Departments of 1 Medicine, 4 Cell Biology, and 5 Physiology, Gastrointestinal Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2195; 2 Department of Physiology, University of Montreal, Montreal, Canada H3C 3J7; and 3 Curie Institute, Paris, France 75231
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ABSTRACT |
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Cell biological approaches were used to examine the location and function of the brush border (BB) Na+/H+ exchanger NHE3 in the opossum kidney (OK) polarized renal proximal tubule cell line. NHE3 epitope tagged with the vesicular stomatitis virus glycoprotein epitope (NHE3V) was stably expressed and called OK-E3V cells. On the basis of cell surface biotinylation studies, these cells had 10-15% of total NHE3 on the BB. Intracellular NHE3V largely colocalized with Rab11 and to a lesser extent with EEA1. The BB location of NHE3V was examined by confocal microscopy relative to the lectins wheat germ aggluttinin (WGA) and phytohemagluttin E (PHA-E), as well as the B subunit of cholera toxin (CTB). The cells were pyramidal, and NHE3 was located in microvilli in the center of the apical surface. In contrast, PHA-E, WGA, and CTB were diffusely distributed on the BB. Detergent extraction showed that total NHE3V was largely soluble in Triton X-100, whereas virtually all surface NHE3V was insoluble. Sucrose density gradient centrifugation demonstrated that total NHE3V migrated at the same size as ~400- and ~900-kDa standards, whereas surface NHE3V was enriched in the ~900-kDa form. Under basal conditions, NHE3 cycled between the cell surface and the recycling pathway through a phosphatidylinositol (PI) 3-kinase-dependent mechanism. Measurements of surface and intracellular pH were obtained by using FITC-WGA. Internalization of FITC-WGA occurred largely into the juxtanuclear compartment that contained Rab11 and NHE3V. pH values on the apical surface and in endosomes in the presence of the NHE3 blocker, S3226, were elevated, showing that NHE3 functioned to acidify both compartments. In conclusion, NHE3V in OK cells exists in distinct domains both in the center of the apical surface and in a juxtanuclear compartment. In the BB fraction, NHE3 is largely in the detergent-insoluble fraction in lipid rafts and/or in large heterogenous complexes ranging from ~400 to ~900 kDa.
sodium/hydrogen exchanger 3; opossum kidney cells; sodium absorption; phosphatidylinositol 3-kinase; recycling endosomes; pH
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INTRODUCTION |
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THERE ARE CURRENTLY SEVEN cloned mammalian Na+/H+ exchangers (NHEs), which differ from each other in tissue distribution, pharmacological inhibitory profiles, organellar subcellular localization, and function (41, 53). Based on a combination of function and cellular localization, one classification of these exchangers has divided them into housekeeping, epithelial, and intracellular isoforms. For instance, by this classification, NHE1 (ubiquitously expressed) and NHE4 (expressed in kidney, stomach, intestine, uterus, kidney, brain, and skeletal muscle) are considered housekeeper isoforms (43, 46, 52). NHE2 and NHE3 are apical membrane exchangers in small intestine colon (NHE2, NHE3), gallbladder (NHE3), proximal tubule (NHE3), thick ascending limb of Henle (NHE2, NHE3), and distal collecting system (NHE2), among other organs (9, 38). NHE6 and NHE7 are present on the membranes of intracellular organelles; NHE7 is present in the trans-Golgi network (TGN), and NHE6 is in the recycling system although initially suggested as being in mitochondria (5, 37, 38).
A weakness of this classification is that NHE3 is not only present on the plasma membrane but is also present in recycling endosomes. In fact, in PS120 and AP-1 fibroblasts, only ~15% of NHE3 is on the plasma membrane, whereas ~85% is intracellular (1, 11, 19, 20). Furthermore, potent agonists such as serum increase plasma membrane NHE3 activity by a maximum of twofold, leading us to question whether intracellular NHE3 may be serving a second function in addition to being a reservoir for plasma membrane recruitable NHE3. The concept of intracellular NHE3 serving an independent function from transcellular Na+ absorption is strengthened by the observations that, under basal conditions, NHE3 continuously cycles from the cell surface to an endosomal compartment through a phosphatidylinositol (PI) 3-kinase-dependent exocytosis mechanism (10, 14). Furthermore, signals within the COOH-terminus of NHE3 are involved in mediating the rapid basal endocytosis of the exchanger (1). In fact, recent work has demonstrated that endosomal NHE3 in opossum kidney (OK) cells may function to both acidify endosomes and to facilitate vesicular fusion (14, 15).
