Am J Physiol Cell Physiol
281: C2039–C2048, 2001.
Half-lives of plasma membrane Na⫹/H⫹ exchangers NHE1–3:
plasma membrane NHE2 has a rapid rate of degradation
MEGAN E. CAVET, SHAFINAZ AKHTER, RAKHILYA MURTAZINA,
FERMIN SANCHEZ DE MEDINA, CHUNG-MING TSE, AND MARK DONOWITZ
Departments of Medicine and Physiology, Gastrointestinal Division, The Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205
Received 18 December 2000; accepted in final form 20 August 2001
⫹ ⫹
THE NA /H EXCHANGERS (NHEs) are a family of membrane transport proteins that catalyze the electroneutral exchange of intracellular H⫹ for extracellular Na⫹.
The “housekeeping” NHE1 is ubiquitously expressed
and is present on the basolateral membrane in virtually all mammalian epithelial cells, where it is involved
in maintaining intracellular pH (pHi) and in cell volume regulation. NHE2 and NHE3 have a more restricted tissue distribution, being predominantly located in the apical membrane of epithelia, and are
highly expressed in the intestine and in the kidney.
They are thought to be involved in Na⫹, bicarbonate,
and water reabsorption (13, 37). However, the relative
roles of these two isoforms in these processes are not
completely understood. Mouse knockout studies demonstrated the importance of NHE3 in fluid and electrolyte balance in the intestine and kidney, but null
NHE2 mice had no perturbations of intestinal or renal
acid-base or Na⫹ homeostasis, and NHE2 was not
upregulated in the NHE3 knockout mouse (31). In
addition, there did not appear to be a difference in
renal proximal tubule function in NHE3 vs. NHE2 plus
NHE3 knockout mice (7). The only abnormal physiology that the NHE2 knockout mice did display was loss
of viability of gastric parietal cells (30). On the other
hand, both NHE2 and NHE3 appear to contribute to
basal rabbit and avian ileal brush border Na⫹/H⫹
exchange (12, 16, 39), and NHE2 is the major Na⫹/H⫹
exchanger in the avian colon, where it is increased by a
low-salt diet (12). NHE2 is also highly expressed in
human skeletal muscle, where its function is unknown,
and in human colon, suggesting that it has a significant role in human colonic Na⫹ absorption (24).
The subcellular distribution and trafficking of the
NHEs give some insights into the physiological roles of
these transport proteins. NHE1 is predominantly on
the cell surface (4, 9) and is regulated by changes in the
sensitivity to intracellular H⫹ (21). In contrast, only
15% of NHE3 is located on the plasma membrane in
PS120 cells (2, 4). The rest is located in a juxtanuclear
recycling compartment from where it undergoes basal
phosphatidylinositol (PI) 3 kinase-dependent cycling to
the plasma membrane (11, 17, 19). The presence of an
intracellular pool of NHE3 has also been demonstrated
in epithelial cells. In the opossum kidney proximal
tubule cell line (OK), the majority of NHE3 is located
intracellularly in a subapical compartment (Akhter
and Donowitz, unpublished observations), whereas in
the colonic adenocarcinoma Caco-2 cells, 80% of NHE3
is located on the plasma membrane, with the rest being
located in a subapical recycling compartment (18). Alteration of NHE3 transport rate usually occurs
through a change in maximal velocity (Vmax), and this
has been shown to occur via changes in the amount of
the protein on the plasma membrane and/or changes in
Address for reprint requests and other correspondence: M. Donowitz, Dept. of Medicine, Division of Gastroenterology, Johns Hopkins
Univ., 925 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD
21205-2195 (E-mail: [email protected]).
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.
sodium absorption; Caco-2 cells; lysosomes; trafficking
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Cavet, Megan E., Shafinaz Akhter, Rakhilya Murtazina, Fermin Sanchez de Medina, Chung-Ming Tse,
and Mark Donowitz. Half-lives of plasma membrane
Na⫹/H⫹ exchangers NHE1–3: plasma membrane NHE2 has
a rapid rate of degradation. Am J Physiol Cell Physiol 281:
C2039–C2048, 2001.—The Na⫹/H⫹ exchangers NHE2 and
NHE3 are involved in epithelial Na⫹ and HCO3⫺ absorption.
