Reciprocal protein kinase A regulatory interactions between cystic fibrosis transmembrane conductance regulator and Na+/H+ exchanger isoform 3 in a renal polarized epithelial cell model

Although Cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to regulate the activity of NHE3, the potential reciprocal interaction of NHE3 to modulate the protein kinase A (PKA)-dependent regulation of CFTR in epithelial cells is still unknown. In the present work, we describe experiments to define the interactions between CFTR and NHE3 with the regulatory, scaffolding protein, NHERF that organize their PKA-dependent regulation in a renal epithelial cell line that expresses endogenous CFTR. The expression of rat NHE3 significantly decreased PKA-dependent activation of CFTR without altering CFTR expression, and this decrease was prevented by mutation of either of the two rat NHE3 PKA target serines to alanine (S552A or S605A). Inhibition of CFTR expression by antisense treatment resulted in an acute decrease in PKA-dependent regulation of NHE3 activity. CFTR, NHE3, and ezrin were recognized by NHERF-2 but not NHERF-1 in glutathione S-transferase pull-down experiments. Ezrin may function as a protein kinase A anchoring protein (AKAP) in this signaling complex, because blocking the binding of PKA to an AKAP by incubation with the S-Ht31 peptide inhibited the PKA-dependent regulation of CFTR in the absence of NHE3. In the A6-NHE3 cells S-Ht31 blocked the PKA regulation of NHE3 whereas it now failed to affect the regulation of CFTR. We conclude that CFTR and NHE3 reciprocally interact via a shared regulatory complex comprised of NHERF-2, ezrin, and PKA.

Although CFTR has been shown to regulate the activity of NHE3, the potential reciprocal interaction of NHE3 to modulate the PKA-dependent regulation of CFTR in epithelial cells is still unknown. In the present work, we describe experiments to define the interactions between CFTR and NHE3 with the regulatory, scaffolding protein, NHERF that organize their PKA-dependent regulation in a renal epithelial cell line that expresses endogenous CFTR. The expression of rat NHE3 significantly decreased PKA-dependent activation of CFTR without altering CFTR expression and this decrease was prevented by mutation of either of the two rat NHE3 PKA target serines to alanine (S552A or S605A). Inhibition of CFTR expression by antisense treatment resulted in an acute decrease in PKA-dependent regulation of NHE3 activity. CFTR, NHE3 and ezrin were recognized by NHERF-2 but not NHERF-1 in GST pull-down experiments. Ezrin may function as a protein kinase A anchoring protein (AKAP) in this signaling complex since blocking the binding of PKA to an AKAP by incubation with the S-Ht31 peptide inhibited the PKA-dependent regulation of CFTR in the absence of NHE3. In the A6-NHE3 cells S-Ht31 blocked the PKA regulation of NHE3 while it now failed to affect the regulation of CFTR. We conclude that CFTR and NHE3 reciprocally interact via a shared regulatory complex comprised of NHERF-2, ezrin and PKA.
Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP activated Clchannel expressed in the luminal membrane of secretory and reabsorptive epithelia (1). In addition to transepithelial chloride transport, CFTR has been shown to influence a large number of cell functions including ion transporters such as outwardly rectifying chloride channels, amiloride-sensitive epithelial sodium channels and renal outer medullary potassium channels (2). Initial hypotheses concerning CFTR function suggested that it may function primarily as a global conductance regulator thus magnifying its role in normal cell function (3). Accordingly, defects in CFTR causing the disease cystic fibrosis (CF) lead not only to disturbances of chloride secretion but also of the transport of other electrolytes. In this context, CFTR has been demonstrated to affect both intracelluar and extracellular pH regulation by alterations in either HCO 3secretion via activation and increased expression of the Cl -/HCO 3antiporter (4,5) or by alterations in Na + /H + exchanger (NHE) activity (6,7).