When considering the role of NHE3 in epithelial cells, it becomes important to identify the subcellular distribution in which NHE3 occurs. For instance, in renal proximal tubule, NHE3 is present in the brush border (BB) and in subapical vesicles, which are likely to represent endosomes, although how much NHE3 exists in each pool and the specific nature of this pool was not reported (4).
In this study, we used proximal tubule-derived polarized OK cells to
examine the subcellular distribution of NHE3 and the function of its
multiple pools. Our results show that, at steady state, the majority of
NHE3 in OK cells resides in an intracellular recycling endosomal
compartment. This pool of NHE3 moves to the surface in a PI
3-kinase-dependent manner. NHE3 functions on both the plasma membrane
as well as in intracellular endosomes. In addition to its role in
transcellular NaCl and HCO
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MATERIALS AND METHODS |
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Cell culture. OK-P cells expressing NHE3 endogenously (generously provided by Dr. O. Moe, University of Texas, Southwestern; Refs. 10, 35, and 55) and OK-E3V cells (stably expressing rat NHE3 epitope tagged on the COOH-terminus with the VSVG epitope as described in Ref. 36; generously provided by Dr. J. Noel, University of Montreal) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 25 mM NaHCO3, 10 mM HEPES, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum in a 5% CO2-95% O2 incubator at 37°C. OK-E3V cells were routinely selected for Na+/H+ exchange activity (every other passage) by exposing cells to an acid load consisting of 50 mM NH4Cl-94 mM NaCl solution for 1 h, followed by an isotonic 2 mM Na+ solution, as described previously (31). Surviving cells were then placed in normal culture medium and allowed to reach confluence. Except where stated, cells were studied after being serum starved for 3 days after reaching confluence, which increases Na+/H+ activity (10).
In some experiments, a fusion protein of NHE3 and enhanced green fluorescent protein (eGFP) at its COOH-terminus was transiently transfected into 50% confluent monolayers of OK-E3V cells using Lipofectamine (Invitrogen, GIBCO BRL), following the manufacturer's recommendations. This NHE3 chimera has been described previously and shown to behave like wild-type NHE3 in terms of percentage expressed on the surface of PS120 cells, basal transport, and fibroblast growth factor-stimulated and wortmannin-inhibited transport (19, 20). Twenty-four hours after transfection, serum was removed, and the cells were studied seventy-two h later.Measurement of Na+/H+ exchange by fluorometry/BCECF. Cells were seeded on glass coverslips and grown until they reached 100% confluency. Transport was measured as described (31) by using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM (BCECF-AM; Molecular Probes). In the wortmannin experiments, the cells were incubated with 100 nM wortmannin for 60 min at 37°C. Control and test experiments were done on the same day by using parallel coverslips.
Fluorescence microscopy for pH measurements of surface and juxtanuclear compartments. A confluent cell monolayer on a glass coverslip was used at room temperature in a chamber mounted on the stage of an inverted microscope (25). Wheat germ agglutinin (WGA) conjugated with FITC was employed as the pH indicator and was initially exposed to OK cells for 30 min at 22°C. Excitation-ratio imaging at 440 and 490 nm was achieved with a 75-W xenon lamp, epifluorescence illumination, a ×100/1.3 N.A. objective lens, and excitation filters (Omega Optical, Brattleboro, VT) on a filter wheel. The microscope output port was connected to an intensified charge-coupled device camera (Hamamatsu). Images were digitized at 8 bits and stored on computer for offline analysis. For each monolayer, two pairs of images from the cell surface and in the juxtanuclear area were collected just superior to the nucleus under control conditions and during perfusion with the NHE3 inhibitor S3226 (100 nM; kindly provided by Dr. H. Lang, Aventis). The fluorescent images were analyzed for pH by choosing five to eight representative cells from each pair of images. The intensity ratio for each cell on the cell surface and the intracellular juxtanuclear region (selected visually) was calculated by using image processing MetaMorph software and compared with a pH calibration curve.