To increase insights into the functions of NHE2 vs. NHE3,
we compared their cellular processing with each other and
with the housekeeping isoform NHE1. Using biotinylated
exchanger, we determined that the half-life of plasma membrane NHE2 was short (3 h) compared with that of NHE1 (24
h) and NHE3 (14 h) in both PS120 fibroblasts and Caco-2
cells. NHE2 transport and plasma membrane levels were
reduced by 3 h of Brefeldin A treatment, whereas NHE1 was
unaffected. NHE2 was degraded by the lysosomes but not
proteosomes, as demonstrated by increasing levels of endocytosed NHE2 protein after inhibition of the lysosomes, but
not with proteosome inhibition. Unlike that of NHE3, basal
NHE2 transport activity was not affected by phosphatidylinositol 3-kinase inhibition and did not appear to be localized
in the juxtanuclear recycling endosome. Therefore, for
NHE2, protein degradation and/or protein synthesis probably play important roles in its basal and regulated states.
These results suggest fundamental differences in the cellular
processing and trafficking of NHE2 and NHE3. These differences may underlie the specialized roles that these exchangers play in epithelial cells.
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HALF-LIVES OF PLASMA MEMBRANE NA⫹/H⫹ EXCHANGERS NHE1–3
MATERIALS AND METHODS
Cell culture. A Chinese hamster lung fibroblast cell line
(PS120) previously selected to lack all endogenous Na⫹/H⫹
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exchangers was stably transfected, using Lipofectin (GIBCO
BRL), with human NHE1 or rabbit NHE2 or NHE3 cDNAs
tagged on the COOH termini with a previously described
11-amino acid epitope of vesicular stomatitis virus G protein
(VSVG) plus an 8-amino acid spacer (NHE1V, NHE2V, or
NHE3V) (41). Cells were grown in medium (DMEM) supplemented with 25 mM NaHCO3, 10 mM HEPES, pH 7.4, 50
IU/ml penicillin, 50 ␮g/ml streptomycin, 10% fetal bovine
serum (FBS), and 800 ␮g/ml G418 in a 5% CO2-95% O2
incubator at 37°C. The colonic adenocarcinoma-derived
Caco-2 clonal cell line TC-7 (6) was stably transfected with
rabbit NHE2V. Clonal NHE2V cells were selected by using
acid loading (see below) and 30 ␮M HOE-694 to inhibit
NHE1. Caco-2 cells were grown in DMEM supplemented
with 0.1 mM nonessential amino acids, 1 mM pyruvate, 50
IU/ml penicillin, 50 ␮g/ml streptomycin, and 10% FBS in a
10% CO2-humidified incubator at 37°C.
Both cell types were acid loaded once a week to maintain a
high level of Na⫹/H⫹ exchange activity, as described (21).
Briefly, cells were placed in NH4Cl solution (in mM: 50
NH4Cl, 70 choline-Cl, 5 KCl, 1 MgCl2, 2 CaCl2, 5 mM glucose,
and 20 HEPES/Tris, pH 7.4) for 1 h, followed by 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.
Cell surface biotinylation to determine half-life of plasma
membrane NHEs. Transfected PS120 cells were grown to
70–80% confluence in 10-cm petri dishes. Cells were then
placed at 4°C. Cells were washed twice in phosphate-buffered
saline (PBS) and once in borate buffer (in mM: 154 NaCl, 10
boric acid, 7.2 KCl, and 1.8 CaCl2, pH 9.0). The 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 sulfo-NHS-LC-biotin (biotinylation solution). An additional 3 ml of the same biotinylation solution was 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-LC-biotin,
and then the cells were washed twice with PBS. Cells were
then returned to complete PS120 medium at 37°C for various
times before being frozen at ⫺80°C until further processing.
Cells were then lysed in 1 ml of N⫹ buffer (in mM: 60
HEPES, pH 7.4, 150 NaCl, 3 KCl, 5 EDTA trisodium, 3
EGTA, and 1% Triton X-100) and sonicated to clarity on ice
with a Branson 450 probe sonicator. The supernatant was
subjected to two consecutive avidin precipitations. The avidin-agarose beads were washed five times in N⫹ buffer, and
bound proteins were solubilized in sample buffer yielding the
surface fraction. Samples were separated on a 9% gel by
SDS-PAGE, and Western analysis was then performed using
anti-VSVG antibody. Bands were visualized with enhanced
chemiluminescence (ECL), exposed to preflashed X-ray film
within the linear range of ECL signal, and quantified using a
densitometer and Imagequant software. Half-life of plasma
membrane NHEs was determined by fitting the data to an
exponential decay curve using Origin software.
