EDITORIAL FOCUS
Na+/H+ exchangers (NHE1-3) have similar turnover numbers but different percentages on the cell surface

Megan E. Cavet, Shafinaz Akhter, Fermin Sanchez de Medina, Mark Donowitz, and Chung-Ming Tse

Gastrointestinal Division, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2195


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NHE1, NHE2, and NHE3 are well-characterized cloned members of the mammalian Na+/H+ exchanger (NHE) gene family. Given the specialized function and regulation of NHE1, NHE2, and NHE3, we compared basal turnover numbers of NHE1, NHE2, and NHE3 measured in the same cell system: PS120 fibroblasts lacking endogenous NHEs. NHE1, NHE2, and NHE3 were epitope tagged with vesicular stomatitis virus glycoprotein (VSVG). The following characteristics were determined on the same passage of cells transfected with NHE1V, NHE2V, or NHE3V: 1) maximal reaction velocity (Vmax) by 22Na+ uptake and fluorometery, 2) total amount of NHE protein by quantitative Western analysis with internal standards of VSVG-tagged maltose-binding protein, and 3) cell surface expression by cell surface biotinylation. Cell surface expression (percentage of total NHE) was 88.8 ± 3.5, 64.6 ± 3.3, 20.0 ± 2.6, and 14.0 ± 1.3 for NHE1V, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despite these divergent cell surface expression levels, turnover numbers for NHE1, NHE2, and NHE3 were similar (80.3 ± 9.6, 92.1 ± 8.6, and 99.2 ± 9.1 s-1, when Vmax was determined using 22Na uptake at 22°C and 742 ± 47, 459 ± 16, and 609 ± 39 s-1 when Vmax was determined using fluorometry at 37°C). These data indicate that, in the same cell system, intrinsic properties that determine turnover number are conserved among NHE1, NHE2, and NHE3.

sodium/hydrogen antiporter; quantitative Western analysis; cell surface biotinylation; PS120 fibroblasts


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE SODIUM-HYDROGEN EXCHANGER (NHE) isoforms NHE1, NHE2, and NHE3 consist of a well-conserved (40-60% identity among isoforms) ~500-amino acid NH2-terminal Na+- and H+-transporting domain and an ~300-amino acid COOH-terminal intracytoplasmic regulatory domain, which shares little homology among isoforms at the amino acid level. Despite the apparent structural similarity among the exchanger isoforms, they differ in tissue localization, regulation, and subcellular distribution, and they serve different physiological functions. NHE1 is ubiquitously expressed in mammalian cells and is present on the basolateral membrane of most epithelial cells, whereas NHE2 and NHE3 are predominantly localized to the apical membrane of renal, intestinal, and salivary epithelia (15, 40). NHE1 is a housekeeper isoform, helping maintain cell volume and intracellular pH and perhaps regulating cell proliferation (33, 39). In contrast, NHE2 and NHE3 are involved in transcellular Na+ absorption. The precise relative roles of NHE2 and NHE3 in the intestinal absorption of Na+ are uncertain. Both isoforms appear to contribute equally to basal rabbit and avian ileal brush-border Na+/H+ exchange (14, 41), whereas NHE3 appears to be the ileal isoform that responds to various pathophysiological conditions such as glucocorticoid treatment (11, 44), hyperthyroidism (4), and chronic acidosis (24). In the dog, NHE3 is responsible for all basal ileal Na+ absorption and the neurohormonal-induced increase in ileal Na+ absorption that occurs after meals (30). NHE3 is also the predominant form explaining basal Na+ absorption in rat proximal colon, with NHE2 and NHE3 activities increasing on Na+ depletion (19). In contrast, NHE2 is the major Na+/H+ exchanger in avian colon, where it is increased by a low-salt diet (14).

The basal and regulated transport rate is dependent on the number of active exchangers expressed on the cell surface and on the turnover number of the individual exchangers. In fibroblasts (PS120 and AP-1) the majority of NHE3 (85-90%) is localized in an intracellular compartment, with 10-15% being present on the cell surface (1, 23). In contrast, in the same cells, NHE1 is almost entirely on the plasma membrane (~90%) (12, 36). The cells in which NHE3 is expressed greatly influence the cellular distribution of NHE3. For instance, in Caco-2 cells and rat proximal tubules, ~70-85% of NHE3 is on the cell surface (16, 20). The cell surface expression of NHE2 has not been reported. It is known that there are major differences in the mechanisms of short-term regulation by protein kinases and growth factors of NHE1 vs. NHE2 and NHE3. Nearly all short-term regulation of NHE1 by protein kinases is by a change in the affinity for intracellular H+ (26). The response of NHE2 and NHE3 to protein kinases is usually by a change in the maximal reaction velocity (Vmax) (26, 27). However, in the case of NHE3, response to the agents squalamine and cAMP also involves a change in the affinity of intracellular H+ (2, 25). Change in Vmax of NHE3 is known to be partially due to a change in the amount of cell surface protein; however, a change in turnover number of NHE3 also appears to be involved (20).