The mechanisms by which CFTR exerts its regulatory role over other epithelial ion transporters is still incompletely understood although several hypotheses are currently being explored. These include indirect regulation via release of ATP through CFTR with a subsequent autocrine activation of luminal purinoceptors and modulation of intracellular calcium which in turn could regulate other transporters (8,9). Another regulatory mechanism involves direct intramembrane or cytoplasmic protein-protein interaction. PDZ domain proteins are emerging as important organizing centers for these regulatory complexes and these scaffold-based regulatory proteins are localized to specific sites in polarized epithelial cells. It has been shown that the PKA-dependent regulation of the NHE isoform located in the apical membrane (NHE3) is mediated via the scaffolding protein, NHE-Regulatory Factor (NHERF), whose PDZ2 domain interacts with the cytoplasmic end of NHE3 (10,11). In vitro binding studies have demonstrated that CFTR can bind to the PDZ1 domain of the NHERF with an intracellular C-terminal domain ending in D-S/T-x-L (12)(13)(14) providing a potential mechanism for the CFTR modulation of the regulation of other membrane proteins such as the NHE3. Recently, using CFTR and NHE3 cotransfection in fibroblasts, it was demonstrated that CFTR modifies the PKAdependent regulation of NHE3 via interaction with the NHERF-1 isoform (7). While some aspects of the mechanism underlying the CFTR-dependent modulation of NHE3 have been established, the existence of the potential reciprocal modulation of PKA-dependent regulation of CFTR by NHE3 remains undescribed. Further, as recently discussed (15) the results of many of the studies on regulation of CFTR have been conducted in non-polarized cells and/or by over-expression of CFTR and, as such, it may be difficult to draw conclusions about the regulation of CFTR during normal epithelial cell function.
The objective of the present work was to elucidate the dynamics and mechanism(s) of the reciprocal alteration of PKA-dependent regulation of CFTR and NHE3 in epithelial cells. To establish a working model of the dynamic interactions in a polarized epithelial cell, we have used an epithelial renal cell line, A6, which when grown on permeable filters forms polarized monolayers with a high transepithelial resistance, an amiloride sensitive sodium transport (16)(17)(18), and that also endogenously expresses wild-type CFTR (19). We have transfected this cell line with: 1) the rat NHE3 (A6-NHE3) which is functionally expressed on the apical membrane and displays the PKA and PKC regulatory pattern charateristic for this isoform when endogenously expressed in epithelial cells (20); 2) antisense oligonucleotides to suppress, transiently, CFTR expression and 3) two mutants of rat NHE3 in which the PKA target serines 552 or 605 have been mutated to alanine. Using these epithelial cell lines, we show that 1) the expression of CFTR is necessary for the PKA-dependent regulation of NHE3 while 2) the expression of NHE3 reduces the PKA-dependent regulation of CFTR; 3) mutation of the PKA substrate serines 552 or 605 of NHE3 relieves this repression of CFTR regulation; 4) both CFTR and NHE3 associate with the NHERF-2 and not the NHERF-1 isoform.
Cell culture A6/C1 cells used for transfection are a subclone of A6-2F3 cells, functionally selected on the basis of high transepithelial resistance and resposiveness to aldosterone (21). Cell cultures are maintained in 0.8 x concentrated DMEM containing 25 mM NaHCO 3 , 10% heat-inactivated fetal bovine serum, 50 IU/ml penicillin and 50 mg/ml streptomycin for a final osmolarity of 220-250 mOsmol. Cells were incubated in a humidified 95% air, 5% CO 2 atmosphere at 28 °C and subcultured weekly by trypsinization using a Ca 2+ /Mg 2+ -free salt solution containing 0.25% (w/v) trypsin and 1 mM EGTA.
