell conductances. Xenopus oocytes were injected with 10 ng of hCFTR or co-injected with 10 ng of hCFTR and 10 ng of mENaC (3.3 ng/subunit). After stimulation with 10 µM forskolin and 100 µM IBMX (white bars, panel A), whole-cell conductances were measured before and after the addition of 50 µM DPC (black bars; panel A) or 200 µM DIDS (panel B) to the bath solution. Whole-cell conductances in oocytes expressing hCFTR (73.2 ± 5.7 µS (DPC), 18.9 ± 6.2 µS (+DPC), n = 10, p < 0.01) or co-expressing hCFTR and mENaC cRNAs (269 ± 37 µS (DPC), 56 ± 13 µS (+DPC), n = 10, p < 0.01) were significantly inhibited by 50 µM DPC. Whole-cell conductances in oocytes expressing hCFTR (89.2 ± 12.5 µS (DIDS), 85.2 ± 11.8 µS (+DIDS), n = 10, p > 0.2) or co-expressing hCFTR and mENaC cRNAs (241 ± 29 µS (DIDS), 226 ± 23 µS (+DIDS), n = 10, p > 0.2) were not inhibited by 200 µM DIDS. In panel B, the white bars indicate CFTR, and the black bars indicate CFTR-ENaC. Data are expressed as the mean ± S.E.

The enhancement of hCFTR Cl conductance observed when hCFTR was co-expressed with mENaC in Xenopus oocytes was dependent on the amount of mENaC cRNA injected. Oocytes co-injected with 10 ng of hCFTR and 2 ng (0.67 ng/subunit) of mENaC cRNAs exhibited a forskolin/IBMX-stimulated conductance (162 ± 27 µS; n = 8) that was only 1.6-fold greater that the forskolin/IBMX-stimulated conductance observed in oocytes injected with 10 ng of hCFTR alone (101 ± 19; n = 8; p < 0.05, CFTR versus CFTR/ENaC; Fig. 8).

View larger version (7K): [in this window] [in a new window]   Fig. 8.   CFTR Cl conductance is enhanced by ENaC. Xenopus oocytes were injected with 10 ng of hCFTR or co-injected with 10 ng of hCFTR and 2 ng of mENaC (0.67 ng/subunit). Whole-cell conductances were measured 30 min after the addition of 10 µM forskolin and 100 µM IBMX to the bath solution. Oocytes co-injected with hCFTR and mENaC cRNAs exhibited a forskolin/IBMX-stimulated conductance (162 ± 27 µS; n = 8) that was 1.6-fold greater than the forskolin/IBMX-stimulated conductance observed in oocytes injected with 10 ng of hCFTR alone (101 ± 19; n = 8; p < 0.05).

Analyses of hCFTR Single Channel Activity in Oocytes Injected with hCFTR cRNA or Co-injected with hCFTR and mENaC cRNAs-- The forskolin/IBMX-stimulated hCFTR Cl conductance increased significantly when hCFTR was co-expressed with mENaC in Xenopus oocytes compared with oocytes expressing hCFTR alone. Increases in whole-cell hCFTR Cl conductance could result from increases in hCFTR single channel conductance, hCFTR open probability, or in the number of hCFTR Cl channels expressed at the plasma membrane. Human CFTR single channel Cl conductance was determined by cell-attached patch clamp analyses of forskolin/IBMX-stimulated hCFTR Cl channels expressed in the presence or absence of mENaC. Analyses of hCFTR single channel current/voltage relationships revealed similar slope conductances (6.7 pS (hCFTR) and 6.9 pS (hCFTR/mENaC)) (Fig. 9). Pipette resistance was similar in both groups (30.0 ± 5.9 megohms (hCFTR, n = 8); 35.1 ± 4.3 megohms (hCFTR/mENaC, n = 9); p > 0.4). Eighteen hCFTR Cl channels were observed in 16 patches in oocytes injected with hCFTR alone, whereas 42 hCFTR Cl channels were observed in 16 patches in oocytes co-expressing hCFTR and mENaC (p < 0.002), suggesting that the number of hCFTR Cl channels expressed at the plasma membrane was increased when hCFTR was co-expressed with mENaC. The average open probability was 0.11 ± 0.02 (n = 11) in oocytes expressing hCFTR alone. This was significantly less than the average open probability of 0.27 ± 0.04 (n = 10) observed in oocytes co-expressing hCFTR and mENaC (p < 0.004, hCFTR versus hCFTR/mENaC (Fig. 9D)). These data suggest that increases in hCFTR Cl whole-cell conductance, when hCFTR is co-expressed with mENaC, occur in conjunction with an increase in both hCFTR open probability and in the number of hCFTR Cl channels expressed at the plasma membrane.