The aim of this study was to define and compare basic transport parameters of NHE1, NHE2, and NHE3, specifically basal turnover number. This was done by determining the amount of NHE1, NHE2, and NHE3 present on the cell surface of PS120 fibroblasts and correlating this amount with the Vmax. The turnover number of a transport protein can be influenced by intrinsic properties of the protein itself and extrinsic properties such as cytoskeletal and signaling molecule interactions (25) and membrane lipid fluidity (7). Because NHE1, NHE2, and NHE3 have different functions, it is important to compare characteristics between isoforms in the same cell system, where extrinsic properties will be constant, allowing comparison of intrinsic properties of the NHEs.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Cell Culture

A Chinese hamster lung fibroblast cell line (PS120) previously selected to lack all endogenous NHEs was stably transfected, using Lipofectin (GIBCO BRL), with human NHE1 or rabbit NHE2 or NHE3 cDNAs epitope tagged on the COOH termini with a previously described 11-amino acid epitope of vesicular stomatitis virus glycoprotein (VSVG) plus an 8-amino acid spacer (NHE1V, NHE2V, and NHE3V) (18). Cells were grown in DMEM supplemented with 25 mM NaHCO3, 10 mM HEPES, pH 7.4, 50 IU/ml penicillin, 50 µg/ml streptomycin, 10% fetal bovine serum, and 800 µg/ml G418 in a 5% CO2-95% O2 incubator at 37°C. Cells were acid loaded once a week to maintain a high level of Na+/H+ exchange activity, as described previously (26). Briefly, cells were placed in NH4Cl solution (in mM: 50 NH4Cl, 70 choline chloride, 5 KCl, 1 MgCl2, 2 CaCl2, 5 glucose, and 20 HEPES-Tris, pH 7.4) for 1 h, then in an isotonic 2 mM Na+ solution for a further 1 h. Surviving cells were then placed in normal culture medium. Cells were used between passages 4 and 25 posttransfection.

Na+/H+ Exchange Vmax by 22Na+ Uptake

Stably transfected PS120/NHE1V, PS120/NHE2V, or PS120/NHE3V cells were seeded on 24-well plates, and Na+/H+ exchange activity was measured when they reached confluency. The cells were washed in PBS, acidified by placement in 50 mM NH4Cl solution for 20 min at 37°C, and subjected to three rapid washes in wash buffer (in mM: 120 choline chloride and 15 HEPES-Tris, pH 7.4). These conditions cause a rapid acidification of cells to pH ~6.0 (35). The wash buffer was then removed and replaced with transport buffer (120 mM choline chloride, 1 mM MgCl2, 2 mM CaCl2, 20 mM HEPES-Tris, pH 7.4, 1 mM ouabain, 0.1 mM bumetanide, and 1 µCi/ml 22NaCl). Na+/H+ exchange was measured at Na+ concentrations ranging from 1 to 60 mM by replacing equimolar choline chloride with NaCl with and without 1 mM amiloride. Na+/H+ exchange was defined as amiloride-sensitive Na+ uptake. At 1 min the buffer was aspirated, and the plates were washed three times rapidly in ice-cold PBS. The cells were lysed with 0.1 mM NaOH for 1 h, and the lysates were assayed for gamma -radiation with a Beckman gamma counter. All transport experiments were carried out at 22°C in triplicate. Vmax was determined using the Eadie-Hoftsee transformation of the amiloride-sensitive Na+ uptake.

Na+/H+ exchange Vmax by Fluorometry

Cells were seeded on glass coverslips and grown until they reached 50-70% confluency. The cells were loaded with 5 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM in "Na+ medium" (in mM: 130 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 1 NaH2PO4, 25 glucose, and 20 HEPES, pH 7.4) for 20 min at 22°C, then washed with "TMA+ medium" (in mM: 130 tetramethylammonium chloride, 5 KCl, 2 CaCl2, 1 MgSO4, 1 NaH2PO4, 25 glucose, and 20 HEPES, pH 7.4) to remove the extracellular dye, and the coverslip was mounted at an angle of 60° in a 100-µl fluorometer cuvette designed for perfusion and thermostated at 37°C. The cells were pulsed with 40 mM NH4Cl in TMA+ medium for 3 min, then in TMA+ medium, which resulted in the acidification of the cells. Na+ medium was then added, and this induced alkalinization of cells.

Na+/H+ exchange (H+ efflux) was calculated, as described previously (26), as the product of Na+-dependent change in intracellular pH (pHi) times the buffering capacity at each pHi and was analyzed using a nonlinear regression data analysis program (Origin) that allowed fitting of data to a general allosteric model described by the Hill equation (v = Vmax · [S]n/K' + [S]n, where [S] is substrate concentration, v is velocity, K' is affinity constant, and n is Hill's number) with estimates for Vmax and the affinity constant for intracellular H+ and their respective standard errors.

Cell Surface Amount of NHE1, NHE2, and NHE3

Maltose-binding fusion proteins. Two fusion proteins, 1) the COOH-terminal 50-amino acid of NHE3V, including the VSVG tag and maltose-binding protein (MBP/E3V), and 2) the last COOH-terminal 47-amino acid of NHE2V, including the VSVG tag and MBP (MBP/E2V), were constructed. The last 180 bp encoding NHE3V and the last 171 bp encoding NHE2V were constructed by PCR amplified from cDNA with use of paired primers (5'-GAATTCCGCCGCCTGGCGCCCTTCC and 3'-CTAGTCTAGATTGGTACCTTAGA for NHE3V and 5'-GAATTCGGCAGACCCAAGCCGCC and 3'-CTAGTCTAGATTGGTACCTTAGA for NHE2V). The fragments were subcloned into pCR2.1 (Invitrogen) and sequenced. pMal-c2 (New England Biolabs) and the NHE2V or NHE3V fragment in pCR2.1 were linearized by digestion with EcoR I and Xba I, and the fragments were inserted into pMal-c2 by unidirectional cloning. The subsequent constructs were confirmed by sequencing and transformed into the Escherichia coli strain BL21 for expression of the fusion protein. As a control, pMal-c2 without an insert was similarly transformed.