These cells express endogenous basolateral Na/H exchange activity (22). Transfected cell lines were generated by stable transfection in A6/C1 cells of the cDNAs encoding 1) full length (wild type) ratNHE3 (A6-NHE3), 2) ratNHE3 mutated at single endogenous serine position on the cytoplasmic tail of NHE3 (A6-NHE3 S552A and A6-NHE3S 605A ) and 3) full length OKNHE3 (NHE3 opossum subtype) (A6-NHE3 OK ). All were subcloned into the pcDNA3.1 vector (Invitrogen, Groningen, Nertherlands) as have been described previously (23). NHE3 constructs all contained a C-terminal 6His tag. For transfection, A6 cells were grown to 20-25% confluence in 35 mm tissue culture dishes and DNA was introduced into cells plated on culture dishes using FuGENE TM (Boehringher, Mannheim, Germany) and 1.5 µg of the construct of interest together with 0.5 µg of the p3SSLacI vector, which allowed us to select on the basis of hygromicin B resistance (450 µg ml -1 culture medium, for details of the p3SSLacI construct see: (20)). Clonal populations of trasfected cell lines obtained by ring cloning were maintaned as described above in hygromycin. Cells generally reached confluency between 7 to 8 days after seeding when the culture medium was changed three times a week. Studies on A6 cells were performed between passage 114 to 128. Experiments on A6-NHE3 cells were carried out on cells from passage 22 to 36 while those performed with the PKA deficient mutants were carried out on cells from passage 34 to 38.

Measurement of intracellular pH (pHi) and Na + /H + -exchange activity
For pH experiments, cells were seeded onto collagen-coated coverslips with a 1.5 mm hole punched in the centre covered by a teflon filter (Millicell-CM, 0.4 µm pore size; Millipore) as described (23). Cells were incubated for 60 min with 5 µM BCECF-AM in Na + -medium containing 50 µM probenecid to minimize possible dye leakage. Coverslips with filters containing confluent monolayers were inserted into a chamber that allowed independent perfusion of the apical and basolateral cell surface with Na + medium and placed on the stage of an inverted microscope (Zeiss IM 35). BCECF was excited sequentially by positioning 390-440 nm and 475-490 nm band pass filters in front of a xenon lamp. The emission light was collected by a 515-565 nm band pass filter. pHi was estimated from the ratio of BCECF fluorescence calibrated by using K + nigericin approach (23). Na + /H + -exchange activity was measured by monitoring pHi recovery after an acid load by using the NH 4 Cl prepulse technique (24). The rate of Na + -dependent alkalinization was determined by linear regression analysis of 15 points taken at 4-sec intervals. The use of nominally CO 2 /HCO 3 free solutions minimizes the likelihood that Na + -dependent HCO 3 transport was responsible for the observed pHi changes.

Fluorescent measurements of apical chloride efflux
Chloride efflux was measured with the aid of the Clsensitive dye, MQAE, using the procedure described before (25,26). In brief, A6 and A6-NHE3 cells were seeded onto collagen-coated cell culture inserts having polyethylene terephthalate (PET) filters (Falcon Becton Dickinson). Monolayers were loaded overnight in culture medium containing 5 mM MQAE at 28 °C in a CO 2 incubator. After several washes the filter containing the confluent monolayers were removed from the plastic insert and inserted into a perfusion cuvette that allowed separate superfusion of apical and basolateral cell surfaces (27). Fluorescence was recorded on a Shimadzu RF 5000 spectrofluorometer using 360 nm Protein extraction and Western blotting.
Total cellular lysates and crude membrane fractions were prepared and their protein content measured as previously described (29). An aliquot of 50 µg protein was separated in 7% SDS-PAGE.
The separated proteins were transferred to Immobilon P (Millipore, DuPont) in a Trans-Blot semidry electrophoretic transfer cell (Bio-Rad) for immunoblotting. Immunocomplexes were detected with ECL reagent (Amersham). The following antibody was used: anti CFTR monoclonal antibody against the carboxyl-terminus (R&D Systems, MAB25031, dilution 1:1000). In the cell lysate this antibody recognizes two bands of approximately 175 kDa and 205 kDa (see Figure 3).