View larger version (33K): [in this window] [in a new window]   Fig. 9.   Analyses of CFTR Cl channels by cell-attached patch clamp. Oocytes were injected with hCFTR cRNA (panel A, left tracing) or co-injected with hCFTR and mENaC cRNAs (panel A, right tracing). The pipette solution contained 96 mM NMDG Cl. The closed state is as indicated in (c). Recordings were performed at 90 mV. Panel B is a representative current tracing from a patch containing multiple hCFTR Cl channels in an oocyte co-injected with hCFTR and mENaC cRNAs (recording was performed at +90 mV to reduce brief channel closures (channel flickering)). Multiple CFTR channels were observed in most patches from oocytes co-injected with hCFTR and mENaC cRNAs. Panel C illustrates single CFTR Cl channel I/V curves from oocytes injected with hCFTR cRNA alone (black squares; n = 3-7 channels) or co-injected with hCFTR and mENaC cRNAs (black diamonds; n = 4-8 channels). Holding potentials were increased stepwise with a 30-mV increment from 90 mV to +90 mV. A linear current voltage relationship was observed for both groups. Slope conductances were 6.7 and 6.9 pS for hCFTR-injected and hCFTR/mENaC co-injected oocytes, respectively. hCFTR Cl channel open probability was determined at holding potentials of 60 mV and is illustrated in panel D. The average open probability was 0.11 ± 0.02 (n = 11) in oocytes expressing hCFTR alone and was 0.27 ± 0.04 (n = 10) in oocytes co-expressing hCFTR and mENaC (p < 0.004).

Effects of Individual mENaC Subunits on CFTR Cl Conductance-- Epithelial Na⁺ channels are composed of at least three structurally related subunits (38). Previous studies demonstrated that amiloride-sensitive Na⁺ currents were detectable in oocytes injected with ENaC cRNA, but at levels that are ~100-fold less than Na⁺ currents observed in oocytes injected with ENaC cRNAs (38, 39). No detectable amiloride-sensitive Na⁺ currents were observed in oocytes injected with ENaC or ENaC cRNAs (38). We examined whether co-expression of CFTR with individual ENaC subunit cRNAs altered the levels of functional CFTR expression (Fig. 10). The forskolin/IBMX-stimulated whole-cell conductance was 49 ± 10 µS in oocytes injected with 5 ng of hCFTR cRNA alone. The forskolin/IBMX-stimulated whole-cell conductance increased to 109 ± 23 µS in oocytes co-injected with 5 ng of hCFTR and of 5 ng mENaC cRNAs (p < 0.03, hCFTR versus hCFTR/mENaC (n = 7)) and to 90 ± 16 µS in oocytes co-injected with hCFTR and mENaC cRNAs (p < 0.05, hCFTR versus hCFTR/mENaC (n = 7)). These data suggested that co-expression of hCFTR with individual mENaC subunits (mENaC or mENaC) enhanced functional hCFTR expression in Xenopus oocytes. Forskolin/IBMX-stimulated whole-cell conductances measured in oocytes injected with hCFTR and mENaC cRNAs (62 ± 13 µS) or in oocytes injected with hCFTR cRNA alone were not significantly different (p > 0.2, hCFTR versus hCFTR/mENaC (n = 7)). These data suggest that the enhancement of functional hCFTR expression by mENaC in Xenopus oocytes is subunit-specific. Moreover, the absence of an effect of mENaC on functional hCFTR expression suggested that the functional activation of hCFTR by mENaC or by selected mENaC subunits is a specific effect and is not simply related to co-injection of an irrelevant cRNA with hCFTR cRNA.