Internalization assay. Cells were pretreated with vehicle
or lysosomal, proteosomal inhibitors. Cells were biotinylated
as described above except for the use of NHS-SS-biotin rather
than sulfo-NHS-LC biotin. Cells were then incubated in complete medium at 37°C for 1 h with and without lysosomal/
proteosomal inhibitors. Cells were then returned to 4 °C, and
all remaining surface biotin was removed by the fresh addition of 10 mM 2-mercaptoethanesulfonic acid (MESNA) for
three 30-min washes in stripping buffer (100 mM NaCl, 1
mM EDTA, 50 mM Tris, pH 8.6, and 2 mg/ml BSA). All
unreacted MESNA was quenched with 100 mM iodoacet-
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the turnover number of the protein by phosphorylation-dependent and -independent mechanisms. Stimulation of NHE3 transport rate by serum and fibroblast
growth factor (FGF) causes an increase in the amount
of NHE3 on the plasma membrane via an increase in
exocytosis in PS120 fibroblasts (2, 17), whereas phorbol
12-myristate 13-acetate inhibits NHE3 by causing an
increase in endocytosis and a change in turnover number in Caco-2 cells (18). In OK cells, activation of NHE3
by acidosis is due to an increase in the exocytosis of
NHE3 and, thus, an increase of NHE3 on the cell
surface (40), whereas parathyroid hormone (PTH) inhibits NHE3 in OK cells by both changing turnover
number (by altering phosphorylation) and later decreasing surface NHE3 (8). In rats, PTH decreases
cortical Na⫹/H⫹ exchange partially through redistribution of NHE3 transporter away from the apical membrane to a nonapical pool (14).
NHE2 exists in two forms in the PS120 cell line, an
85-kDa glycosylated form that is predominantly located on the plasma membrane and an unglycosylated
75-kDa form that is predominantly located intracellularly (4, 36). Little is known about the intracellular
trafficking and location of NHE2 in either the basal or
regulated state. Functionally, NHE2 responds to agonists through a change in Vmax (21); whether this is due
to changes in turnover number and/or the percentage
of transporter on the plasma membrane has yet to be
determined.
To gain insight into the relative functional roles of
NHE2 and NHE3, we designed this study to investigate the cellular trafficking and processing of NHE2
compared with NHE3 and NHE1. Initially, we studied
the plasma membrane half-life of the NHEs and determined that NHE2 has a short half-life compared with
NHE1 and NHE3. Because changing synthesis and
degradation rates for a protein with a short half-life is
much more effective at regulating protein levels than
for a protein with a long half-life, we then investigated
the involvement of synthesis and proteolytic pathways
in the regulation of the amount of cellular NHE2.
Inhibiting the synthetic pathway with Brefeldin A decreases the amount of plasma membrane NHE2. There
are two main proteolytic systems within cells for the
intracellular degradation of proteins, the lysosomal
system and the proteosomal system (3, 25, 34). We
demonstrated that inhibition of the lysosomes causes
an increase in the amount of endocytosed NHE2, suggesting that NHE2 is degraded by lysosomes. In contrast to NHE3, NHE2 does not appear to undergo basal
recycling. This suggests that for NHE2, protein degradation and/or protein synthesis probably plays an important role in basal and regulated states. The cellular
pathways of trafficking of NHE2 and NHE3 expressed
in the same cell type appear to be fundamentally different, and this may underlie the specialized role of
NHE2 vs. NHE3.
HALF-LIVES OF PLASMA MEMBRANE NA⫹/H⫹ EXCHANGERS NHE1–3
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0.1% saponin/PBS. Cells were then incubated with secondary
antibodies (Alexa conjugates; Molecular Probes) and Hoechst
33342 for 1 h. After three 10-min washes in 0.1% saponin/
PBS, cells were mounted in Prolong antifade reagent before
being viewed on a Zeiss LSM 410 confocal microscope. Images were analyzed using Metamorph software (Universal
Imaging).