Biotinylation of apical membrane proteins
To further characterize the molecular weight and expression levels of the form of CFTR located in the apical membrane, apical cell surface biotinylation experiments were performed. A6 and A6-NHE3 cells were grown on 60-mm Petri dishes and at confluence were washed with ice-cold Ringer NaCl and incubated with 2 mg/ml sulfo-NHS-biotin in Ringer NaCl for 30 min at 4°C. Free sulfo-NHS-biotin was blocked by washing cells twice at 4°C with 0.1 M glycine in Ringer NaCl and then with ice-cold Ringer NaCl. Cells were lysed in buffer lysis (0.4% sodium deoxycholate, 1% Igepal CA-630 (SIGMA), 50 mM EGTA, 10 mM Tris-HCl pH 7.4 plus protease inhibitor cocktails) centrifuged for 10 min (13,000 x g) and the pellet was discarded. 30 µl of Streptavidin-agarose beads was added to the lysates (500 µl) and the mixture was incubated with gentle mixing at 4°C overnight.
Streptavidin-bound complexes were pelleted (13,000 x g) and washed three times with 500 µl of lysis buffer. Biotinylated proteins were eluted in Laemmli buffer by boiling for 10 min, resolved by SDS-PAGE, elettroblotted onto Immobilon-P, and immunoblotted with the C-terminal -CFTR antibody (1:1000 dilution).

Pull-down Experiments
The GST-NHERF-1 and GST-NHERF-2 fusion protein homogenates were obtained and the experiments were performed as previously described (29). In brief, equal amounts of GST-NHERF-1 and 2 fusion proteins (≥ 2 µg) were incubated with 25 µl of pre-equilibrated glutathione-agarose beads (Sigma G-4510, 50% slurry) in a total volume of 500 µl of binding buffer (50 mM Tris-HCl, pH 8, 120 mM NaCl, 0.5% Igepal, 5 mM dithiothreitol), by rocking at 4°C for 1 h. After absorption, beads were collected by brief centrifugation at 12,000 rpm for 10 sec (4° C) and gently washed three times with 500 µl of binding buffer containing 0.075% SDS. Pull-down experiments were performed by incubation of these beads with total cellular lysate from A6 and A6-NHE3 cells.
Cellular lysates were prepared from 100 mm-diameter confluent plates. Cells were washed, scraped using 1 ml of binding buffer (50 mM Tris-HCl, pH 8, 120 mM NaCl, 0.5% Igepal-CA-630 and then subjected three times to pulsed sonication for 30 sec on ice. Aliquots were cleared at 12,000 rpm for 2 min (4°C) and ≈ 3 mg protein of these lysates were incubated for 1 h at 4°C with GST-NHERF (1 and 2) immobilized beads. The samples were then washed three times: firstly, with 500 µl of binding buffer without SDS, secondly with 500 µl of binding buffer, diluted 1:2 with same buffer without detergent and, finally, with 500 µl of binding buffer further diluted 1:2. The resulting pellet was extracted in Laemmly buffer and used for SDS-PAGE electrophoresis. Western blotting was performed with either the monoclonal anti-human CFTR antibody directed against the C-terminus or a monoclonal anti-human Ezrin antibody (BD Transduction Laboratories) or the anti-rat NHE3 antibody 1568 (gift of Prof. O. W. Moe, Univ. Texas) obtained as described (30), and immunoreactive bands were detected by ECL using a secondary HRP-coupled IgG (Sigma A-8924).

Data presentation
Results are presented as mean ± SE. Statistical comparisons were made using the paired and unpaired data Student's t test and p< <0.05 was considered statistically significant.  The Na + /K + /2Clcotransporter, which is responsible for chloride loading of the cells, has been demonstrated to be stimulated by cAMP (33). In the experiments summarized in Figure 2, we treated the basolateral side of the monolayer with bumetanide (5 µM) for five minutes before each stimulation with FSK to avoid the possibility that the observed increase of chloride efflux induced by FSK could be due in part to the stimulation of the Na + /K + /2Clcotransporter. However, the glibenclamide-sensitive chloride transport obtained in presence of bumetanide was not significantly different from that obtained in a parallel series of experiments in which we did not pretreat the cells with bumetanide (glibenclamide sensitive Clefflux: 0.0202 ± 0.002 (F/F 0 )/min, n=7 vs 0.0169 ± 0.002 (F/F 0 )/min, n=9 n.s, in the absence or presence of bumetanide, respectively).