View larger version (20K): [in this window] [in a new window]   Fig. 10.   Forskolin/IBMX-activated whole-cell conductances in oocytes expressing hCFTR or co-expressing hCFTR and individual mENaC subunit cRNAs. Oocytes were injected with 5 ng of hCFTR cRNA alone or co-injected with 5 ng of hCFTR and 5 ng of -, -, or ENaC cRNA. Whole-cell conductances were measured 30 min after the addition of 10 µM forskolin and 100 µM IBMX to the bath solution. The forskolin/IBMX-stimulated whole-cell conductance in oocytes injected with 5 ng of hCFTR cRNA was 49 ± 10 µS (n = 7). The forskolin/IBMX-stimulated whole-cell conductance in oocytes co-injected with hCFTR and mENaC cRNAs (109 ± 23 µS) was 2.2 times as large as that observed in oocytes injected with hCFTR cRNA alone (n = 7, p < 0.03). The forskolin/IBMX-stimulated whole-cell conductance was 90 ± 16 µS in oocytes co-injected with 5 ng of hCFTR and of 5 ng of mENaC cRNAs (n = 7, p < 0.05). Similar forskolin/IBMX-stimulated whole-cell conductances were observed in oocytes expressing hCFTR and co-expressing hCFTR and mENaC (62 ± 13 µS, n = 7, p > 0.2). Data are expressed as mean ± S.E.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In addition to its primary function as a Cl channel, CFTR functions as a regulator of several other epithelial ion channels (26, 40), including an outwardly rectifying Cl channel (41-44), the epithelial Na⁺ channel (23-27, 29-31), and a renal K⁺ channel (45-47). Through these interactions, it appears that CFTR has an important role in regulation of the content and volume of fluids in airways, intestines, pancreas, sweat ducts, and renal tubules.

Our results confirm previous studies demonstrating that the functional expression of CFTR is associated with an inhibition of functional ENaC expression in Xenopus oocytes (25, 29, 34). The mechanisms by which CFTR regulates ENaC are being defined. CFTR-mediated inhibition of ENaC is associated with a decrease in Na⁺ channel open probability (16, 24, 30) and not with changes in levels of ENaC mRNA or protein expression (8, 24). CFTR-mediated inhibition of ENaC may involve autocrine or paracrine signaling and G protein-coupled signaling mechanisms, changes in intracellular Cl concentration that may alter ENaC activity, or perhaps direct interactions between CFTR and ENaC (25, 48, 49). The inhibition of ENaC by CFTR may be dependent on an intact first nucleotide binding domain and does not require expression of a full-length CFTR protein (33). The ability of CFTR mutants to transport Cl correlates with their ability to inhibit ENaC; i.e. CFTR mutants that express low levels of Cl currents in Xenopus oocytes are inefficient in inhibiting functional expression of ENaC (34); however, CFTR-mediated inhibition of ENaC is not necessarily dependent on the magnitude of Cl current (29).

The present study demonstrates that interactions between the CFTR and ENaC are more complex than suggested previously. 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 a recent observation by Chabot and co-workers (29). This enhanced conductance observed in oocytes co-expressing CFTR and ENaC was blocked by the CFTR inhibitor DPC (35, 37), was insensitive to DIDS, a stilbene derivative that does not block CFTR but inhibits other anion transporters (35, 36), and was not dependent on the presence of a permeant extracellular cation (i.e. Na⁺, see Figs. 4 and 7). These data suggest that this enhanced conductance in forskolin/IBMX-stimulated oocytes co-expressing CFTR and ENaC is a result of increased activity of CFTR Cl channels.

The increase in CFTR Cl conductance observed in oocytes co-expressing -mENaC was dependent on the amount of ENaC cRNA injected into oocytes (Figs. 5 and 8). Co-expression of CFTR with individual ENaC subunits enhanced Cl conductance but in a subunit-specific manner (Fig. 10). An enhanced activation of CFTR occurred with co-expression of either the - or -subunits of ENaC but not with the  subunit. This enhanced activation of CFTR observed with individual - or -mENaC subunits (2.2- fold and 1.8-fold, respectively) was considerably less than that observed with -mENaC (3.7-fold). The enhanced functional expression of CFTR Cl channels which we observed with co-expression of ENaC was not dependent on expression of functional Na⁺ channels. Low levels of expression of amiloride-sensitive Na⁺ currents have been reported in Xenopus oocytes expressing ENaC alone (38, 39), and no amiloride-sensitive Na⁺ currents have been observed in oocytes expressing ENaC alone (38). When ENaC subunits are expressed individually in Xenopus oocytes, the subunits are found primarily within intracellular compartments (50). These results suggest that the co-expression of individual ENaCs with CFTR might result in increased functional CFTR expression by altering CFTR maturation or intracellular trafficking, leading to an enhanced cell surface expression of CFTR (see below).