The fusion proteins were affinity purified using amylose resin, as described previously (41). Briefly, 1 liter of transformed bacteria was grown in 2XYT medium (per liter: 16 g tryptone, 10 g yeast extract, and 5 g NaCl) at 1:100 dilution from an overnight culture until the optical density at 600 nm was 0.5 and was induced with 0.3 mM isopropyl-beta -thiogalactoside for 2 h. The cells were collected by centrifugation for 20 min at 4,000 g. Cells were resuspended in 50 ml of column buffer (in mM: 30 NaCl, 10 HEPES-Tris, pH 7.2) plus protease inhibitors (100 µM phenylmethylsulfonyl fluoride and 1 mM iodoacetamide). Then the cells were sonicated to clarity on ice with use of a Branson 450 probe sonicator. The cell suspension was centrifuged for 30 min at 9,000 g, and then the supernatant was applied to an amylose-agarose column and washed extensively with column buffer. The bound fusion proteins were eluted with column buffer and 10 mM maltose. After being separated on a 10% SDS-polyacrylamide gel, the protein of interest was excised, electroeluted to remove degraded products, and then dialyzed against PBS.

Preparation of PS120 total crude lysates. Cells grown to confluency on six-well plates were washed with PBS (in mM: 150 NaCl and 20 Na2HPO4, pH 7.4) and then scraped in 1 ml of PBS. After a brief centrifugation, the cells were lysed with 100 µl of lysis buffer (60 mM Tris and 1% SDS, pH 7.5) to obtain crude lysates.

SDS-PAGE and quantitative Western analysis. Crude lysates of NHE1V, NHE2V, NHE3V, MBP/E3V, and MBP/E2V were resolved on 9% SDS-polyacrylamide gels. The samples were run at 150 V for 1.3 h (running buffer was 14.4 g/l glycine, 3 g/l Tris, and 1 g/l SDS). The gel was electrotransferred onto nitrocellulose at 100 V for 1 h (transfer buffer was 200 ml/l methanol, 14.4 g/l glycine, and 3 g/l Tris). Then the blot was blocked using 5% nonfat dry milk in Tris-buffered saline (in mM: 150 NaCl and 13 Tris, pH 8.0) for 1 h at room temperature. After it was blocked, the blot was incubated with monoclonal anti-VSVG P5D4 antibody overnight at 4°C. After five washes with Tris-buffered saline plus 0.02% Triton X-100, the blot was incubated with donkey anti-mouse secondary antibody (Jackson Laboratory) for 1 h at room temperature. The blot was washed again, and the bound secondary antibody was detected by enhanced chemiluminescence (ECL; Renaissance, New England Nuclear) and exposed to preflashed X-ray film. The developed X-ray films were scanned, and densitometry was performed using ImageQuant software.

To determine the absolute amount of NHE1V, NHE2V, and NHE3V protein per milligram of PS120 protein, a dilution series of MBP/E3V and crude lysates of NHE1V, NHE2V, or NHE3V was run on the same gel. It was assumed that transfer efficiency was equal across the entire gel surface onto the nitrocellulose membrane, inasmuch as the transfer of protein appeared to be complete by staining the gel with Coomassie blue after transfer. A standard curve of amount of fusion protein loaded vs. intensity of the bands was determined, and amounts of NHE1V, NHE2V, or NHE3V were determined within the linear range of the standard curve, as described elsewhere (41). Similar quantitation results were obtained using MBP/E2V fusion protein as a standard.

Cell surface biotinylation and quantitation. Cell surface biotinylation was used to determine the percentage of total NHE1V, NHE2V, and NHE3V protein that is present on the cell surface of PS120 cells. Transfected PS120 cells were grown to 70-80% confluency in 10-cm petri dishes. All subsequent manipulations were performed at 4°C. Cells were washed twice in PBS and once in borate buffer (in mM: 154 NaCl, 10 boric acid, 7.2 KCl, and 1.8 CaCl2, pH 9.0). The surface plasma membrane proteins were then biotinylated by gently shaking the cells for 20 min with 3 ml of borate buffer containing 1.5 mg of N-hydroxysulfosuccinimydyl-S,S-biotin (NHS-SS-biotin; biotinylation solution). An additional 3 ml of the same biotinylation solution were then added, and the cells were rocked for an additional 20 min. The cells were washed extensively with quenching buffer (in mM: 120 NaCl and 20 Tris, pH 7.4) to remove excess NHS-SS-biotin, and the cells were washed twice with PBS. Cells were scraped and solubilized with 1 ml of N+ buffer (in mM: 60 HEPES, pH 7.4, 150 NaCl, 3 KCl, 5 trisodium EDTA, 3 EGTA, and 1% Triton X-100), sonicated for 20 s, agitated on a rotating rocker at 4°C for 30 min, and centrifuged at 12,000 g for 30 min to remove insoluble cellular debris. A portion of the resulting supernatant was retained as the total fraction, and the remainder was then incubated with avidin-agarose. After two consecutive avidin precipitations, the remaining supernatant was retained as the intracellular fraction. The avidin-agarose beads were washed five times in N+ buffer, and bound proteins were solubilized in equivalent volumes (to the volume initially added to beads) of sample buffer (110 mM Tris · HCl, 0.9% SDS, 0.8% EDTA, 5% glycerol, 1% 2-mercaptoethanol, and bromphenol blue), yielding the surface fraction.