Influence of NHE3 on CFTR activity
The ability of active CFTR to influence the PKA-dependent regulation of NHE3 activity in fibroblasts co-transfected with CFTR and NHE3 via coupled interaction of the two transporters with NHERF has been recently reported (7) and, thus, it could be hypothesized that NHE3 can affect CFTR regulation. We, therefore, next examined whether there exists the reciprocal modulation by NHE3 on PKA-dependent regulation of CFTR activity. To accomplish this, we utilized A6 cells stably transfected with cDNA encoding the rat subtype of NHE3 (A6-NHE3 cells). As previously reported, the transfected NHE3 is expressed on the apical membrane and is inhibited ( -38.14 ± 2.72, n=6  These data further support that the presence of functional NHE3 negatively modulates (ie. decreases) the regulation of CFTR activity by PKA.
While the mechanism by which PKA phosphorylation of NHE3 leads to its inhibition is still unknown, it is clear that this phosphorylation is necessary for its functional regulation (38). A critical question in the context of the current study is if the PKA-dependent phosphorylation of NHE3 is necessary for its modulation of PKA-induced CFTR regulation. To this purpose, we analyzed the CFTR-dependent apical secretion in transfected A6 cells with rat NHE3 in which either of the PKA target serines 552 or 605 was mutated to alanine (37). Previous studies, in fact, have demonstrated that these two serines are PKA phosphorylation substrates in rat NHE3 and are necessary for its PKA dependent regulation (38 ,39 ,40). We found that both the S552A and S605A mutations completely pHi/min in A6-NHE3 S605A , n=4, n.s.). As illustrated in Figure 5A, the expression of membrane CFTR did not change in any of these transfected cell lines. The cell monolayers with the mutated NHE3 were then stimulated with forskolin and CFTR-dependent apical chloride efflux analyzed by fluorescence measurements as in Figure 2. Figure 5B shows that when PKA can no longer phosphorylate either of these two serines, the regulation of CFTR activity by forskolin returned to levels not significantly different from that observed in the wild-type A6 cells. These results demonstrate that endogenous NHE3 phosphorylation by PKA is an absolute requirement for its modulation of PKA-dependent regulation of CFTR. Altogether, these data imply that the PKAdependent regulation of CFTR but not its expression is negatively modulated by functional NHE3.

Inhibition of CFTR expression results in a decrease of the PKA-dependent regulation of apical NHE3
activity.
As we found a negative modulating effect of NHE3 expression on PKA-dependent regulation of CFTR activity, we felt it necessary to validate the positive effect of CFTR on NHE3 regulation that was previously demonstrated in fibroblasts (7) in the A6-NHE3 cells which express CFTR endogenously on the apical membrane. Inhibition of CFTR expression by an antisense oligonucleotide (ODN) against the CFTR start site has been previously utilized in cells expressing endogenous CFTR to demonstrate that CFTR modulates the activity and regulation of the ENaC sodium channels (19).
Based on these observations, we synthetized a 21 mer antisense ODN or its missense against the Xenopus laevis CFTR start site in order to confirm the occurrence and pattern of modulation of the PKAdependent regulation of apical NHE3 activity by endogenous, apically located CFTR. Incubation of A6-NHE3 cells with 10 µM of antisense for 48 hrs led to a marked reduction of both CFTR protein expression (Fig. 6A) and of the forskolin-dependent stimulation of CFTR activity (the mean reduction of the CFTR chloride efflux by antisense treatment was -49.4 ± 2.8%, n=3, p< 0.02). This is a slightly higher level of inhibition of CFTR activity by the antisense ODN treatment than that reported by Ling et al. (19).