An increase of CFTR Cl conductance was not observed in oocytes co-expressing CFTR and ENaC, suggesting that specific domains within the - or -subunits of ENaC are likely important in conferring the ability of ENaC to regulate the functional expression of CFTR. Consistent with this notion, previous work examining ENaC interactions with CFTR using a yeast two-hybrid approach also suggested that limited domains within ENaC were capable of binding CFTR through its NBD1/R domain (25). Additional studies are needed to define the mechanism(s) by which - or ENaC alters functional CFTR Cl channel expression.

The increase in the whole-cell CFTR Cl conductance observed in oocytes co-expressing CFTR and ENaC reflects changes in one or more of the following variables: single channel conductance, open probability, and the number of channels at the cell surface. Single channel analyses of CFTR Cl channels indicated that CFTR single channel conductances were nearly identical in oocytes expressing CFTR alone and in oocytes co-expressing CFTR and ENaC (Fig. 9). The open probability of CFTR Cl channels was increased significantly when CFTR was co-expressed with ENaC. However, this 2.5-fold increase in CFTR Cl channel open probability did not account for the 3.7-fold increase in whole-cell conductance which we observed when CFTR Cl channels were co-expressed with ENaC, suggesting that the number of CFTR Cl channels at the plasma membrane of the oocytes was increased as well. Consistent with this notion, a significantly greater number of CFTR Cl channels was observed in cell-attached patches of oocytes co-expressing CFTR and ENaC (42 channels in 16 patches) than were observed in oocytes expressing CFTR alone (18 channels in 16 patches). Additional studies using biochemical approaches to determine CFTR surface expression are needed to confirm this observation and to determine mechanisms by which ENaC enhances plasma membrane expression of CFTR. Increases in levels of plasma membrane expression of CFTR might arise from changes in CFTR biosynthesis, maturation, recruitment to the plasma membrane, or retrieval from the plasma membrane.

Although the concept that one ion channel species (i.e. CFTR) regulates other ion channels is well established, it is unclear whether this phenomenon is broad or limited in scope. Coordinate regulation of Na⁺ and Cl transport proteins in the apical plasma membrane of epithelia provides a mechanism by which epithelial cells can modulate rates of NaCl reabsorption in response to agonists. This is particularly important within selected regions of the lung, sweat duct, salivary gland, and kidney sites where epithelial cells reabsorb NaCl and express both ENaC and CFTR (19-22). Protein kinase A-mediated activation of CFTR in epithelia expressing both CFTR and ENaC results in increased transepithelial Cl reabsorption as well as an inhibition of epithelial Na⁺ channels as a result of reduced Na⁺ channel open probability (16, 19-22, 24). In the absence of CFTR expression, ENaC is activated by agonists that activate protein kinase A because of increases in Na⁺ channel open probability or in the number of channels expressed at the plasma membrane (16, 17, 24, 51). We propose that the regulatory interactions between CFTR Cl channels and epithelial Na⁺ channels provide a mechanism by which selected epithelia can modulate net NaCl entry across the apical membrane in response to agonists that would otherwise activate both CFTR and ENaC. In this regard, an enhanced activity of CFTR might augment CFTR-mediated inhibition of ENaC. These interactions provide a cellular mechanism by which certain epithelia limit increases in cell volume which might otherwise occur if CFTR and ENaC were activated in parallel. In summary, our results indicate that, in addition to acting as an ion channel and a regulator of other ion channels (including ENaC), CFTR activity is regulated by ENaC. Simply stated, Na⁺ channels are not innocent bystanders; ENaCs are active participants in this regulatory phenomenon.

	ACKNOWLEDGEMENTS

We thank Dr. Don-on Mak for help with two-electrode voltage clamp experiments and related data analyses.

	FOOTNOTES

^* This work was supported in part by grants from the Cystic Fibrosis Foundation (to J. K. F. and T. R. K.) and by National Institutes of Health Grant DK56305.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.

^§ Recipients of postdoctoral fellowship awards from the Cystic Fibrosis Foundation.

^** To whom correspondence should be addressed: Renal Division, University of Pennsylvania, 700 CRB, 415 Curie Blvd., Philadelphia, PA 19104-6144. Tel.: 215-573-1848; Fax: 215-898-0189; E-mail: kleyman@mail.med.upenn.edu.

	ABBREVIATIONS

The abbreviations used are: ENaC(s), epithelial Na⁺ channel(s); CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; IBMX, 3-isobutyl-1-methylxanthine; V_m, transmembrane potential; I, transmembrane current; NMDG, N-methyl-D-glucamine; DPC, diphenylamine-2-carboxylic acid; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; E_rev, reversal potential; pS, picosiemens; µS, microsiemens.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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