A pH calibration curve was generated by measuring the ratio of fluorescence emission intensities with the 440- and 490-nm excitation of WGA-FITC dissolved in buffer solutions with known pH values. A 20-µl drop of solution was placed on a coverslip for each of five different pH calibration standards from pH 5.5 to 8.0, a pair of images was taken, and fluorescence intensity ratio was determined.Cell surface biotinylation. OK cells were grown to 100% confluence in 10-cm petri dishes. For wortmannin studies, 100 nM wortmannin were added for 60 min at 37°C. All subsequent manipulations were performed at 4°C. Cells were washed twice in phosphate-buffered saline (PBS; 150 mM NaCl and 20 mM Na2HPO4, pH 7.4) and once in borate buffer (154 mM NaCl, 10 mM boric acid, 7.2 mM KCl, and 1.8 mM CaCl2, pH 9.0). The surface plasma membrane proteins were then biotinylated by gently shaking the cells for 40 min with 3 ml of borate buffer containing NHS-SS-biotin (0.75 mg/ml; Pierce), with a fresh 3 ml of the same biotinylation solution added at 20 min. The cells were then washed extensively with the quenching buffer (20 mM Tris and 120 mM NaCl, pH 7.4) to scavenge the unreacted biotin and subsequently washed twice with PBS.
Cells were scraped and solubilized with 1 ml of N+ buffer (60 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM KCl, 5 mM EDTA trisodium, 3 mM EGTA, 1 mM Na3VO4, and 1% Triton X-100), sonicated for 20 s, agitated on a rotating rocker at 4°C for 30 min, and then centrifuged at 5,000 g for 10 min to remove insoluble cellular debris. To determine the percentage of cell surface NHE3V, biotinylated surface proteins were precipitated by two 2-h incubations with streptavidin-agarose beads at 4°C. The depletion of biotinylated proteins was ascertained by the absence of proteins in a further avidin precipitation. Biotinylated proteins bound to the avidin-agarose beads were eluted by heating in Laemmli sample buffer and were subsequently analyzed in an immunoblot (1). Biotinylated NHE3V was quantitated by using monoclonal antibodies to the VSVG epitope (P5D4 hybridoma tissue culture supernatant). The amount of surface NHE3 was expressed as a percentage of total cellular NHE3, which was determined by using Western analysis of whole cell lysates (1). In some experiments, NHE3V was immunoprecipitated from the biotinylated cell extract by using anti-VSVG monoclonal antibodies and visualized through Western analysis. Surface NHE3V was recognized by using horseradish peroxidase-conjugated streptavidin (HRP-streptavidin; Molecular Probes), whereas total NHE3V was recognized by using anti-VSVG monoclonal antibodies followed by exposure to enhanced chemiluminescent solution.Cell fractionation into detergent-soluble and detergent-insoluble fractions. Surface NHE3 was separated into detergent-soluble (DS) and detergent-insoluble (DI) fractions. Cells were grown to confluency in 10-cm Petri dishes and biotinylated at 4°C, as described in Cell surface biotinylation. Cells were then solubilized in 50 mM MES buffer, pH 6.4, containing 60 mM NaCl, 3 mM EGTA, 5 mM MgCl2, 1% Triton X-100, and 1 mM vanadate plus 1:1,000 protein-inhibitor cocktail (Sigma), sonicated, and spun briefly to remove unbroken cells, nuclei, and cell debris. The resulting supernatant was subsequently centrifuged at 100,000 g for 30 min to recover the DI fraction, while the supernatant was designated as the DS fraction. The DI pellet was solubilized in RIPA buffer [150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris · HCl, pH 8.0]. Surface NHE3V was avidin precipitated as described in Cell surface biotinylation. Equal volumes of surface DS and DI fractions and total DS/DI fractions were separated by SDS-PAGE.
Sucrose gradient centrifugation.
Cells were grown to confluency in 10-cm petri dishes. Cells were then
solubilized in 1 ml of N+ buffer (see Cell surface
biotinylation), sonicated, and spun briefly to remove unbroken
cells, nuclei, and cell debris. Solubilized cell extracts (4°C or
1°C in 1% Triton X-100) were applied to the top of discontinuous
5-25% sucrose gradients (increasing at increments of 2.5%
sucrose containing 0.1% Triton X-100). After centrifugation for
16-18 h at 4°C at 150,000 g in an Sw41 rotor, the
gradients were fractionated (0.6 ml) from the bottom with a pump and
NHE3 immunoprecipitated with anti-VSVG monoclonal antibody from each
fraction. Complex size was estimated by comparison to standard proteins
(-amylase = 200 kDa, apoferritin = 443 kDa, and
thyroglobulin = 669 kDa) run on parallel identical sucrose gradients.