We then determined the effect of this missense and antisense treatment on the PKA-dependent regulation of the apical NHE3 in the A6-NHE3 cell line. As can be seen in Figure 6B, this antisenseinduced reduction in CFTR expression had no effect on basal transfected, apical NHE3 activities

Role of NHERF-2 in the reciprocal PKA-dependent regulation of CFTR and NHE3 activity.
It has been demonstrated that both NHE3 and CFTR can associate with either the NHERF-1 (11,41) or the NHERF-2 (14) isoform. In the renal cortical collecting duct the NHERF-2 isoform is more strongly expressed (42), suggesting that in the A6 cells NHERF-2 could be the relevant isoform.
We therefore examined, via GST-fusion protein pull-down assays, the association of these two NHERF isoforms with CFTR and NHE3 in the A6 and A6-NHE3 cell lines. Figure 7 shows that, indeed, CFTR (Fig. 7A) was pulled-down from total cellular lysate by NHERF-2 but not NHERF-1 in both cell lines. It is noteworthy that, although both mature and immature CFTR are present in the lysate (see Fig. 3), only the immature form was pulled-down by NHERF-2 (see arrowhead). Similar experiments performed in A6-NHE3 OK cells confirmed that the low molecular weight, immature form of CFTR associates preferentially with NHERF-2 (data not shown).
As can seen in Fig. 7B, NHE3 was pulled-down by only NHERF-2 and only in the A6-NHE3 cell line. In brush border membrane fractions from rat kidney, used as positive controls, NHE3 was also recognized almost exclusively by the NHERF-2 fusion protein.
The NHERF directed PKA phosphorylation of target proteins has been demonstrated to be mediated by the association of the AKAP protein, ezrin, to NHERF in a wide variety of cell contexts (14,43). In order to determine if ezrin forms a part of this signaling complex in our cell model, we probed NHERF-1 and NHERF-2 pull-downs with an anti-ezrin antibody. Indeed, as can be seen in Figure 7C, ezrin was found to associate with the two NHERF isoforms in both cell lines.

Role of the AKAP protein ezrin in the reciprocal PKA-dependent regulation of CFTR and NHE3
activity.
Recent work has demonstrated that A-kinase anchoring proteins (AKAPs) play a fundamental role in governing the cellular compartimentalization of PKA in order to localize it in proximity of the target substrate. This appears to be due to the binding of the regulatory PKA subunits RII to the AKAPs at a specific amino acid consensus sequence that can be blocked by the synthetic peptide Ht31 (44).
Recently, it has been demonstrated that PKA associates with CFTR by the AKAP protein, ezrin (45).
To determine if PKA-dependent regulation of CFTR is mediated by anchorage to an AKAP in our cell models and how this is altered by the presence of NHE3, we preincubated monolayers of either A6 or A6-NHE3 cells for 30 minutes with Ht31, an amphipathic peptide that corresponds to the RII binding motif of a human thyroid AKAP (46) or with its inert analog, Ht31-P containing prolines at positions 502 and 507 (47) and measured the PKA-dependent stimulation of CFTR-dependent chloride efflux in both cell lines and inhibition of NHE3 in the A6-NHE3 cell line. We have used Ht31 and Ht31-P coupled to stearate residues (S-Ht31 and S-Ht31-P) and thus rendered membrane-permeable (47). As can be seen in Figure 7, preincubation with S-Ht31 almost completely eliminated forskolin-dependent stimulation of apical CFTR-dependent chloride transport in A6 cells. In contrast, S-Ht31 treatment had no effect on the CFTR-dependent efflux in the A6-NHE3 cells (Fig.8) while it completely prevented the PKA-dependent inhibition of NHE3 (Fig. 9). The inert analogue, S-Ht31-P, had no effect on the PKA-dependent regulation of either CFTR (Fig. 8) or NHE3 (Fig. 9) transport activity.