Western analysis was then performed on dilutions of the total, intracellular, and surface fractions run on the same gel described above. Bands were visualized using ECL, exposed to preflashed X-ray film, and quantified using a densitometer and ImageQuant software. Values for the bands were plotted as arbitrary densitometric units vs. sample volume, and numbers for each fraction were compared with each other only when they fell on the linear part of all three curves (total, intracellular, and supernatant) simultaneously (see Fig. 8B). Results were discarded when the total recovery of protein in the intracellular and surface fractions was <85% of total.

Protein Determination

Protein concentration was determined using a bicinchoninic acid protein assay (Sigma Chemical).

Materials

Human NHE1 was a kind gift from Dr. J. Noel (Dept. of Physiology, University of Montreal) and Dr. J. P. Pouyssegur (Centre de Biochimie-CNRS, Université de Nice). 22Na+ was obtained from DuPont NEN, NHS-SS-biotin and avidin-agarose were acquired from Pierce. All other chemicals were purchased from Sigma Chemical unless stated otherwise.

Statistical Analysis

Values are means ± SE. Statistical significance was evaluated using one-way ANOVA with Bonferroni post tests to account for multiple comparisons. Significance was accepted at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Vmax Measurement of NHE1V, NHE2V, and NHE3V

To measure Vmax of NHE1V, NHE2V, and NHE3V, amiloride-sensitive 22Na+ uptake was determined as a function of extracellular Na+ concentration. The acidification protocol has been shown to give maximal transport rates with pHi ~6 (26, 35). The uptake for all NHEs conformed to simple Michealis-Menten kinetics, and the Eadie-Hoftsee analysis (V vs. V/[S]) was used to determine the Vmax and Na+ affinity constant [Km(Na+)] of the three isoforms at 22°C. Measurements of Vmax were determined in the same passage that was used to quantify the amount of NHE protein on the cell surface (see below), since the Vmax is dependent on the amount of NHE in the plasma membrane.

Analysis of the y-intercept of the Eadie-Hoftsee transformation of the transport data for NHE1V in a single experiment gave a Vmax of 147.0 nmol · mg protein-1 · min-1, and the determination of the negative slope gave an apparent Km(Na+) of 10.2 mM (Fig. 1A). Study of NHE2V gave a Vmax of 129.6 nmol · mg protein-1 · min-1 and an apparent Km of 11.6 mM (Fig. 1B). For NHE3V, Vmax was 51.8 nmol · mg protein-1 · min-1 and apparent Km was 16.6 mM (Fig. 1C). The apparent Km values for NHE1V, NHE2V, and NHE3V were similar to those determined by fluorometry in PS120 cells in a previous study (26).


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Fig. 1.   Maximal velocity (Vmax) of Na+/H+ exchangers (NHEs) stably expressed in PS120 cells. Cells were acidified with NH4Cl, and rates of H+-activated amiloride-sensitive 22Na+ uptake were measured at increasing concentrations of extracellular Na+. Uptake is expressed as amiloride-sensitive 22Na+ uptake. Vmax and apparent affinity constant (Km) for extracellular Na+ were calculated from linear transformation of data according to Eadie-Hoftsee analysis (insets), where V is velocity and [S] is substrate concentration. A: NHE1; B: NHE2; C: NHE3. Results are from a representative study from 3-5 identical experiments for each NHE, and means ± SE of triplicate estimates per extracellular NaCl concentration ([NaCl]o) are shown.

The Vmax of NHE1, NHE2, and NHE3 were also determined as previously described using fluorometry at 37°C (26). Vmax (H+ efflux rate, in µM/s) was 4,148 ± 115, 1,589 ± 293, and 1,433 ± 245 for NHE1, NHE2, and NHE3, respectively.

Fusion Proteins MBP/E3V and MBP/E2V Are Equivalently Recognized by Anti-VSVG Antibody P5D4

To determine the turnover number of NHE1, NHE2, and NHE3 in PS120 cells, the total amount of each NHE protein expressed by the cells was determined. To do this, a quantitative Western approach was used, in which dilutions of MBP/E3V fusion protein are separated by SDS-PAGE on the same gel as the NHE protein to be quantified. This method relies on the antibody used to recognize the NHE protein and the purified fusion protein equivalently. Two fusion proteins, MBP/E2V and MBP/E3V, were constructed from the last 30 and 33 amino acids, respectively, of the COOH termini of NHE2 and NHE3 plus the linker and VSVG sequence (17 amino acids). Figure 2A demonstrates the production of MBP/E3V and MBP/E2V. The fusion proteins were purified on an amylose resin column. This yielded more than one protein for both MBP/E2V and MBP/E3V, one of which was the size of the induced protein before purification (50 kDa), and other smaller products, which were probably partially degraded fusion protein or other contaminating bacterial proteins. The 50-kDa fusion proteins can be recognized with P5D4 VSVG antibody, whereas other small products did not cross-react (data not shown). To remove the latter proteins, electroelution was performed, after which both fusion proteins were homogeneous and consisted only of the 50-kDa protein. Figure 2B shows that although MBP/E3V is recognized by monoclonal anti-VSVG antibody P5D4, an equal amount of MBP is not recognized by P5D4. This confirms that P5D4 anti-VSVG antibody does not cross-react with MBP, and therefore MBP does not interfere with the quantitative Western blotting.