Immunofluorescence. Cells were seeded on glass coverslips, and grown until they reached 100% confluency, and then studied 72 h after serum was removed. Cells were fixed in 4% paraformaldehyde/PBS for 10 min at room temperature. The residual paraformaldehyde was neutralized with 20 mM glycine in PBS for 10 min. Cells were then permeabilized for 30 min in 0.1% saponin/PBS before being blocked for 30 min in 1% BSA/PBS supplemented with 10% FBS. Cells were incubated in primary antibody (at the indicated concentrations) in PBS for 60 min at room temperature. After three 10-min washes in 0.1% saponin/PBS, secondary antibodies (Alexa conjugates, Molecular Probes) were added at 1:100 in PBS, incubated for 30 min, and again washed three times for 10 min. Cells were then mounted on slides with Prolong (Molecular Probes) antifade reagent and viewed on a Zeiss LSM 410 confocal fluorescent microscope. For lectin experiments, cells were labeled at 4°C with 100 µg/ml fluorescently labeled lectins for 30 min before fixation [WGA, phytohemagluttin E (PHA-E), cholera toxin B (CTB) subunit, or peanut agglutinin; Sigma].
Antibodies. Anti-VSVG monoclonal antibodies were generated from P5D4 hybridoma supernatant (26) and used at a dilution of 1:5 in PBS for immunoprecipitation. Polyclonal anti-VSVG antibodies 1648 were as described (14) and used at a dilution of 1:1,000. Polyclonal antibodies to endogenous OK NHE3 (Ab 5683) were a generous gift from Dr. Moe and were used at a dilution of 1:2,000 (10). Rab11 (monoclonal) and EEA1 (polyclonal) antibodies were purchased from Transduction Laboratories (Lexington, KY) and used at dilutions of 1:1,000. Monoclonal antibodies to Na+-K+-ATPase were a gift from Dr. Fambrough (Department of Biology, Johns Hopkins University School of Medicine) (13) and were used at a dilution of 1:1,000. Monoclonal anti-Biotin conjugated with HRP was from Jackson ImmunoResearch Lab (200-032-096).
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RESULTS |
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NHE3V in OK-E3V cells is expressed at a similar level as endogenous
NHE3 in OK-P cells.
OK-P cells express endogenous NHE3 as their only NHE isoform. In
preliminary immunofluorescence experiments, antibodies to endogenous
NHE3 (Ab 5683) did not give a clean signal. Accordingly, we studied OK
cells stably expressing epitope-tagged NHE3 (OK-E3V), which was
identified with anti-VSVG monoclonal antibodies (P5D4). OK-E3V cells
were derived as previously described (36). Briefly, a
population of OK cells (OK-Tina) was subjected to repeated cycles of
acid suicide to reduce expression of endogenous NHE3. Transport experiments demonstrated that expression of endogenous NHE3 was reduced
to ~10% of the wild-type cell line. The low-expressing cells were
subsequently stably transfected with COOH-terminal VSVG-tagged rat NHE3
(NHE3V). Western analysis of OK-P and OK-E3V cell lysates using
antibodies to endogenous NHE3 demonstrated that endogenous OK NHE3
migrated as a 90-kDa band, whereas NHE3V migrated as a slightly smaller
85-kDa band. Only the smaller band was recognized by antibodies to the
VSVG epitope (Fig. 1A).
Comparisons of the NHE3 expression levels in the two cell lines
showed that OK-P and OK-E3V cells expressed approximately equal levels
of NHE3 (see Fig. 1A).
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A minor fraction of both endogenous NHE3 and NHE3V targeted to the
OK apical membrane-BB NHE3V is predominantly present in a central
apical surface location.