Materials. Human NHE1 was a kind gift from Drs. J. Noel
and J. P. Pouyssegur (Dept. of Physiology, University of
Montreal, Canada, and Dept. Biochemistry, University of
Nice, France). [35S]methionine/cysteine cell-labeling mix was
from Amersham, and NHS-SS-biotin, NHS-LC-biotin, and
avidin-agarose were from Pierce. All other chemicals were
from Sigma unless stated otherwise.
Statistical analysis. Results are expressed as means ⫾ SE.
Statistical significance was evaluated using Student’s t-test.
Significance was accepted at P ⬍ 0.05.
RESULTS
Half-life of plasma membrane NHEs. Half-lives of
Na⫹/H⫹ exchanger isoforms NHE1–3 appeared in
PS120 fibroblasts that were initially on the plasma membrane and were compared by determining the amount of
biotinylated NHE proteins remaining within the cells
0–24 h after initial biotinylation of surface proteins.
These results (Fig. 1) demonstrate that plasma membrane NHEs have different degradation rates. The 110kDa form of NHE1 had a half-life of 23.1 ⫾ 2.3 h (n ⫽ 4)
(Fig. 1A). NHE3 also had a long half-life of 13.8 ⫾ 1.6 h
(n ⫽ 4) (Fig. 1C). In contrast, the half-life of the 85-kDa
form of NHE2 was much shorter at 3.2 ⫾ 0.3 h (n ⫽ 5).
The 75-kDa form of NHE2 had a similar short half-life of
3.1 ⫾ 0.2 h (n ⫽ 5) (Fig. 1B).
NHE1 exists in two forms, a mature 110-kDa form
that is almost entirely on the plasma membrane (90%)
and an 85-kDa form that is core glycosylated and
intracellular (4). Because the 110-kDa NHE1 is mainly
on the cell surface (9), as a comparison for the biotinylation method we measured half-life for NHE1 using
the pulse-chase technique. PS120/NHE1 cells were
pulse-labeled with [35S]methionine/cysteine and
chased in medium containing unlabeled methionine
and cysteine. Quantitation of radioactivity associated
with the 110-kDa NHE1 protein from 0 to 24 h after
pulse-labeling gave a half-life of 20.3 ⫾ 1.7 h (n ⫽ 3)
(Fig. 2), which was not significantly different from that
obtained using the biotinylation method (23.1 ⫾ 2.3 h).
The 85-kDa core-glycosylated form of NHE1 was
present at 2.5 h of chase but was not detected at 14 h
of chase. These data are similar to those from a previous study in A6 cells, in which it was determined that
the core-glycosylated NHE1 was present 0–5 h into the
chase period and the 110-kDa NHE1 was present 1–24
h into the chase period (10).
NHE2 is expressed normally in epithelial cells
rather than in fibroblasts. Therefore, studies were performed to determine whether NHE2 also had a short
half-life when stably expressed in epithelial cells. We
verified the short half-life of plasma membrane NHE2
in the intestinal epithelial cell Caco-2. Figure 3 shows
a comparison of plasma membrane NHE2 half-life in
PS120 cells (A) and in Caco-2 cells (B) measured in the
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amide in PBS for 10 min. One sample of cells was stripped
without a 37°C incubation to check stripping efficiency,
which was ⱖ90%. Cells were then lysed and treated as
described above.
Pulse-chase labeling of cultured cells. Cells were rinsed
once and incubated with methionine- and cysteine-free
DMEM for 1 h to deplete intracellular pools. The cells were
then incubated with methionine- and cysteine-free DMEM
containing 0.2 mCi/ml [35S]methionine/cysteine cell-labeling
mix (Amersham) for 4 h. To chase, we aspirated this labeling
solution and rinsed the cells four times with PBS. Cells were
then incubated with DMEM containing 10% serum, 2 mM
methionine, and 2 mM cysteine for between 2 and 26 h. The
cells were then rinsed four times in ice-cold PBS and stored
at ⫺70°C until further processing. Cells were lysed in 1 ml of
N⫹ buffer, homogenized through a 26-gauge needle, and
agitated on a rotating rocker at 4°C for 30 min, followed by
centrifugation at 12,000 g for 30 min, to remove insoluble
cellular debris. The supernatant was precleared by using 20
␮l of protein A-Sepharose beads cross-linked to anti-mouse
IgG (PAM; Jackson). NHE1V was then immunoprecipitated
from the precleared supernatant by incubation with antiVSVG P5D4 monoclonal antibody, followed by two rounds of
incubation with PAM. Immunoprecipitated pellets were
washed five times with N⫹ buffer. The precipitated proteins
were eluted from the beads with SDS-sample buffer (110 mM
Tris 䡠 HCl, 0.9% SDS, 0.8% EDTA, 5% glycerol, 1% 2-mercaptoethanol, and bromphenol blue) by incubation at 95°C for 3
min, followed by SDS-PAGE on 9% gels. Proteins were transferred to nitrocellulose and exposed to Amersham Hyperfilm
for 2 days. The nitrocellulose was then subjected to Western
analysis with anti-VSVG antibody to ensure that an equal
amount of NHE protein was precipitated at each time point.
