Gastrointestinal Division, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2195
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ABSTRACT |
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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 s1, 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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 forNa+/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-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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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
protein1 · 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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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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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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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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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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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
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DISCUSSION |
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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 s1 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 s1 (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 s1, 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.
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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.
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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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