Modulation of Na+/H+ exchange activity by Clminus

Orit Aharonovitz1, András Kapus2, Katalin Szászi1, Natasha Coady-Osberg1, Tim Jancelewicz2, John Orlowski3, and Sergio Grinstein1

1 Cell Biology Program, Hospital for Sick Children, Toronto M5G 1X8, and 2 Department of Surgery, Toronto Hospital and University of Toronto, Toronto, Ontario M5G 1L7; and 3 Department of Physiology, McGill University, Montreal, Quebec, Canada H3G 1Y6


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Na+/H+ exchanger (NHE) activity is exquisitely dependent on the intra- and extracellular concentrations of Na+ and H+. In addition, Cl- ions have been suggested to modulate NHE activity, but little is known about the underlying mechanism, and the Cl- sensitivity of the individual isoforms has not been established. To explore their Cl- sensitivity, types 1, 2, and 3 Na+/H+ exchangers (NHE1, NHE2, and NHE3) were heterologously expressed in antiport-deficient cells. Bilateral replacement of Cl- with nitrate or thiocyanate inhibited the activity of all isoforms. Cl- depletion did not affect cell volume or the cellular ATP content, which could have indirectly altered NHE activity. The number of plasmalemmal exchangers was unaffected by Cl- removal, implying that inhibition was due to a decrease in the intrinsic activity of individual exchangers. Analysis of truncated mutants of NHE1 revealed that the anion sensitivity resides, at least in part, in the COOH-terminal domain of the exchanger. Moreover, readdition of Cl- into the extracellular medium failed to restore normal transport, suggesting that intracellular Cl- is critical for activity. Thus interaction of intracellular Cl- with the COOH terminus of NHE1 or with an associated protein is essential for optimal activity.

antiport; type 1 Na+/H+ exchanger; anion dependence; osmotic activation; volume regulation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SODIUM/HYDROGEN EXCHANGERS (NHEs) are integral membrane proteins found in all eukaryotic cells studied to date. Several isoforms have been identified, and some reside in endomembranes (e.g., type 6 Na+/H+ exchanger), while others are exclusively or predominantly plasmalemmal [e.g., types 1-3 Na+/H+ exchanger (NHE1-3)] (for review, see Refs. 28 and 39). The latter transporters promote the electroneutral 1:1 exchange of intracellular H+ for extracellular Na+ across the surface membrane, thereby eliminating excess acid generated by actively metabolizing cells. Exchange of Na+ for H+ is also crucial for the regulation of cellular volume and for the (re)absorption of NaCl across renal, intestinal, and other epithelia. The hydropathy profiles of all members of the NHE family suggest that they share a similar transmembrane organization: an NH2-terminal hydrophobic domain composed of 12 membrane-spanning helices and a COOH-terminal hydrophilic cytoplasmic domain. Recent structural studies support this topological paradigm. The membrane-embedded region is highly homologous among the various isoforms and was shown to be both necessary and sufficient to catalyze ion exchange (28, 39). In contrast, greater divergence is observed between the cytosolic tails, which are thought to confer to the individual isoforms their characteristic pattern of regulation by hormones and growth factors (11, 28, 39).

Because NHE plays a central role in salt and water homeostasis, feedback regulation would ensure accurate control of these parameters. Accordingly, NHE activity is not only dictated by the concentration of its physiological substrates, Na+ and H+, but is also exquisitely sensitive to the cell volume (4). In addition, evidence exists that anions are also involved in NHE regulation. The first definitive indication that Cl- affects the activation of the antiporter was provided by Parker (29), who observed that in dog erythrocytes, NHE failed to respond to cell shrinkage when Cl- was replaced with either thiocyanate or nitrate. A similar effect of anionic replacement on osmotic activation of NHE was subsequently reported in rabbit red blood cells (18) and in barnacle muscle fibers (9). More recently, a Cl--dependent Na+/H+ exchange, presumably catalyzed by an amiloride-resistant form of NHE, was also detected in the apical membrane of colonic crypt cells (31). In the latter case, however, the basal activity, as opposed to the osmotic activation, was affected by Cl-. In contrast, in salivary acinar cells, the presence of Cl- was reported to inhibit Na+/H+ exchange (32). In this tissue, carbachol induces the opening of Cl- channels, leading to Cl- efflux and cell shrinkage. Both the decreased volume and the reduced intracellular Cl- concentration were shown to contribute to the carbachol-evoked activation of the Na+/H+ exchange (12, 32).

The source of the differential Cl- responsiveness of the individual biological systems tested to date remains unclear. In view of the divergent sensitivity of the individual isoforms to other regulatory factors, it is conceivable that the reported differences toward Cl- are attributable to differential expression of distinct isoforms of the NHE in the tissues examined. The analysis may have been confounded further by the simultaneous expression of multiple isoforms in a single cell type, particularly in the case of epithelia. Finally, there is evidence that the same isoform may behave differently depending on the cellular context in which it is expressed (23, 27).