Surface expression of endogenous NHE3 and transfected NHE3V in OK cells
was monitored by using cell surface biotinylation. Western analysis of
total and surface biotinylated proteins using both endogenous NHE3
antibodies and anti-VSVG antibodies showed that only a minor fraction
of both NHE3 and NHE3V (~10-15%) was successfully biotinylated
(Fig. 2A).
Because we could not eliminate the possibility that surface
biotinylation in OK cells was incomplete, we next examined the cellular
location of NHE3 with confocal microscopy. To mark the BB,
fluorescently labeled lectins and CTB subunit were applied to the
apical surface of live cells at 4°C. OK cells grown on glass
coverslips were polarized with BB facing the free solution. These cells
mimicked the binding specificities of native proximal tubule cells in
that their apical membranes stained positively with PHA-E, CTB subunit,
and WGA but not peanut agglutinin (data not shown) (Fig. 2,
B, D, and F; Ref. 8). The
positive lectins and CTB exhibited different surface-binding patterns.
PHA-E and CTB labeled the microvilli diffusely and also labeled the
junctional areas of cells (Fig. 2, B and D). WGA
had less junctional labeling and less staining of the most lateral
microvilli compared with PHA-E and CTB but also otherwise labeled
microvilli diffusely. NHE3 was visualized in the same cells (Fig. 2,
C, E, and G). NHE3 was present in the
BB where it had predominantly a central apical location, although some
diffuse apical NHE3 was present. The fact that NHE3 was not diffusely
present in the microvilli is shown in Fig. 2, C,
F, and G, in which peripheral microvilli as
marked by lectins and CTB lack NHE3.
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Characteristics of surface NHE3 in OK-E3V cells: surface NHE3 is in
a large DI complex.
The central clustered distribution of BB NHE3V suggested that it might
be tethered to prevent diffuse BB distribution. Studies of renal
proximal tubule have shown that endogenous NHE3 has limited solubility
with various detergents, further suggesting the link between NHE3 and
cytoskeleton (3). To investigate these potential interactions, the solubility characteristics of both total and surface
NHE3V in OK cells were examined. Surface NHE3V was biotinylated before
separating DS and DI fractions. Figure 4
demonstrates that, whereas the majority of total NHE3V is in the DS
pool, surface NHE3 is only ~10% in the DS (~90% in the DI)
fraction.
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Wortmannin treatment reduces both transport rates and surface
levels of NHE3.
NHE3 stably transfected into both PS120 and AP-1 fibroblasts is
found both in an intracellular recycling endosome compartment as well
as on the plasma membrane. Under basal conditions, the exchanger
rapidly trafficks between the recycling endosomes and the cell surface
in a PI 3-kinase-dependent manner. In fibroblasts, wortmannin, a potent
inhibitor of PI 3-kinase, reduces the transport rates and surface
levels of NHE3 by inhibiting the exocytosis of the exchanger back to
the plasma membrane (19, 27). Because NHE3V in OK cells
also appeared to be targeted to both cell surface and intracellular
sites, it was determined whether NHE3, in the context of a polarized
epithelial cell, also underwent rapid basal recycling. Wortmannin
treatment (100 nM, 60 min, 37°C) significantly reduced
Na+/H+ exchange rates in both OK-P and OK-E3V
cells (Fig. 6). Wortmannin reduced NHE3
transport by 57% in OK-P cells and 51% in OK-E3V cells. Cell surface
biotinylation indicated that the reduction in transport rates was
accompanied by a corresponding reduction in surface NHE3 levels.
Surface levels of endogenous NHE3 in OK-P cells were reduced by ~50%
with 1 h of wortmannin treatment. Surface levels of NHE3V in
OK-E3V cells showed a similar reduction (Fig. 6).
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NHE3V functions in both surface and intracellular locations.
Studies were undertaken to determine whether NHE3 functioned in
both its major cellular locations, on the cell surface and in an
intracellular Rab11-positive endosomal pool. pH in these compartments
was measured by exposing the OK cells to FITC-WGA at 22°C for 60 min,
allowing the cells to take up the WGA. Of note, after 60 min at 22°C,
WGA, NHE3V, and Rab11 had similar locations based on confocal
colocalization studies (compare Figs. 3C and 7A).
To ascertain the role of functional NHE3V, the exchanger was inhibited
by exposure to the specific blocker S3226 (100 nM; 48), and the pH on
the surface as well as inside the cell was compared before and after
NHE3V inhibition (Fig. 7B).