Na⫹/H⫹ exchange Vmax by fluorimetry. Cells were seeded
on glass coverslips and grown until they reached 50–70%
confluency. The cells were loaded with the acetoxymethyl
ester of 2⬘,7⬘-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF-AM; 5 ␮M) 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 and 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-␮l fluorometer cuvette designed for perfusion and thermostatted at
37°C. The cells were pulsed with 40 mM NH4Cl in TMA⫹
medium for 3 min, followed by TMA⫹ medium, which resulted in the acidification of the cells. Na⫹ medium was then
added, which induced alkalinization of the cells.
Na⫹/H⫹ exchange rates (H⫹ efflux) were calculated, as
described previously (21), as the product of Na⫹-dependent
change in pHi and the buffering capacity at each pHi and
were analyzed with the use of a nonlinear regression data
analysis program (Origin) that allowed fitting of data to a
general allosteric model described by the Hill equation (v ⫽
Vmax 䡠 [S]napp/K⬘⫹[S]napp, where [S] is substrate concentration,
v is velocity, K is affinity constant, and napp is apparent Hill
coefficient.) with estimates for Vmax and K⬘[H⫹]i and their
respective errors (SE).
Immunofluorescence. Cells were seeded on glass coverslips
until they reached 80% confluency. Cells were fixed in 3%
paraformaldehyde/PBS for 30 min at 4°C, and the paraformaldehyde was neutralized in 20 mM glycine for 10 min.
Cells were permeabilized for 30 min in 0.1% saponin/PBS
and then blocked for 30 min in 1% BSA/PBS supplemented
with 10% FBS. The cells were incubated in anti-VSVG P5D4
monoclonal antibody for 1 h, followed by three washes in
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HALF-LIVES OF PLASMA MEMBRANE NA⫹/H⫹ EXCHANGERS NHE1–3
same experiment. The half-life of plasma membrane
NHE2 transfected into Caco-2 cells also was short for
both the 85-kDa (3.6 ⫾ 0.2 h, n ⫽ 3) and the 75-kDa
form of NHE2 (3.5 ⫾ 0.3 h) and was not significantly
different from that in PS120 cells. As a comparison, the
half-life of endogenous plasma membrane NHE1 was
measured simultaneously in Caco-2 cells and was
20.2 ⫾ 2.0 h (Fig. 3C), not significantly different from
that in transfected PS120 cells (23.1 ⫾ 2.3 h). Therefore, the short half-life of plasma membrane NHE2 is
not just a feature of expression in PS120 cells and also
occurs in an epithelial cell model. Similarly, plasma
membrane NHE1 had a similar half-life in both fibro-
blasts and in epithelial cells. The half-life of endogenous NHE3 was not determined because of low expression levels of this protein in the Caco-2 cells we
studied.
NHE2 and NHE3 have divergent subcellular distribution and trafficking mechanisms in PS120 cells. The
significantly faster degradation rate of plasma membrane NHE2 compared with NHE3 was of interest and
suggests a fundamental difference in the cellular processing of these isoforms. NHE2 and NHE3 are both
epithelial isoforms whose relative roles in Na⫹ absorption are not well understood. The current study has
demonstrated that one fundamental difference be-
Fig. 2. Half-life of NHE1 determined with the pulse-chase method. PS120 cells expressing NHE1 were pulselabeled with 0.2 mCi/ml of [35S]methionine/cysteine cell-labeling mix for 4 h and chased in medium containing
methionine/cysteine at varying time points. After solubilization, NHE1 was immunoprecipitated and subjected to
SDS-PAGE. Proteins were transferred to nitrocellulose, and autoradiography was performed (left). Densitometric
analysis was performed using Imagequant software. Graph (right) shows quantitation of autoradiograph for
110-kDa NHE1. Half-life was estimated to be 22.2 h. A representative experiment of 3 similar experiments is
shown.