The primary aim of the present study, therefore, was to explore the effect of Cl- on the activity of the three major plasmalemmal isoforms of NHE (NHE1, NHE2, and NHE3) and to further our understanding of the underlying mechanism(s). To eliminate the ambiguity introduced by the coexistence of multiple isoforms and to circumvent differences induced by cellular context, individual isoforms from a defined species were expressed singly in the same cell type. Rat NHE1-3 were transfected into an antiport-deficient line of Chinese hamster ovary (CHO) cells termed AP-1 (27). The effect of Cl- and its sidedness and the responsiveness to osmotic challenge in the presence and absence of the anion and structure-function analysis were performed in such heterologous transfectants.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Materials and solutions. Nigericin and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) were obtained from Molecular Probes (Eugene, OR). Chymotrypsin, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), and cytochalasin B were from Sigma Chemical (St. Louis, MO). Mouse monoclonal antibodies to influenza virus hemagglutinin (HA) were obtained from BAbCo (Berkeley, CA). Horseradish peroxidase-coupled goat anti-mouse antibodies were from Jackson ImmunoResearch Laboratories (Mississauga, ON). Alexa 488-conjugated goat anti-mouse antibody was from Molecular Probes. Enhanced chemiluminescence reagents were from Amersham (Buckinghamshire, England). 125I-labeled goat anti-mouse IgG was from ICN Pharmaceuticals (Irvine, CA). 3-O-[3H]methyl-D-glucose was from NEN (Boston, MA). Trypsin-EDTA was purchased from Life Technologies (Burlington, ON). The ATP assay kit was from Calbiochem (San Diego, CA). All other chemicals were of analytical grade and were obtained from Aldrich Chemical (Milwaukee, WI).

The isotonic Na+-rich medium contained (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4. PBS contained (in mM) 150 NaCl, 10 KCl, 8 sodium phosphate, and 2 potassium phosphate, pH 7.4. Isotonic Na+-free medium contained (in mM) 140 KCl, 1 CaCl2, 1 MgSO4, 5.5 glucose, and 25 N-methyl-D-glucammonium-HEPES, pH 7.4. Isotonic Cl--free media had the same composition as the Na+-rich medium, except that Cl- was replaced by nitrate or thiocyanate. Laemmli sample buffer contained 0.8% SDS (wt/vol), 0.0015% bromphenol blue (wt/vol), 5% 2-mercaptoethanol (vol/vol), 8% glycerol (vol/vol), and 50 mM Tris · HCl, pH 6.8.

Cells. WT5 is a subline of wild-type CHO cells. AP-1 cells, which are devoid of endogenous Na+/H+ exchange activity, were derived from mutagenized WT5 cells as previously described (34). AP-1/NHE1, AP-1/NHE2, and AP-1/NHE3 cells were obtained by stable transfection of AP-1 cells with the complete coding region of the rat NHE1, NHE2, or NHE3, respectively, as described in detail elsewhere (27). For immunological detection of the proteins, epitope-tagged versions were used. In AP-1/NHE1HA cells, one copy of an influenza virus HA peptide (YPYDVPDYA) was appended to the COOH cytoplasmic tail of the coding region of the NHE1 (35). In AP-1/NHE2'813HA, the HA epitope, preceded by a single glycine linker, was inserted at the COOH terminus of NHE2 (after amino acid 813) (6). In AP-1/NHE3'38HA3 cells, a triple HA epitope was inserted into the coding region within the first predicted extracellular loop of NHE3 (21).

Intracellular Cl- depletion and determination. For depletion of Cl-, cells were incubated for the indicated time at 37°C in isotonic Na+-rich Cl--free medium. Cellular Cl- content was determined according to the method of Zall et al. (40) with slight modifications. Briefly, after the appropriate preincubations, cells were washed three times with large volumes of ice-cold Cl--free medium. Next, the cells were lysed with 1 mM nitric acid, scraped off the wells with a rubber policeman, and sedimented. The supernatant was collected and mixed with an equal volume of the photometric determination mixture (1 part of mercuric thiocyanate in 0.417% methanol solution, 1 part of 20.2% ferric nitrate, and 13 parts of water). The absorbance of the test samples and Cl- standards was then determined at 480 nm using a Hitachi U-2000 spectrophotometer.

Cell volume measurement. Cells grown on multiwell plates were incubated in isotonic Na+-rich medium in the presence of tritiated 3-O-[3H]methyl-D-glucose (1 mCi/ml) for 20 min. This time is sufficed for equilibration of 3-O-[3H]methyl-D-glucose across the membrane, as revealed by previous determinations. The cells were then washed three times with ice-cold Na+-rich medium containing 2 µM cytochalasin B, and finally 0.5 ml of 1% SDS was added. The solubilized cells were collected from the wells and counted in an LKB 1217 beta counter. Intracellular volume was calculated from the specific activity of the loading solution, measured in parallel. Sizing of cells was also performed electronically using a model ZM Coulter counter and C1000 Channelyzer, as described earlier (14).