The surface pH after S3226 exposure as measured by FITC-WGA was
alkalinized, suggesting that NHE3 activity contributed to the low pH of
the BB surface. The intracellular compartment marked with WGA also alkalinized with S3226 exposure. Because FITC-WGA was inside this intracellular compartment, this result indicated that the function of
NHE3 in that compartment was to acidify it.
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DISCUSSION |
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Although BB membrane Na+/H+ exchange in epithelial cells has been partially characterized, studies have not specifically used cell biological approaches to examine the subcellular localization of the apical Na+/H+ exchanger isoform, NHE3. Studies of NHE3 transfected into fibroblasts have shown that the protein is expressed in a surprising pattern for an exchanger postulated to work only on the plasma membrane. Specifically, only a minor fraction of NHE3 in two unrelated fibroblast lines is targeted to the plasma membrane, whereas the vast majority is retained in a recycling endosome compartment (11, 19). There are other reasons to suspect that NHE3 is present in multiple subcellular locations. We have previously shown that the COOH terminus of NHE3 contains at least two independent signals that determine the endocytic rate and, consequently, the plasma membrane levels of the exchanger (1). Furthermore, a number of studies have demonstrated a role for membrane trafficking in the regulation of NHE3 function in both polarized epithelial cells and native tissue (18, 21, 42, 55-58). Finally, electron microscopy of NHE3 in kidney proximal tubule has shown some vesicular staining in addition to BB membrane and intermicrovillar cleft populations (3, 4). Despite the evidence suggesting the existence of multiple pools of NHE3 in kidney and intestine, little has been reported on the subcellular distribution of NHE3 in physiological settings. Unlike the situation of other transport proteins such as GLUT4, gastric H+-K+ ATPase, or aquaporin-2, which are present largely in intracellular pools under basal conditions and respond to physiological stimuli by massive mobilization to the plasma membrane (12, 17, 40), the more subtle peak stimulation of NHE3 (amount of plasma membrane NHE3 increases ~2 times) prompted us to question what other function the intracellular exchanger might have (1, 19, 32).
For these studies, the OK polarized proximal tubule cell line was chosen because these cells express endogenous NHE3 that is correctly targeted to the apical membrane and is sensitive to regulation by such physiological agents as dopamine, endothelin, and parathyroid hormone (PTH; Refs. 10, 18, 29, and 36). Furthermore, the existence of stably transfected NHE3V-expressing cells allowed us to study the expression patterns of the exchanger in a setting that demonstrated similar transport rates to the endogenous exchanger (Fig. 1). Both cell surface biotinylation studies and confocal microscopy experiments showed that the minority of total cell NHE3V is on the BB (10-15% of total), whereas the majority of NHE3V is intracellular, with most in the Rab11-associated recycling compartment (Figs. 2A and 3, A and B). Moreover, as also seen in the fibroblast cell lines, NHE3V cycles under basal conditions from the cell surface to an endosomal population through a mechanism that is at least partially regulated by PI 3-kinase dependent exocytosis. Inhibition of PI 3-kinase by wortmannin decreases both the percentage of NHE3V on the plasma membrane and the transport rates of the protein (Fig. 6). The PI 3-kinase-dependent cycling of NHE3 under basal conditions has been found in all cell types studied, polarized and symmetrical, and appears to be a general property of NHE3.
Although some NHE3V colocalized with EEA1, a marker for early endosomes, the distribution of NHE3V more closely mirrored that of Rab11, a marker for recycling endosomes (Fig. 3B). Although NHE3 is endocytosed under basal conditions, the greater colocalization with Rab11 would suggest that NHE3 is either preferentially retained in the Rab11-positive compartment or internalized through a population of EEA1-negative endosomes. Studies with the apical Na+-phosphate cotransporters NaPi-2 and NaPi-4 in OK cells have shown that both proteins are rapidly internalized with PTH stimulation and then degraded in subapical lysosomes (23, 24). It remains to be determined what signals mediate why endocytosed NHE3 is targeted to be recycled, whereas NaPi-2/4 are degraded.