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Fig. 1. Half-life of plasma membrane Na⫹/H⫹ exchanger isoforms NHE1–3 in PS120 fibroblasts. Plasma membrane NHE proteins were biotinylated at 4°C using sulfo-NHS-LC-biotin and, after incubation at 37°C, were
harvested at varying time points. After solubilization, biotinylated protein was recovered with streptavidinagarose from equal amounts of total cellular protein. Densitometric analysis was performed using Imagequant
software. Graphs (right) show quantitation of Western blots (left) for NHE1 (A), NHE2 (B; 85 kDa only), and NHE3
(C). Half-lives of these experiments were estimated as 24.4 h for NHE1, 3.2 h for 85-kDa NHE2, 3.0 h for 75-kDa
NHE2 (degradation curve not shown), and 11.8 h for NHE3. A single representative experiment of 3–5 similar
experiments is shown.
HALF-LIVES OF PLASMA MEMBRANE NA⫹/H⫹ EXCHANGERS NHE1–3
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tween NHE2 and NHE3 is the short half-life of NHE2.
One possible explanation for differences in plasma
membrane half-life is differences in the rates of degradation. Differences in degradation rates may suggest
differences in the cellular processing and trafficking of
these two isoforms. Whereas NHE3 is known to undergo a basal PI-3 kinase-dependent recycling between
the cell surface and an intracellular compartment (11,
17, 19), subcellular trafficking of NHE2 has not been
described. We studied the effect of the PI-3 kinase
inhibitor wortmannin (100 nM, 1 h) on basal NHE2
transport in PS120 cells. The Vmax of H⫹ efflux for
NHE2 was not affected by wortmannin [control:
3,790 ⫾ 90 ␮M/s, wortmannin: 3,533 ⫾ 90 ␮M/s, P ⫽
not significant (NS), Fig. 4A], in contrast to the 33%
inhibition obtained with NHE3 (control: 4,095 ⫾ 92
␮M/s, wortmannin: 2,754 ⫾ 112 ␮M/s, P ⬍ 0.0001, Fig.
4B). This finding demonstrates that NHE2 is not undergoing PI-3 kinase-dependent basal recycling. Furthermore, immunolocalization demonstrates that the
subcellular distribution of NHE2 and NHE3 is different. The subcellular distribution of NHE2 and NHE3
was compared in PS120 fibroblasts. While NHE3 is
predominantly localized in a juxtanuclear compartment and on the plasma membrane as previously described (Fig. 5A) (11), the pattern of staining for NHE2
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was different. Intracellular NHE2 (Fig. 5B) had a more
diffuse cytoplasmic and perinuclear localization.
The amount of recoverable endocytosed plasma membrane NHE2 is increased by inhibiting the lysosomes.
Proteins with a short half-life are much more effectively regulated by changing synthesis and degradation rates than are proteins with a long half-life. Therefore, we investigated the role of the lysosomes in the
short-term degradation of NHE2 in PS120 cells. The
amount of endocytosed plasma membrane NHE2 protein detectable by Western blotting was compared in
the presence and absence of lysosomal inhibitor. Degradation by the lysosomal pathway was inhibited using
the inhibitor leupeptin (100 ␮g/ml) (22) and the lysosomotropic agents ammonium chloride (20 mM) and
chloroquine (50 ␮M) (29, 32). Cells expressing NHE2
were treated for 3 h with each blocker, after which cell
surface proteins were labeled with biotin and allowed
to internalize for 1 h in the continued presence of
lysosomal inhibitor. Residual cell surface biotinylated
protein was stripped with the reducing agent, which is
efficient in removing ⬎90% of surface biotin in PS120
fibroblasts (2) (data not shown), after which the
amount of endocytosed NHE protein was determined.