The fact that Ht31 did not affect forskolin-mediated activation of CFTR in A6-NHE3 cells suggests that when NHE3 is coexpressed with CFTR the protein complex ezrin-NHERF2 might anchor the Type II regulatory subunits of PKA (PKA II) in proximity to NHE3. In this way the residual forskolin activation of CFTR could regulated predominantely by the cytosolic, non-anchored Type I PKA. Singh et al. (48) have reported that CFTR can be regulated by both Type I and II PKA in T84 cells. In support of this hypothesis, we found that 10 µM of the pan-specific PKA inhibitor, H89, was able to almost completely inhibit the CFTR-dependent chloride efflux in A6-NHE3 cells by 86 ± 6% (n=4, p<0.001).

Discussion
In addition to transepithelial chloride transport, CFTR has been shown to influence a large number of cell functions including the transport of other electrolytes (3). By influencing these electrolyte transports it appears that CFTR plays a fundamental role in regulating cell content and volume of fluids. As a modulator of transepithelial sodium transport, CFTR has been demonstrated to play a modulating role in the PKA-dependent regulation of the activity of both sodium channel (ENaC) (49) and Na + /H + exchanger isoform 3 (NHE3) (7). The mechanism underlying the PKAdependent regulation of NHE3 involves direct interaction with PDZ-containing scaffolding proteins such as the NHERF in which the NHERF can function to link NHE3 with ezrin, a protein kinase A anchoring protein, creating a multi-protein complex and, thereby, mediating the PKA-dependent regulation of NHE3 (10,43,50). There is evidence demonstrating that either NHERF isoform can also associate with CFTR via the PDZ1 domain (12,41) to confer, via direct, ezrin-mediated phosphorylation, PKA-dependent regulation of its activity (14).
This co-ordination of PKA-dependent regulation of either NHE3 or CFTR in the apical plasma membrane of epithelia by NHERF suggests a mechanism by which CFTR could regulate the NHE3 through the joint association with NHERF. Indeed, it has been recently demonstrated, by cotransfection of NHE3 and CFTR in fibroblasts, that CFTR modifies the PKA-dependent regulation of NHE3 via interaction with the NHERF-1 isoform (7). Conversely, the joint interaction of CFTR and NHE3 with NHERF suggests the existence of a reverse, reciprocal modulating effect of NHE3 on CFTR regulation. Along this line, recent work examining the relationship between CFTR and ENaC sodium channels demonstrated such a reciprocal interaction between CFTR and ENaC: CFTR not only acts as a regulator of ENaC but is, in turn, regulated by ENaC (51).
In the present study, we have considered the possibility of the existence of a potential reciprocal modulation of PKA-dependent regulation of CFTR by NHE3. In order to study this reciprocal interaction between CFTR and NHE3 in a highly polarized monolayer, we used a cell line expressing an endogenous CFTR (19) and a transfected rat NHE3 on the apical membrane (20). CFTR and ezrin associated with NHERF-2 in both cell lines and NHE3 associated with NHERF-2 in the A6-NHE3 cell line (Fig. 7). In these cell lines, we verified that in CFTR antisense-treated A6-NHE3 monolayers, forskolin was no longer able to inhibit the NHE3 activity. These data confirm that CFTR is required for the PKA-dependent inhibition of Na + absorption driven by NHE3 as reported in both heterologous double transfected fibroblasts (7) and in mouse intestine (52) and support the hypothesis that CFTR and NHE3 could interact via a common regulatory scaffold protein.
The most significant finding of the present study was that the PKA-dependent regulation of CFTR is also, in turn, negatively modulated by the presence and activity of NHE3. The PKAdependent regulation of CFTR-mediated Clsecretion was lower in A6-NHE3 than in A6 cells without a change in either CFTR protein expression (Fig. 3) or association of CFTR with NHERF-2 ( Fig. 7). The same pattern of modulation of the PKA-dependent regulation of CFTR-mediated Clsecretion by NHE3 expression and association of CFTR with NHERF-2 was observed in an A6 cell line that had been stably transfected with the opossum subtype of NHE3 (A6-NHE3 OK ).