We characterized the BB expression of NHE3 in OK cell monolayers. Several fluorescent-tagged lectins and CTB were used to label the microvillar surface. PHA-E and CTB both showed a diffuse surface distribution with clear labeling of the junctional areas. In contrast, although WGA also labeled microvilli, it appeared to be excluded from peripheral microvilli and the junctional areas. Staining of NHE3V in the same 0.5-µM X-Y confocal sections showed that the exchanger was not distributed diffusely on the OK apical surface. Rather, NHE3 seems to be in a cluster of centrally localized microvilli on the apical surface of most OK cells (Fig. 2, B--G). In a small fraction of cells, NHE3 demonstrated a more uniform distribution in clumped microvilli, an observation also made by Moe and colleagues for NHE3 and similar to distribution of NaPi-2 and NaPi-4 in OK cells (Refs. 18, 22; S. Akhter and M. Donowitz, unpublished results). Although NHE3 was concentrated toward the center of the apical cell surface, there was detectable staining throughout the BB, including toward the junctional complexes. Of note, the difference in the microvillar distribution of NHE3 compared with the lectins and CTB showed that it is NHE3 that is in a centralized location rather than the centralized location of the microvilli. The surface expression of NHE3V may reflect subdomains of protein expression on the BB of OK cells. Thus epithelial cell proteins may be targeted not only to the apical or basolateral domains but may be more specifically delivered to or retained at physical subdomains within these larger areas. Several other transport proteins have been shown to have restricted distributions on the surface of epithelial cells. 1) Functional studies by Oberleithner demonstrated that Na+ channels in Madin-Darby canine kidney (MDCK) cells had such a central localization (36, 39); 2) K+ channel, Kv1.4, clustered at the leading edge of migrating MDCK cells (47); 3) NHE1 is localized to pseudopodial protrusions in invasive MDCK cells (28); and 4) Sorribas et al. (50) showed that NaPi-2 and NaPi-4 in OK cells were expressed only in the clumped microvilli and were largely excluded from the intermicrovillar clefts. The existence of plasma membrane subdomains has been clearly demonstrated in neurons. Although the mechanism underlying these plasma membrane subdomains in epithelial cells in unknown in neurons, isoform-specific targeting of K+ channels in the dendrite is mediated through such diverse mechanisms as interactions with binding proteins, associations with the cytoskeleton, or targeting to LRs or caveolae (33, 34).
On a biochemical level as well, BB NHE3 was shown to be made up of multiple pools and not to consist of a single population of exchanger. This was demonstrated by 1) the fact that NHE3 exists in OK cells in DS and DI populations; although total NHE3V is largely soluble in detergent, surface NHE3V is almost entirely insoluble (Fig. 4); and 2) sucrose density gradient centrifugation of total cell protein. The total NHE3V is present in complexes of multiple sizes, with two predominant peaks of ~900 and ~400 kDa. Of interest, total NHE3V consists almost entirely of the 400-kDa pool, whereas surface NHE3V was fairly evenly distributed between the 900- and 400-kDa populations (Fig. 5A). Whether this represents NHE3 in multiprotein complexes and/or NHE3 presence in LRs, as we have documented occurs (32), is not known. The preliminary studies reported in Fig. 5B comparing complex size in apical surface NHE3 exposed to Triton X-100 at 4 and 15°C (difference interpreted as LR contribution) did not reveal a role for LRs in large surface NHE3 complexes under basal conditions. However, it will be important to extend these studies at least by using other ways to define LR and with stimulated NHE3.
In addition, enrichment of NHE3V in large DI complexes on the apical surface of OK cells suggests that the different pools of NHE3 may fulfill distinct functional roles. The endocytosis of albumin in the kidney proximal tubule, for instance, is mediated through binding such large scavenger receptors as megalin and cubilin (7). Independent studies have shown that 1) inhibition of NHE3 virtually eliminates receptor-mediated albumin uptake by OK cells (14, 15), and 2) proximal tubule NHE3 tightly associates with megalin, denoting a megalin-associated, inactive form of the exchanger which is targeted to the intermicrovillar microdomain of the apical plasma membrane (2, 3). Whether the 900-kDa surface NHE3 in OK cells also include a scavenger receptor isoform and whether it is this association that mediates the NHE3-dependent albumin uptake is not known.