Measurement of NHE activity. Stably transfected cells grown to 60-70% confluence on glass coverslips were incubated for 30 min at 37°C in Na+-rich medium with or without Cl-. BCECF-AM (2 µg/ml) was added during the last 15 min of this incubation. In experiments in which the Na+-dependent recovery of intracellular pH (pHi) was measured, an acute acid load was imposed by the NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-prepulse technique (33). To this end, cells were incubated with 20 mM [NH4]2SO4 for 10 min, washed rapidly and repeatedly with NH<UP><SUB>4</SUB><SUP>+</SUP></UP>- and Na+-free solution, and perfused with this medium while establishing a baseline pH recording. Where indicated, isotonic Na+-rich medium was added to initiate Na+/H+ exchange. Readdition of Cl- to depleted cells was made at this stage, together with Na+. To measure the fluorescence of BCECF, the coverslip was placed in a thermostatted Leiden holder on the stage of a Nikon TMD-Diaphot microscope equipped with a Nikon Fluor oil-immersion objective. A chopping mirror was used to direct the excitation light alternately to two excitation filters (500 ± 10 nm and 440 ± 10 nm) in front of a xenon lamp. To minimize dye bleaching and photodynamic damage, neutral density filters were used to reduce the intensity of the excitation light reaching the cells. The excitation light was directed to the cells via a 510-nm dichroic mirror, and fluorescence emission was collected through a 535 ± 25 nm band-pass filter. Photometric data were continuously acquired at 1 Hz using Felix software (Photon Technologies, South Brunswick, NJ).

Calibration of the fluorescence ratio vs. pH was performed for each experiment by equilibrating the cells in isotonic K+-rich medium buffered to varying pH values (between 6.3 and 7.7) and adding the K+/H+ ionophore nigericin (10 µg/ml). Calibration curves were constructed by plotting the extracellular pH, which is assumed to be identical to the pHi, against the corresponding fluorescence ratio (36).

Quantification of surface NHE1, NHE2, and NHE3. AP-1/NHE1HA, AP-1/NHE2'813HA3, or AP-1/NHE3'38HA3 cells grown on six-well plates were preequilibrated for 30 min at 37°C with Na+-rich medium containing Cl-, nitrate, or thiocyanate. Surface NHE1 was quantified using a previously established procedure (35). Briefly, AP-1/NHE1HA cells were incubated for 5 min at 37°C in the presence or absence of chymotrypsin (100 U/ml). The cells were then scraped off with a rubber policeman, washed twice using Na+-rich medium containing 100 µM TPCK, resuspended in twice-concentrated Laemmli sample buffer, and boiled for 5 min. Samples were subjected to electrophoresis in 7.5% polyacrylamide gels and transferred to nitrocellulose. Blots were blocked with 5% nonfat dried milk and exposed to a 1:5,000 dilution of mouse monoclonal antibodies against HA. The secondary antibody, goat anti-mouse coupled to horseradish peroxidase, was used at 1:5,000 dilution. Immunoreactive bands were visualized using enhanced chemiluminescence.

To quantify surface NHE2, AP-1/NHE2'813HA3 cells grown on glass coverslips were left untreated or incubated in isotonic Cl--free medium (containing NO<UP><SUB>3</SUB><SUP>−</SUP></UP> or SCN- instead of Cl-) for 30 min. After depletion, cells were fixed for 15 min at room temperature using 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS for 20 min. After blocking with 5% milk in PBS, the coverslips were incubated with monoclonal anti-HA antibody (1:3,000 dilution) for 1 h. After washing, the cells were incubated with Alexa 488-conjugated goat anti-mouse antibody (1:3,000 dilution) for 1 h, followed by extensive washing with PBS. The coverslips were mounted onto glass slides using Dako fluorescence mounting medium (Dako, Carpinteria, CA), and the cells were visualized with a Zeiss LSM 510 confocal microscope. Serial optical slices (0.5 µm thick) were acquired, and a stacked image was composed using LSM 510 software. The middle slice of the stack was used to quantify the surface staining. Plasmalemmal and total fluorescence were quantified after background subtraction using Scion software.

To estimate surface NHE3, AP-1/NHE3'38HA3 cells were washed three times with cold PBS and incubated with a 1:1,000 dilution of mouse monoclonal antibodies against HA for 1 h at 4°C, to prevent endocytosis. After washing the cells three times with cold PBS, cells were blocked with 10% goat serum for 30 min, and each well incubated for 1 h with 0.24 µCi of 125I-labeled goat anti-mouse IgG. The wells were washed three more times with large volumes of cold PBS, and 1 ml of trypsin-EDTA was added. Finally, the cells were collected and counted.

Other methods. Cellular ATP content was determined using the Calbiochem assay kit. Cells (~5 × 105) were extracted with 0.4 ml of 8% perchloric acid and placed on ice. The extract was then neutralized with 1 M K2CO3, debris were sedimented, and aliquots of the supernatant (10 µl) were mixed with the buffer and luciferin-luciferase mixture provided by the kit manufacturer. Sample luminescence was determined using a Beckman LS7000 counter and compared with ATP standards. Unless otherwise specified, all experiments were carried out at 37°C. Data are presented as means ± SE of the number of experiments specified.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Depletion of intracellular Cl-. To examine the impact of Cl- on NHE activity, CHO cells expressing individual isoforms of the antiporter were depleted of Cl-. Reduction of the intracellular Cl- concentration was carried out by incubation of the cells in a solution in which this anion was substituted by equimolar NO<UP><SUB>3</SUB><SUP>−</SUP></UP> or SCN-. As illustrated in Fig. 1, this procedure resulted in a rapid decrease in intracellular Cl- content. Nearly 50% of the intracellular Cl- was lost within 5 min, while longer incubation time resulted in further loss of Cl-, reaching ~70% after 30 min. The residual component may represent a slowly exchangeable pool of intracellular Cl-. Alternatively, cellular components other than Cl- may produce a spurious reading in the photometric assay. In either case, depletion of the cytosolic Cl- pool is in all likelihood nearly complete after 30 min. This time was chosen for all subsequent experiments.