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Fig. 2.   A: Coomassie blue staining of maltose-binding proteins MBP/E3V and MBP/E2V. Bacteria containing MBP/E3V and MBP/E2V were induced with 0.3 mM isopropyl-beta -thiogalactoside to express fusion protein, which was then purified on an amylose column. Arrow at 50 kDa points to MBP/E3V and MBP/E2V after purification on an amylose column and subsequent electroelution. Molecular mass markers (kDa) are shown on left. B: monoclonal P5D4 antibody recognizes MBP/E3V, but not MBP alone. Equal amounts (10 ng) of MBP/E3V and MBP alone were separated on a 9% SDS-polyacrylamide gel and transferred to nitrocellulose. Western analysis was performed using P5D4. Arrow at 50 kDa points to MBP/E3V.

To determine whether the COOH termini of NHEs alter the recognition of the VSVG epitope by the antibody, Western blotting of equal amounts of MBP/E2V and MBP/E3V was performed (Fig. 3A). The signals generated by each fusion protein were then quantified using ImageQuant software, and a standard curve was generated. This yielded a linear relationship between amounts of fusion protein separated by SDS-PAGE and magnitude of the ECL signal (Fig. 3B). The magnitude of ECL was similar for the two fusion proteins at equal loading. Determination of the mean ratio of the E3V to the E2V ECL signal from four experiments showed that the ratio was close to 1 (Fig. 3C). This suggests that the immediate environment of the protein (last 30 amino acids of NHE2 vs. last 33 amino acids of NHE3) does not affect the recognition of the VSVG epitope by P5D4. Because there was no difference between recognition of MBP/E2V and MBP/E3V by P5D4 antibody, subsequently MBP/E3V was used as a standard for the quantitative Western blotting.


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Fig. 3.   P5D4 monoclonal vesicular stomatitis virus glycoprotein (VSVG) antibody recognizes MBP/E3V and MBP/E2V equally. A: equal amounts of fusion proteins MBP/E3V and MBP/E2V were separated on a 9% SDS-polyacrylamide gel and transferred to nitrocellulose. Western analysis was performed using monoclonal P5D4 antibody. A representative Western blot is shown. Arrow, size of fusion protein (50 kDa). B: MBP/E3V (black-triangle) and MBP/E2V () band density determined with ImageQuant software from Western blot in A. C: ratio of MBP/E3V to MBP/E2V band density obtained with various amounts of fusion proteins. Values are means ± SE of 4 experiments.

Determination of the Amount of NHE1, NHE2, and NHE3 in the Plasma Membrane

Quantitative Western analysis was used to determine the amounts of NHE1, NHE2, and NHE3 expressed in PS120 cells, with the MBP/E3V fusion protein as an internal standard. A standard curve of MBP/E3V protein separated by SDS-PAGE and magnitude of ECL signal was generated for each NHE isoform. From this calibration curve, picomoles of NHE present in the PS120 crude lysates were determined by linear regression analysis.

Figure 4A shows a representative Western blot to determine the amount of NHE1V protein per milligram of PS120 total protein. Determination of the density of the ECL signal for MBP/E3V gave the standard curve shown in Fig. 4B. From this it was determined that there were 35.3 pmol (0.39% wt/wt) per milligram of total PS120 protein of the 110-kDa band of NHE1 present in this passage of cells. The 85-kDa protein was not quantified, since this is the intracellular form of the protein (see below) (12, 36). The same analysis was repeated for NHE2 and NHE3. Figure 5 shows a representative experiment for NHE3V: Fig. 5A shows a representative Western blot for NHE3, and Fig. 5B shows the standard curve for this Western blot. This passage of NHE3V has 62.9 pmol (0.53% wt/wt) per milligram of PS120 protein. NHE2 expresses 24.3 pmol (0.21% wt/wt) of the 85-kDa form and 20.6 pmol (0.15% wt/wt) of the 75-kDa form per milligram of PS120 protein in a representative passage of PS120 cells (Fig. 6). The average amount of NHE per milligram of total cell protein was 26.3 ± 2.8 pmol (n = 4), 20.0 ± 4.3 pmol (n = 3), 19.4 ± 1.2 pmol (n = 3), and 70.0 ± 7.9 pmol (n = 5) for NHE1, NHE2 85-kDa form, NHE2 75-kDa form, and NHE3, respectively.


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Fig. 4.   Amount of NHE1 epitope tagged with VSVG (NHE1V) present in PS120 cells as determined by quantitative Western blot. E3V/MBP was used as an internal standard to quantify amount of NHE1V present in passage of PS120 cells used for determination of Vmax. A: 3 amounts of PS120 crude lysates expressing NHE1V were separated on same 9% gel used to separate 5 amounts of MBP/E3V. Western analysis was performed using anti-VSVG P5D4 monoclonal antibody. B: MBP/E3V band density determined from densitometry with ImageQuant software was linearly correlated with amount of separated and transferred MBP/E3V. Smallest amount of MBP/E3V could not be quantified. Amount of NHE1V protein in PS120 cells was determined from this standard curve by linear regression analysis. For NHE1V, only top band was used in quantitation. Results are representative of 4 identical experiments.