The mutation of either of the two PKA phosphorylation substrate serines, 552 or 605, to alanine (40) prevented the forskolin-induced inhibition of NHE3 and significantly relieved the negative modulating effect of NHE3 on PKA-dependent regulation of CFTR activity without a change in CFTR expression (Fig. 5), demonstrating that it is not the presence of NHE3 but the phosphorylation by PKA that is required for the negative influence of NHE3 on CFTR regulation. All together, the data suggest either that NHE3 has a higher affinity for associating with the regulatory NHERF-ezrin-PKA complex or that CFTR functions to direct PKA-dependent regulation to NHE3 when the two transporters are functionally co-expressed. That is, when only CFTR is expressed it is the substrate for the NHERFezrin-PKA complex while when NHE3 is co-expressed, CFTR becomes a component in a new complex for NHE3 regulation as was previously suggested (7). This could explain how CFTR can function either as a PKA-regulated chloride channel or as a transmembrane regulatory protein for other transporters: it is the relative expression of the various components of this regulatory module that determines which function CFTR will have. A significant confirmation for this hypothesis came from the experiments in which we pretreated A6 and A6-NHE3 cells with S-Ht31 which prevents the binding between AKAPs and Type II regulatory subunits of PKA (47). Indeed, S-Ht31 interfered with the PKA-mediated activation of CFTR in A6 cells, suggesting that the PKA-CFTR interaction is mediated by an AKAP as recently was observed in Calu-3 airway cells (45). In the A6-NHE3 cells, S-Ht31 completely blocked the forskolin-mediated inhibition of NHE3 activity (Fig. 9) while it was no longer able to block the activation of CFTR chloride efflux by forskolin (Fig. 8), suggesting that the co-expression of NHE3 led to the targeting of the multiprotein complex ezrin-PKAII-NHERF-2 to the proximity of NHE3 rather than to CFTR.
A possible complementary mechanism for this last hypothesis could be that an interaction configuration of dimeric CFTR would give rise to more active channels when activated by PKA, thus modulation of the intermolecular CFTR interaction is an attractive mechanism for the potentiation of its activity by PKA (54). The same mechanism was recently demonstrated for the adapter protein, CAP70 (55), in which the CFTR channel is switched to a more active conductive state via an interaction with the two CAP70 PDZ domains, suggesting that this mechanism is widespread.
In conclusion, the novel finding of this study was that there is a reciprocal interaction between CFTR and NHE3 for PKA-dependent regulation.
To determine the precise mechanism whereby NHE3 co-expression influences the regulation of CFTR by PKA will require further investigation. Thus, NHE3, like ENaC, is not simply a passive recipient of CFTR regulatory action but plays an active role by altering, in turn, CFTR function.   Glibenclamide was applied for five minutes before apical anion substitution. The basolateral side was perfused with a chloride solution containing 5 µM bumetanide for 5 minutes before each stimulation with FSK to block the cAMP sensitive basolateral Na + /K + /2Clcotransporter. B) Summary of these data from 9 independent experiments where the glibenclamide-sensitive Clefflux rates across the apical membrane (empty bar) were calculated as the difference in the F/F 0 ratio per minute ((F/F 0 )/min) in the absence of (light grey bar) and presence of (dark bar) glibenclamide. Each bar represents the mean ± S.E.. In the same monolayer we first followed the rate of Clefflux after forskolin treatment and then always in the same monolayer we analyzed the effect of glibenclamide; this permitted the use of two-tailed, paired Students t-test analysis of the data.    Figure 3. B) The glibenclamide-sensitive chloride efflux was measured as in Figure 2 in A6, A6-NHE3, A6-NHE3 S552A and A6-NHE3 S605A cell lines.