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Fig. 1.   Time course of depletion of intracellular Cl-. Depletion was initiated at time 0 by bathing cells in Cl--free medium (NO<UP><SUB>3</SUB><SUP>−</SUP></UP> substitution). Intracellular Cl- content was determined spectrophotometrically, as described in MATERIALS AND METHODS. The cell volume, estimated isotopically using 3-O-[3H]methyl-D-glucose, was used to calculate the intracellular Cl- concentration (ordinate, in mM). Results are means ± SE of 4 determinations. Where absent, error bars are smaller than the symbol.

Because cell volume is known to affect the activity of NHE isoforms, it was essential to assess the effect of anion depletion on this parameter. Two separate methods were used: isotopic equilibration of 3-O-[3H]methyl-D-glucose and electronic cell sizing. The results obtained with both procedures were internally consistent and showed that substitution of cellular Cl- by either NO<UP><SUB>3</SUB><SUP>−</SUP></UP> or SCN- did not affect cell volume. The ratio of the volumes of control to Cl--depleted cells was 1.02 ± 0.07 (means ± SE, n = 16), implying that intracellular Cl- must have been replaced by an equivalent concentration of the substitute anions, in agreement with the known ability of NO<UP><SUB>3</SUB><SUP>−</SUP></UP> or SCN- to enter mammalian cells.

Effect of Cl- depletion on Na+/H+ exchange mediated by NHE1, NHE2, and NHE3. Though Cl- is not transported by the NHEs (20), depletion of this anion was reported to reduce the rate of cation exchange in a variety of cells, while stimulation was reported in one instance. Expression of different isoforms in these systems may account for the apparent discrepancies. We therefore compared the effect of Cl- depletion on NHE activity in cells transfected with the individual isoforms. To assess the effect of anions over a wide pHi range, control or Cl--depleted cells were initially acid loaded by the NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-prepulse technique, and the Na+-induced pHi recovery was then monitored.

As shown in Fig. 2A, bilateral (intra- and extracellular) substitution of Cl- by NO<UP><SUB>3</SUB><SUP>−</SUP></UP> substantially reduced the rate of recovery from an acid load in AP-1/NHE1 cells. Importantly, the depletion procedure did not alter the intracellular buffering capacity, so the rates of pHi recovery could be directly compared in Cl--replete and -depleted cells. Moreover, although the absolute rates of transport vary between isoforms, omission of Cl- similarly depressed the activity of NHE2 (Fig. 2B) and NHE3 (Fig. 2C). In all cases, inhibition was apparent at all the pHi levels tested.


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Fig. 2.   Na+/H+ exchange activity in antiport-deficient Chinese hamster ovary cells transfected with rat types 1, 2, or 3 Na+/H+ exchanger (NHE1, NHE2, or NHE3): effect of Cl- substitution. Cells grown on coverslips were incubated for 30 min in Cl- or NO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing medium. The cells were loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and acidified by prepulsing with NH<UP><SUB>4</SUB><SUP>+</SUP></UP> during the final stages of depletion. To assess NHE activity, the Na+-induced recovery of intracellular pH (pHi) from the acid load was measured fluorimetrically. The rates of alkalinization recorded in cells preincubated with Cl- (open circle ) or NO<UP><SUB>3</SUB><SUP>−</SUP></UP> () are summarized as means ± SE of at least 7 determinations for NHE1 (A), NHE2 (B), and NHE3 (C). Where absent, error bars are smaller than the symbol. Inset (A): representative pHi determination in AP-1/NHE1 cells.

The observed inhibition could be an indication of a requirement for Cl- but may instead reflect an inhibitory effect of NO<UP><SUB>3</SUB><SUP>−</SUP></UP>. We therefore tested the effects of a chemically unrelated, but equally permeant substitute anion, namely SCN-. As illustrated in Fig. 3A, NHE1 activity was also inhibited when Cl- was replaced by SCN-. In contrast, replacing Cl- with another halide, namely I-, did not inhibit but instead caused a significant activation of NHE1 (not shown). These observations can be explained most simply by postulating that optimal NHE activity is halide dependent. Moreover, addition of NO<UP><SUB>3</SUB><SUP>−</SUP></UP> or SCN- in the presence of Cl- does not affect the transport, confirming that these ions are not inhibitory.