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Fig. 5.   Amount of NHE3 epitope tagged with VSVG (NHE3V) present in PS120 cells as determined by quantitative Western blot. MBP/E3V was used as an internal standard to quantify amount of NHE3V present in same passage of PS120 cells used for determination of Vmax. A: 3 amounts of PS120 crude lysates expressing NHE3V were separated on same 9% gel used to separate 5 amounts of MBP/E3V. Western analysis was performed using anti-VSVG P5D4 monoclonal antibody. B: MBP/E3V band density determined from densitometry with ImageQuant software was linearly correlated with amount of separated and transferred MBP/E3V. Smallest amount of MBP/E3 could not be quantified. Amount of NHE3V protein in PS120 cells was determined from this standard curve by linear regression analysis. Results are representative of 5 identical experiments.



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Fig. 6.   Amount of NHE2 epitope tagged with VSVG (NHE2V) present in PS120 cells as determined by quantitative Western blot. MBP/E3V was used as an internal standard to quantify amount of NHE2V present in same passage of PS120 cells used for determination of Vmax. A: 4 amounts of PS120 crude lysates expressing NHE2V were separated on same 9% gel used to separate 4 amounts of MBP/E3V. Western analysis was performed using anti-VSVG P5D4 monoclonal antibody. B: MBP/E3V band density determined from densitometry with ImageQuant software was linearly correlated with amount of separated and transferred MBP/E3V. Amount of NHE2V protein in PS120 cells was determined from this standard curve by linear regression analysis. Results are representative of 3 identical experiments.

Percentage of NHE1, NHE2, and NHE3 Present on the Cell Surface

To determine the basal turnover number for NHE1, NHE2, and NHE3, it is necessary to determine the amount of NHE protein on the cell surface under basal conditions. Cell surface biotinylation was used to determine the percentage of NHE protein at the cell surface (see MATERIALS AND METHODS). With this method, the surface proteins are labeled with biotin, which is then precipitated with avidin-agarose. Multiple rounds of avidin precipitation demonstrated that one avidin precipitation was sufficient to completely deplete the supernatant of biotinylated protein (Fig. 7). In subsequent experiments, two rounds of avidin precipitation were used to ensure complete absorption of biotinylated proteins onto avidin-agarose. Figure 8A shows representative Western blots of NHE1, NHE2, and NHE3 total protein, intracellular protein, and surface protein after cell surface biotinylation. Bands were analyzed by ImageQuant software for intensity of ECL signal, and volume of sample loaded was plotted against density of bands. An example of quantitation of percent cell surface expression is shown for NHE3V (Fig. 8B). The volume of sample required to give an identical densitometry value for all three samples (total, intracellular, and surface) when they were in their linear range was determined. There was 15.3% of total in the avidin fraction and 70.1% of total in the intracellular fraction, with a recovery of 85.4%. Figure 8C shows a summary of the percentage of NHE1, NHE2, and NHE3 on the cell surface. For NHE1, 88.8 ± 3.5% of the 110-kDa form was present on the cell surface, whereas the entire 85-kDa form was present in the supernatant (intracellular fraction, n = 4). In contrast, 14.0 ± 1.3% of NHE3 was present on the cell surface, whereas 83.0 ± 5.2% was present in the supernatant (n = 13). For NHE2, both bands were present on the cell surface: for the 85-kDa form, 64.6 ± 3.3% was located on the cell surface and 40.1 ± 7.6% was located intracellularly, whereas for the 75-kDa form, 20.0 ± 2.6% was located on the cell surface and 78.6 ± 2.7% was located intracellularly (n = 3).


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Fig. 7.   Precipitation of biotinylated NHE3 by avidin-agarose. PS120 cells expressing NHE3 were subjected to cell surface biotinylation with N-hydroxysulfosuccinimydyl-S,S-biotin, and biotinylated protein was recovered with consecutive avidin-agarose precipitations. Lane 1, Western analysis of crude membrane preparation of PS120/NHE3V; lane 2, avidin precipitation of biotinylated NHE3. Lanes 3 and 4 show no further biotinylated NHE3 precipitated, despite further exposure to avidin-agarose. Results are representative of 3 identical experiments.




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Fig. 8.   Cell surface expression of NHE1, NHE2, and NHE3 in PS120 fibroblasts. PS120 cells expressing NHE1, NHE2, or NHE3 were subjected to cell surface biotinylation with N-hydroxysulfosuccinimydyl-S,S-biotin, biotinylated protein was recovered with streptavidin-agarose, and dilutions of total, intracellular, and surface fractions were analyzed by SDS-PAGE. Bands were then quantified using ImageQuant software. A: representative Western blots of NHE1, NHE2, and NHE3 biotinylation experiments. B: NHE3 biotinylation data from A showing volume of sample loaded against arbitrary densitometry units for total (), intracellular (black-triangle), and surface () fractions. C: percent surface and intracellular fractions of total fraction expressed as mean ± SE for NHE1 (n = 4), 85- and 75-kDa forms of NHE2 (n = 3), and NHE3 (n = 13).