A postulated function for the presence of BB NHE3 in large restricted complexes and/or LRs is suggested by the functional association between NHE3 and H+/peptide transport. Thwaites et al. (51) have demonstrated that inhibition of NHE3 in intestinal Caco-2 cells leads to a reduction in the rates of H+/peptide uptake, suggesting a coordinated activity of H+-solute symport with apical Na+/H+ exchange. Such an association, if correlated with physical proximity of the exchangers, would allow the creation of acidic microdomains on the surface of epithelial cells and, consequently, would optimize the efficient absorption of nutrients and Na+ while maintaining intracellular pH homeostasis and the ionic gradients involved in driving transport. Our measurements of surface pH using FITC-WGA did, in fact, verify that NHE3 substantially reduced the pH on the microvillar surface (Fig. 7B). Measurements of surface pH in both CaCo-2 monolayers and native tissue have shown the presence of an acidic environment, and in Caco-2 cells, this environment is significantly alkalinized in the presence of blockers of NHE3 (16, 24). The level of resolution of our measurements did not allow us to determine whether the BB central NHE3-rich area of OK cell surface was more acidic than the periphery. It also remains to be determined whether H+-solute transporters or other transport proteins driven by a H+ gradient are associated with NHE3 in the large surface complexes.
For both plasma membrane and intracellular NHEs, the directionality of Na+ and H+ transport is determined by the gradients of the ions across the phospholipid membrane. In most physiological settings, plasma membrane NHEs extrude intracellular H+ coupled to the inward movement of Na+ down its concentration gradient. The transport function of vesicular NHE3 would be expected to be dependent on the cytoplasmic and intravesicular Na+ and H+ concentrations. Although the intraendosomal pH concentration is expected to be acidic because of V-ATPase function, previous reports of endosomal NHE3 function in fibroblasts and OK cells have demonstrated that NHE3 works to further acidify the endosome (11, 14, 15). The required levels of intravesicular Na+ to drive H+ uptake could be derived either from the transport of Na+ via functional Na+-K+-ATPase in the endosomal membrane or through the delivery of Na+ via endocytic vesicles carrying Na+-rich extracellular fluid. In polarized epithelial cells, Na+-K+-ATPase is a basolateral membrane protein and would not be expected to be present in the apical membrane or to be available in the apical recycling endosomes. In this study, we measured intravesicular pH using internalized FITC-WGA and found that inhibition of NHE3 by S3226 raised endosomal pH, further supporting the role of NHE3 in acidifying endosomes (Fig. 7). WGA was chosen because it behaves similarly to NHE3, being endocytosed and distributing in the recycling endosome before trafficking back to the surface but does not distribute significantly in lysosomes (45, 49). Gekle et al. (15) have shown that inhibition of endosomal NHE3 in OK cells functions not only to alkalinize endosomes but also to reduce the fusion of vesicles early in the endocytic process. It is unclear whether NHE3 works to acidify both early and recycling endosomes. Because our measurement of vesicular pH by WGA targeted all vesicular compartments labeled during 30 min of uptake at room temperature, it is conceivable that the change in pH we detected was restricted to the early endosome population. Although it seems unlikely that endosomes later in the membrane trafficking system would retain a sufficient gradient of Na+ to fuel the uptake of H+, we cannot rule out this possibility without measurements of the Na+ concentration within these vesicles.
In conclusion, the presence of NHE3 in multiple apical plasma membrane subdomains and in multiprotein complexes of multiple sizes and/or in LRs suggests that a single transport protein may be present in several separate subcellular and functional pools. How this separation occurs and the specific functional role for these distinct NHE3 populations will be important to understand.
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ACKNOWLEDGEMENTS |
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We acknowledge the expert editorial assistance of H. McCann.
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FOOTNOTES |
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These studies were supported in part by National Institute of Diabetes and Digestive and Kidney Diseases RO1 DK-26523, PO1 DK-44484, T32 DK-07632, the Wellcome Foundation, the Hopkins Center for Epithelial Disorders, and the Hopkins CMM Graduate Program.
Portions of this paper are presented in S. Akhter's PhD dissertation.
Address for reprint requests and other correspondence: M. Donowitz, Johns Hopkins Univ. School of Medicine, 925 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205-2195 (E-mail: mdonowit{at}jhmi.edu).
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.
May 22, 2002;10.1152/ajpcell.00613.2001
Received 26 December 2001; accepted in final form 13 May 2002.
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