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Fig. 3.   Anion sensitivity of NHE1 and NHE3. AP-1/NHE1 (A) or AP-1/NHE3 (B) cells containing intracellular Cl- (Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>), NO<UP><SUB>3</SUB><SUP>−</SUP></UP> (NO<UP><SUB>3i</SUB><SUP>−</SUP></UP>), or SCN- (SCN<UP><SUB>i</SUB><SUP>−</SUP></UP>) were prepared by preequilibration with the appropriate solution. The cells were loaded with BCECF and acidified as described for Fig. 2. pH recovery was induced by adding Na+ in the presence of extracellular Cl- (Cl<UP><SUB>o</SUB><SUP>−</SUP></UP>) or in the absence of extracellular Cl- using NO<UP><SUB>3</SUB><SUP>−</SUP></UP> (NO<UP><SUB>3o</SUB><SUP>−</SUP></UP>) or SCN- (SCN<UP><SUB>o</SUB><SUP>−</SUP></UP>) as substitutes, as specified. Results are means ± SE of at least 3 determinations of the rate of recovery measured at pHi 6.6.

The effects of ion replacement were not restricted to the NHE1 isoform. As illustrated in Fig. 3B, NHE3 is also inhibited when the intracellular Cl- is exchanged for NO<UP><SUB>3</SUB><SUP>−</SUP></UP> or SCN-.

The sidedness of the Cl- requirement. In the above experiments, Cl- was depleted bilaterally. To establish whether intra- and/or extracellular Cl- is required for the maintenance of optimal NHE activity, we depleted intracellular Cl- and reintroduced this anion to the bathing medium at the time of Na+ readdition. Figure 3A shows that when measured at pHi 6.6, inhibition of NHE1 activity in Cl--depleted cells was similar in the absence (NO<UP><SUB>3i</SUB><SUP>−</SUP></UP>/NO<UP><SUB>3o</SUB><SUP>−</SUP></UP> or SCN<UP><SUB>i</SUB><SUP>−</SUP></UP>/SCN<UP><SUB>o</SUB><SUP>−</SUP></UP>) or presence (NO<UP><SUB>3i</SUB><SUP>−</SUP></UP>/Cl<UP><SUB>o</SUB><SUP>−</SUP></UP> or SCN<UP><SUB>i</SUB><SUP>−</SUP></UP>/Cl<UP><SUB>o</SUB><SUP>−</SUP></UP>) of extracellular Cl-. The fact that readdition of Cl- to the extracellular space during the recovery phase failed to restore NHE activity suggests that intracellular Cl- is the critical determinant of NHE1 activity. Similar results were obtained with NHE3 (Fig. 3B).

To test the reversibility of the inhibitory effect caused by depletion of cellular Cl-, we readded Cl- to the extracellular medium of cells that had been previously depleted in NO<UP><SUB>3</SUB><SUP>−</SUP></UP> medium, and the cells were allowed to reequilibrate with Cl- for different time periods before activating NHE by adding Na+. The activity of the exchanger had recovered completely within 5 min of readdition of Cl- (not shown), demonstrating that the inhibition is fully reversible.

Effect of Cl- depletion on ATP content. Though ATP is not hydrolyzed during the Na+/H+ exchange cycle, depletion of this nucleotide drastically reduces the rate of transport in a variety of cells (3, 5). In cells transfected with individual isoforms, NHE1, NHE2, and NHE3 all proved to be sensitive to metabolic depletion (19, 23). The reduced NHE activity of Cl--depleted cells superficially resembles the inhibition evoked by ATP depletion. It was therefore important to establish whether Cl- depletion alters the cellular ATP content, since such an effect could contribute to the observed inhibition. Direct measurements using the luciferin-luciferase assay indicated that the ATP content of Cl--depleted cells was identical to that of Cl--containing controls, irrespective of the anion used for depletion (99 ± 6% and 90 ± 9% of the control level for depletion in NO<UP><SUB>3</SUB><SUP>−</SUP></UP> and SCN- medium, respectively; data are means ± SE of 6 determinations). Thus the inhibition of NHE induced by anion substitution is not mediated by alteration in the content of ATP.

Quantitation of plasmalemmal NHE1, NHE2, and NHE3. The observed decrease in the rate of transport throughout the pHi range studied is consistent with a reduction in the number of exchangers exposed on the cell surface. In this respect, it is noteworthy that NHE3 has been shown to be present not only in the plasmalemma but also in an intracellular vesicular compartment (10) and that a net reduction in transport can result from redistribution of antiporters between these pools (21). It was, therefore, important to test whether the density of plasmalemmal antiporters is altered upon depletion of Cl-. To this end, we assessed the number of exchangers present at the cell surface both before and after anion substitution. Quantitation of NHE1 was based on the chymotrypsin sensitivity of the surface-exposed exchangers, whereas antibodies were used to estimate the surface density of NHE2 and NHE3, which were tagged with the HA epitope.

As reported earlier (35), when whole cell lysates obtained from AP-1/NHE1HA cells are subjected to Western blotting, two immunoreactive bands can be observed (Fig. 4A). The smaller of these polypeptides is thought to be an incompletely glycosylated form of NHE1 that resides in the endoplasmic reticulum, whereas the larger one represents the mature, fully glycosylated molecule that is located in the plasma membrane (7). Accordingly, the latter can be virtually eliminated by brief exposure to chymotrypsin, under conditions where the protease remains exclusively outside the cells (Fig. 4A) (35). Therefore, sensitivity to chymotrypsin can be used to quantify the fraction of NHE1 exposed extracellularly. Using this approach, we compared the fraction of NHE1 present in the plasmalemma in Cl--containing and -depleted cells. As illustrated in Fig. 4A, almost all of the mature NHE1 was exposed to the surface in all instances. Therefore, the reduction of transport associated with Cl- depletion cannot be attributed to internalization of plasmalemmal exchangers.