Calculation of Turnover Number for NHE1, NHE2, and NHE3

To calculate the turnover number of NHE1, NHE2, and NHE3, it was assumed that all the NHE present on the cell surface is active in Na+/H+ exchange. It was also assumed that the plasma membrane 75- and 85-kDa forms of NHE2 contribute equally to Na+/H+ exchange for the calculation of turnover number, because glycosylation does not appear to be important for the function of NHE2 (38). The amount of surface NHE in picomoles can be determined from the total amount expressed and the percentage on the cell surface. Vmax and amount of NHE expressed on the cell surface were measured in the same cell passage for the calculation of turnover number with use of the following formula: turnover number (cycles/s) Vmax of Na+/H+ exchange (pmol · mg protein-1 · s-1)/amount of NHE molecules on the cell surface (pmol/mg protein). Table 1 shows the calculated turnover numbers for NHE1, NHE2, and NHE3, with correlation of amount and Vmax from a particular passage. The turnover numbers of the three isoforms are not significantly different from each other when measured by Na+ uptake at 22°C (P > 0.05) and ranged from 80 to 99 s-1. When fluorometry at 37°C was used to measure Vmax, turnover numbers were as shown in Table 2: 742 ± 47, 459 ± 16, and 609 ± 39 s-1 for NHE1, NHE2, and NHE3, respectively. Although the turnover numbers were again very similar to each other and were within onefold difference, statistically the turnover number was significantly lower for NHE2 than for NHE1.

                              
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Table 1.   Turnover number of NHE1, NHE2, and NHE3 calculated from Vmax, measured by Na+ uptake at 22°C, and amount of NHE at cell surface of PS120 fibroblasts


                              
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Table 2.   Turnover number of NHE1, NHE2, and NHE3 calculated from Vmax, measured by fluorometry at 37°C, and amount of NHE at cell surface of PS120 fibroblasts


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study we have determined the turnover numbers of NHE1, NHE2, and NHE3 expressed in the same cell type (PS120) by correlating the Vmax (as measured by 22Na+ uptake kinetic studies and fluorometry) with the amount of NHE protein expressed at the cell surface. Previous kinetic studies in PS120 cells by use of fluorometry have determined the Km(Na+) to be 15-18 mM for rabbit NHE1, NHE2, and NHE3 (26), similar to values determined in this study by Na+ uptake (10-17 mM). The Km(Na+) for rat NHE1 and NHE3 in Chinese hamster ovary cells is also similar (4.7 and 10 mM, respectively) (32). However, rat NHE2 has a much higher Km(Na+) (50 mM) (43). This suggests a species variation between rat and rabbit NHE2 for extracellular Na+ affinity.

A quantitative Western analysis approach was used to determine the amount of NHE protein expressed in PS120 cells. This was based on the use of a fusion protein standard that contained the epitope with which all the NHE proteins to be quantified were tagged. An advantage of this technique is that the same antibody is used to quantify the amount of NHE1V, NHE2V, or NHE3V expressed. This technique relies on the ability of anti-VSVG antibody to recognize the VSVG epitope independently of the parent molecule (NHE1V, NHE2V, NHE3V, MBP/E2V, or MBP/E3V). To demonstrate that this was likely to be the case, we constructed two MBP fusion proteins of the COOH termini (including the VSVG epitope tag) of NHE3V and NHE2V. These two fusion proteins gave similar densitometry units at equal amounts of fusion protein, analyzed by SDS-PAGE (Fig. 3). This suggests that the rest of the protein does not affect the recognition of the antibody to the VSVG epitope. In addition, the antibody used in this study was raised against the VSVG epitope alone (22). Therefore, it is assumed that the VSVG epitope is recognized equally by P5D4 antibody for NHE1V, NHE2V, NHE3V, MBP/E2V, and MBP/E3V. We then quantified the amount of NHE protein present in PS120 cells by comparing the density of the bands of a series of dilutions of known amounts of the fusion protein MBP/E3V with the density of the bands of NHE1V, NHE2V, and NHE3V. The NHE proteins contributed 0.37-0.53% (wt/wt) of total PS120 protein. Cell surface biotinylation was used to determine the percentage of total NHE protein present on the cell surface, because this is the NHE pool involved in basal Na+/H+ exchange. This method requires that 1) there is high efficiency of biotinylation, 2) the density of bands obtained by Western analysis be within the linear range and not saturated, and 3) recovery of the intracellular plus surface fractions should be ~100%. We set our criteria that recovery of intracellular and surface fractions be >85% to be certain that all NHE was accounted for as surface or intracellular forms. Three dilutions of each sample (total, intracellular, and surface) were used to establish that band density was within the linear range and saturation had not been reached. The majority of the 110-kDa mature glycosylated form of NHE1 (88%) was located on the cell surface, as has been determined previously (12, 36). This demonstrates that the efficiency of NHE biotinylation is >= 88%. The 85-kDa core glycosylated form, which has previously been shown to be present only intracellularly (12), is only present in the intracellular fraction, which demonstrates that only cell surface proteins are biotinylated. NHE3 was located predominantly intracellularly, as shown previously (1, 23). It has previously been shown that NHE2 transfected in PS120 cells exists in two forms: a 75-kDa form that is neither N- nor O-glycosylated and an 85-kDa form that is O-glycosylated (38). In this study we demonstrated that the predominant form of NHE2 on the cell surface is the 85-kDa form (64% of total 85-kDa form), whereas only 20% of the 75-kDa form was on the cell surface. The cell surface expression of the 85-kDa glycosylated form is similar to the cell surface expression of NHE1. In contrast, the unglycosylated 75-kDa form is predominantly intracellular, similar to NHE3. NHE3 expressed in PS120 cells is also neither N- nor O-glycosylated (5, 6). Protein kinases and growth factors regulate NHE2 by a change in Vmax (26, 27). It is not known whether this Vmax change involves changes in the cell surface expression of the predominantly intracellular 75-kDa form, as seen with NHE3 (20), or whether 75- and 85-kDa forms are involved. To determine the amount of NHE2 on the cell surface, it was necessary to quantify the 75- and 85-kDa bands on the assumption that both forms on the cell surface contribute to the transport of Na+ and H+.