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Fig. 4.   Quantification of surface NHE1, NHE2, and NHE3. A: chymotryptic cleavage of NHE1. AP-1/NHE1HA cells were equilibrated with Cl--, NO<UP><SUB>3</SUB><SUP>−</SUP></UP>-, or SCN--containing medium, and an aliquot of each group of cells was then treated with chymotrypsin (100 U/ml, 5 min). The cells were then lysed and subjected to electrophoresis and blotting onto nitrocellulose. The blots were probed with anti-hemagglutinin (HA) antibody using enhanced chemiluminescence. The blot is representative of 3 similar experiments. B: quantification of plasmalemmal NHE2 by immunostaining. AP-1/NHE2813HA cells grown on coverslips were fixed and stained to detect the HA epitope, as described in MATERIALS AND METHODS. Fluorescence was visualized with a Zeiss LSM510 confocal microscope, and a typical image of an equatorial optical section is shown (inset). Resulting digital images were quantified using Scion software (see MATERIALS AND METHODS), and the fluorescence at the surface is shown expressed as a percentage of the total fluorescence. Data are means ± SE of 20 determinations for each treatment. C: radioisotopic quantification of NHE3. AP-1/NHE3'38HA3 cells were equilibrated with Cl--, NO<UP><SUB>3</SUB><SUP>−</SUP></UP>-, or SCN--containing medium. The cells were then chilled and incubated with monoclonal anti-HA antibody (1:1,000 dilution, 1 h, 4°C). After washing and blocking, the cells were incubated with 125I-labeled goat anti-mouse IgG (0.24 µCi/well, 1 h, 4°C). Next, the cells were detached from the wells, and radioactivity bound to surface NHE3 was quantified. Results are means ± SE of 9 determinations from 4 experiments.

Next we analyzed the effect of Cl- depletion on the surface distribution of NHE2. Control and Cl--depleted AP-1/NHE2'813HA cells were immunostained with anti-HA antibody, followed by a labeled secondary antibody to visualize the exchanger. Confocal fluorescence images were acquired and analyzed digitally to quantify the density of exchangers at the cell surface. As shown in the inset of Fig. 4B, which illustrates an equatorial optical section, more than half of the NHE2 is localized at the plasma membrane in control cells. The remainder is localized in a juxtanuclear vesicular complex. This distribution was not significantly altered when intracellular Cl- was depleted, using either NO<UP><SUB>3</SUB><SUP>−</SUP></UP> or SCN- as substitutes (Fig. 4B). Thus as in the case of NHE1, the inhibition of NHE2 caused by Cl- depletion cannot be attributed to changes in the subcellular distribution of transporters.

To test the effect of Cl- depletion on NHE3, AP-1/NHE3'38HA3 cells were preincubated with or without Cl-. The amount of NHE3 at the plasma membrane was quantified using an antibody to the exofacial HA epitope, followed by a radiolabeled secondary antibody. Because treatment with the antibodies was performed in the cold, only surface exchangers were detected under these conditions. The data presented in Fig. 4C show that the surface density of NHE3 does not change as a result of Cl- depletion. Together, these results indicate that Cl- depletion does not significantly alter NHE1, NHE2, or NHE3 distribution and that the inhibition of transport results from a diminution in the intrinsic activity of the transporters.

Effect of Cl- depletion on the osmotic responsiveness of the NHE isoforms. Activation of Na+/H+ exchange is an important mechanism in the restoration of cell volume in osmotically shrunken cells (4). NHE1 and NHE2 can fulfill this role, since both of these isoforms are activated upon cell shrinkage (2, 19; but see Ref. 25 for discrepant view). In contrast, the epithelial brush border isoform NHE3 is inhibited when the cell shrinks (19), perhaps guarding the organism against solute (re)absorption under abnormally hypertonic conditions.

It was shown earlier that the hypertonic activation of NHE normally observed in dog erythrocytes (29), rabbit red blood cells (18), and barnacle muscle fibers (9) was absent when intracellular Cl- was depleted. Because the specific isoforms involved in these responses were not defined, we analyzed the responsiveness of NHE1, NHE2, and NHE3 in the heterologous transfectants. In agreement with earlier determinations (2), the resting pHi of AP-1/NHE1 cells in Cl--containing Na+ solution was found to average 7.33 ± 0.05 (n = 7). In nominally bicarbonate-free medium, depletion of Cl- from these cells reduced the resting pHi only marginally (7.27 ± 0.05; n = 8). As illustrated in Fig. 5A, elevation of the medium osmolarity by addition of an extra 100 mM NaCl resulted in a sizable and sustained alkalinization that averaged 0.26 ± 0.02 pH units (see also Fig. 5B). Unexpectedly, we found that pHi also increased when Cl--depleted cells were made hypertonic by the addition of 100 mM NaNO3. As reported earlier (19), NHE2-transfected cells also responded to hypertonicity with a significant alkalinization. As observed for NHE1, the osmotic activation of NHE2 persisted following depletion of Cl- (Fig. 5B).


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Fig. 5.   Hypertonic activation of NHE1 and NHE2: effect of Cl- depletion. AP-1/NHE1 or AP-1/NHE2 cells were preequilibrated with Cl--containing or Cl--free (NO<UP><SUB>3</SUB><SUP>−</SUP></UP> substituted) medium and loaded with BCECF. After measuring the basal pHi, the medium was made hypertonic by addition of 100 mM NaCl or NaNO3, respectively, and recording continued until a new steady state was attained. A: representative pHi determination in Cl--depleted AP-1/NHE1 cells. Arrow indicates the time when osmolarity was increased. B: summary of experiments like that in A, using NHE1- and NHE2-transfected cells preincubated with Cl- (open bars) or NO<UP><SUB>3</SUB><SUP>−</SUP></UP> (hatched bars). Maximal change in pHi induced by hypertonicity (Delta pH) was determined after the new steady state was attained. Results are means ± SE of at least 5 determinations.

Because NHE3, unlike the other two isoforms, is inhibited by hypertonicity, its osmotic responsiveness cannot be analyzed by measuring the effects of osmotic challenge on the resting (basal) pHi. Therefore, to monitor the effect of hypertonicity on this isoform, NHE3-expressing cells were first acid loaded, and then their Na+-dependent pHi recovery was followed under iso- and hypertonic conditions. In agreement with our previous results, the Na+-induced alkalinization was considerably slower in hypertonic than in isotonic solution (Fig. 6A, inset). The pHi dependence of the transport rate, as determined from a series of similar measurements, is summarized in Fig. 6A. Next, the pHi recovery of Cl--depleted cells was compared in iso- and hypertonic NaNO3 medium. As illustrated in Fig. 6B, the NHE3 activity remaining in these cells was not further inhibited by the hypertonic challenge. It is not clear if this implies that changes in Cl- concentration are required to signal the hypertonic inhibition of NHE3 or whether the exchanger is already maximally suppressed and not amenable to further inhibition.


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Fig. 6.   NHE3 activity under iso- and hyperosmotic conditions: effect of Cl- depletion. AP-1/NHE3'38HA3 cells were equilibrated with Cl- (A) or NO<UP><SUB>3</SUB><SUP>−</SUP></UP> (B). The cells were then loaded with BCECF, and the Na+-induced recovery from an acid load was recorded fluorimetrically as in Fig. 2. Where indicated, the medium was made hypertonic by addition of 100 mM NaCl (A) or NaNO3 (B). Rates of H+ extrusion measured in isotonic (open circle ) or hypertonic () conditions are presented. Data are means ± SE of at least 6 determinations of each type. Where absent, error bars are smaller than the symbol. Insets: representative pHi determinations.

Role of the cytosolic tail in conferring Cl- sensitivity to the exchanger. The cytosolic COOH-terminal domain of the antiporters is thought to be essential for regulation of activity by hormones, growth factors, and cell volume (see Refs. 28 and 39 for review). On the other hand, the transport and (part of) the allosteric H+-sensitive sites are thought to reside in the transmembrane moiety. To define the region(s) of the protein responsible for the sensitivity to anions, we compared the Cl- sensitivity of the full-length exchanger with that of a truncated mutant (NHE1Delta 582) lacking a large portion of the cytoplasmic tail. At pHi 6.4-6.8, Cl- depletion resulted in a 62 ± 4% decrease in the activity of the wild-type NHE1 (n = 40), whereas it caused only a 31 ± 3% drop in the activity of Delta 582 (n = 15). The effect of further truncations could not be assessed accurately, since their basal activity was marginal.

Truncation of the cytosolic domain also affected the inhibition of NHE3 by Cl- depletion. In fact, in this isoform, the consequences of truncation were even more pronounced. In full-length NHE3, omission of Cl- decreased the activity by 72 ± 3% (at pHi 6.4-6.8; n = 21), whereas in NHE3Delta 579, anion substitution had no detectable effect (0.05 ± 10% decrease in the activity in the absence of Cl-; n = 18). These findings suggest that the effect of Cl- is exerted at least in part through the cytosolic tail of the NHE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cl- was initially shown to be involved in the regulation of NHE in red blood cells. Using dog erythrocytes, Parker (29) reported that replacement of Cl- with NO<UP><SUB>3</SUB><SUP>−</SUP></UP> or SCN- abolished the shrinkage-induced activation of the antiporter. If the cells were first shrunken in the presence of Cl- and then briefly treated with low concentrations of the mild fixative, glutaraldehyde, the exchanger remained in the activated state, in spite of the subsequent removal of Cl- or the restoration of isotonic cell volume (30). These findings were interpreted to mean that the activation, rather than the function, of the exchanger was sensitive to the anion composition. Similarly, Motais et al. (26) found that Cl- was required for the cAMP-induced stimulation of the exchanger of trout red blood cells. As in the previous case, however, once the trout antiport was activated, the anion sensitivity was lost.