A few studies have quantified the turnover number of the NHEs. However, this is the first study in which all three isoforms have been studied simultaneously, allowing direct comparison in the same cell system. This allows comparison of intrinsic properties of the exchanger, while the characteristics of the cell environment such as membrane lipid composition, cytoskeletal properties, and other associated proteins, which may affect the turnover number, remain constant. For example, an increase in membrane fluidity resulted in a decrease in the transport activity of NHE1 and NHE3 in fibroblasts, partially by directly or indirectly altering the active state of the NHE protein (7). In the present study, all three NHE isoforms had a turnover rate of ~80-100 s-1 when Na+ uptake was used to measure Vmax at 22°C and a turnover number of 459-742 s-1 when fluorometry at 37°C was used. NHEs in lymphocytes (likely to be NHE1) have a high temperature coefficient (Q10) of 3.1 (28). When this is taken into account, the estimated turnover number for NHE1 determined by fluorometry at 22°C is 142 s-1, which is close to that measured by 22Na+ uptake (80 s-1; Table 1). The Q10 for NHE2 and NHE3 is not known, and thus comparison cannot be made. A possible explanation for our observation that NHE2 has a significantly lower turnover number at 37°C is that NHE2 might have a smaller Q10 than NHE1. NHE3 and NHE1 may have similar Q10 values, since the turnover numbers are not significantly different from each other at 22 and 37°C.

The turnover numbers determined in the present study contrast with previous estimations of turnover for NHE1 of 2,000-3,000 s-1 (13, 36, 37). Differences in cell type may result in differences in turnover number. It has been demonstrated that virally transforming lymphocytes or fibroblasts increases the turnover of NHE1 by about threefold (36). Other studies of nonpump transmembrane transport proteins have shown turnovers in a range similar to NHE in this study. The mammalian Na+-glucose cotransporter SGLT1 has a reported turnover number of 19-38 s-1 (17, 34, 42), the plant H+-hexose cotransporter (STP1) has a turnover number of 59 s-1 (9) (all measured at 22°C), and a mammalian Na+-K+-2Cl- transporter has a turnover of 255 s-1 at 37°C (21). A plant H+-amino acid transporter has a slightly higher transport rate of 300-800 s-1 at 22°C (8), as does the mitochondrial K+/H+ antiporter (700 s-1 at 37°C) (31). Therefore, the value obtained in the present study for the transport of NHE is comparable to most other cotransporter or exchanger transmembrane transport proteins. One exception is that of erythrocyte band 3 protein, an anion exchanger that has a turnover number estimated to be 50,000 s-1 at 37°C (10).

The turnover numbers for the NHE isoforms determined in PS120 fibroblasts in this study are similar. Given the different cell and subcellular localizations, cell surface vs. intracellular expression, functions, and differences in the kinetics of regulation of the Na+/H+ exchange isoforms, it was expected that they would also have different basal turnover numbers. For example, isoforms of the GLUT transporter family have been shown to have different functions and different corresponding turnover rates. Although GLUT-1, which is ubiquitously expressed in mammalian cells, has a turnover number of 123 s-1, GLUT-3, which is localized to the brain and plays a role in maintaining brain glucose concentration, has a higher turnover number of 823 s-1, both measured at 25°C (29). The results in the present study suggest that, in an identical lipid/protein environment, the NHEs have conserved among them regions that determine turnover number. This is despite the fact that they have very divergent COOH-terminal intracytoplasmic tails, which are known to interact with the NH2 terminus during basal and regulated states (45). In epithelial cells, turnover numbers of NHE1, NHE2, and NHE3 may be different from those determined in the present study, since extrinsic properties such as cytoskeleton, the cellular membrane lipid environment, and proteins associated with the COOH terminus of the NHE will be more variable. Using a quantitative Western approach similar to that used here and a previously published value of Vmax measured at 25°C (41), our group previously estimated the turnover number of NHE3 to be 458 s-1 in ileal brush border. The regions of the NHEs that determine turnover number, which will give insight into the mechanism by which Na+ is exchanged for H+, remain to be identified.


    ACKNOWLEDGEMENTS

M. E. Cavet was supported by a Wellcome International Travelling Fellowship (United Kingdom). F. S. De Medina was supported by a grant from the Spanish Ministry of Education. These studies were also supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-26523, PO1-DK-44484, and DK-51116, the Meyerhoff Digestive Diseases Center, and the Hopkins Center for Epithelial Disorders.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C.-M. Tse, Ross 925, Gastrointestinal Div., Dept. of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205-2195 (E-mail: mtse{at}welchlink.welch.jhu.edu).

Received 29 March 1999; accepted in final form 4 August 1999.


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