The results in these studies differ from those reported here in two significant aspects. First, mammalian NHE1-3 were found to be anion dependent in unstimulated cells, i.e., in isotonic media devoid of hormonal or other agonists. Second, in the cases of NHE1 and NHE2, the volume-induced response persisted in the absence of Cl-, in contrast to the results in erythrocytes. The explanation for this divergent behavior may lie in the pHi dependence of the different systems, which in turn reflects their primary function. In unstimulated red blood cells, NHE activity is negligible at or near physiological pH. In these cells, stabilization of the pHi is accomplished through the exchange of Cl- for bicarbonate, by far the predominant transport system in their membrane. Therefore, the primary role of Na+/H+ exchange in red blood cells is thought to be in the regulation of cellular volume (4). This contrasts with the exchangers of nucleated mammalian cells, where NHE activity is mainly required for pH regulation or transepithelial electrolyte transport. For these functions, NHE must be active at or very near the physiological pH. The factor(s) responsible for this basal activity, which may be related to those stimulated by shrinkage or cAMP in red blood cells, could be the target of Cl- ions.

This explanation is consistent with the reported effects of Cl- in barnacle muscle fibers. Like red blood cells, barnacle fibers have negligible NHE activity near the resting pHi but show a significant activation by hyperosmolarity (9). This activation was similarly found to depend on Cl-, an effect that was attributed to coupling of NHE with a heterotrimeric GTP-binding protein (8, 9). Subsequently, Hogan and colleagues (15) found that the barnacle exchanger could also be activated by a profound acidification. Because this acid-induced effect was also sensitive to anion replacement, it was suggested that regulation by the GTP-binding protein may be exerted tonically. A similar mechanism may operate in mammalian cells. G protein control of NHE1 in salivary cells has been reported (17), although no such evidence exists at present for the other isoforms of the exchanger.

The precise mechanism of action of Cl- ions remains undefined. The data in Fig. 4 imply that the number of transporters available at the membrane is unaffected. Thus it is most likely that the intrinsic activity of each exchanger is depressed when Cl- is removed. NHE1 is known to possess an autoinhibitory domain that constitutively represses the activity of the transport site (37). The effect exerted by this domain can be modulated by calcium/calmodulin and may also be sensitive to Cl- ions. However, part of the effect of Cl- ions persisted in NHE1Delta 582, a truncated form lacking the autoinhibitory domain. Therefore, other mechanisms must be invoked. For instance, Cl- may alter the sensitivity of the exchangers to intracellular H+ ions, an explanation consistent with the leftward shift in pHi dependence noted in Fig. 2. The latter is dictated primarily by an allosteric "modifier" site that confers cooperativity to the H+ concentration dependence (1). The modifier was initially postulated to be a discrete site located in the transmembrane domain (38), but an important contribution of the cytosolic tail was suggested more recently (16). Regardless of its precise composition, the modifier site is generally accepted to face the cytoplasmic milieu, compatible with the finding that, at least in the case of NHE1, the site of action of Cl- is intracellular.

Another important volume-dependent ion transporter, the Na+-K+-Cl- cotransporter, has also been proposed to be regulated by intracellular Cl- (22, 24; however, see Ref. 13 for alternative view). In this system, it has been suggested that changes in intracellular Cl- concentration regulate the activity of serine/threonine kinase(s) that directly phosphorylate and thereby activate the cotransporter. This, together with the earlier reports in red blood cells, raised the possibility that cellular effectors, including NHE, detect volume changes by sensing the associated alterations in Cl- concentration. However, the hyperosmotic activation of NHE1 and NHE2 persisted following bilateral Cl- removal. An important implication of this finding is that the shrinkage-induced rise in intracellular Cl- concentration cannot itself be the trigger for the osmotic stimulation of NHE. Since the intracellular Cl- content was reduced by at least 70%, and the addition of 100 mM NaNO3 decreased the cell volume by not more than 40%, the Cl- concentration in the depleted, shrunken cells would remain well below (<50%) the resting intracellular level under isotonic conditions. Therefore, other volume sensors must relay the information to activate NHE.

In summary, we found that anion sensitivity is a common feature of the three major NHE isoforms. In all cases, the presence of Cl- was found to be essential for optimal activity and, at least in the case of NHE1, the site of action of the anion is intracellular. Accordingly, deletion of part of the cytosolic tail reduced the anion dependence of transport. The results can be most readily explained by direct binding of the anion to the COOH-terminal region of the exchanger or to an ancillary protein that in turn interacts with the cytosolic aspect of the exchanger.


    ACKNOWLEDGEMENTS

This work was supported by the Canadian Cystic Fibrosis Foundation and the Canadian Institutes for Health Research (CIHR). O. Aharonovitz was supported by the Arthritis Society of Canada and the Canadian Cystic Fibrosis Foundation.


    FOOTNOTES

A. Kapus is a CIHR Scholar. K. Szászi is supported by a CIHR fellowship. J. Orlowski is supported by a Scientist Award from CIHR. S. Grinstein is supported by a Distinguished Scientist Award from the CIHR and is cross-appointed to the Department of Biochemistry of the University of Toronto. S. Grinstein is an International Scholar of the Howard Hughes Medical Institute and is the current holder of the Pitblado Chair in Cell Biology.

Address for reprint requests and other correspondence: S. Grinstein, Cell Biology Program, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8 (E-mail: sga{at}sickkids.on.ca).

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.

Received 5 May 2000; accepted in final form 13 February 2001.


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