Treatment of cells with lysosomal inhibitors (leupeptin, chloroquine, and NH4Cl) led to an increase in
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Fig. 3. Comparison of half-life of plasma membrane NHE2 in Caco-2 cells and PS120 fibroblasts. Plasma
membrane NHE2 proteins were biotinylated at 4°C using sulfo-NHS-LC-biotin and, after incubation in cell
medium at 37°C, were harvested at varying time points. After solubilization, biotinylated protein was recovered
with streptavidin-agarose from equal amounts of total cellular protein. Densitometric analysis was performed
using Imagequant software. Graphs (right) show quantitation of Western blots (left). A: half-life of transfected
85-kDa NHE2 in PS120 cells, estimated as 3.0 h. B: half-life of transfected 85-kDa NHE2 in Caco-2 cells, estimated
to be 3.4 h. C: half-life of endogenous NHE1 in Caco-2 cells, estimated to be 22.1 h. A representative experiment
of 3 similar experiments is shown.
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HALF-LIVES OF PLASMA MEMBRANE NA⫹/H⫹ EXCHANGERS NHE1–3
NHE2 (both 85 and 75 kDa) as detected by Western
blotting (Fig. 6, A–C). Densitometric analysis revealed
that treatment of NHE2 with lysosomal inhibitors led
to an ⬃100% increase in NHE2 internalized protein. In
contrast, internalized NHE3 was not significantly altered after treatment with leupeptin (Fig. 6D).
The other major pathway for the degradation of
proteins is the proteosome. This system appears to be
primarily for the breakdown of misfolded protein retained in the endoplasmic reticulum (ER) but also has
Fig. 5. Distribution of NHE3 and
NHE2 in PS120 fibroblasts. PS120
cells stably expressing either epitopetagged rabbit NHE3 (NHE3V; left) or
NHE2 cDNAs (NHE2V; right) were
fixed, permeabilized, and stained with
anti-vesicular stomatitis virus G protein (anti-VSVG) P5D4 monoclonal antibody (green) and Hoechst 33342 nuclear stain (blue). Cells were then
visualized with a Zeiss confocal microscope. Representative x-y scans at and
above the plane of the nucleus are
shown. Images are representative of 5
experiments.
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Fig. 4. Effect of the phosphatidylinositol (PI) 3-kinase inhibitor
wortmannin on NHE2 and NHE3 Na⫹/H⫹ exchange rate. PS120/
NHE2 (A) and PS120/NHE3 (B) cells were grown to subconfluence on
glass coverslips. The rate of Na⫹/H⫹ exchange was measured by
determining the rate of intracellular pH (pHi) recovery after an acute
NH4Cl-induced acid load using 2⬘,7⬘-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). H⫹ efflux rates, equivalent to Na⫹/H⫹
exchange, were plotted against intracellular [H⫹]. Na⫹/H⫹ efflux
rates were calculated at various pHi, lines were fit to the data by an
allosteric model, and kinetic parameters [maximal velocity (Vmax),
K⬘[H⫹]i, and apparent Hill coefficient (napp)] were estimated (21).
Cells were exposed to vehicle (squares) or 100 nM wortmannin
(circles) for 1 h at 37°C before measurement of Na⫹/H⫹ exchange
rate (n ⫽ 4 experiments).
been shown to be involved in the internalization and
degradation of plasma membrane proteins (3, 34). We
initially investigated the potential role of the proteosome in the degradation of NHE2 by determining
whether NHE2 was ubiquitinated. Ubiquitination can
be a targeting mechanism to both the proteosome and
the lysosome. NHE2 is not recognized by anti-ubiquitin
antibody and does not appear as a laddered protein,
unlike the ubiquitinated transporter proteins cystic
fibrosis transmembrane conductance regulator (38)
and epithelial Na⫹ channel (ENaC) (1, 33) (data not
shown). In addition, the amount of internalized NHE2
detected was not increased in cells pretreated with the
proteosomal blocker lactacystin (50 ␮M, 3 h) (15) (Fig.
6E). Thus proteosomal degradation does not appear to
contribute to the rapid breakdown of NHE2.
Brefeldin A, an inhibitor of the synthetic pathway,
inhibits NHE2 transport. We used Brefeldin A (BFA),
which blocks Golgi-to-plasma membrane trafficking by
causing tubulation and redistribution of the Golgi into
the ER (23, 26), to determine the importance of the
synthetic pathway in determining plasma membrane
levels of NHE2. PS120 cells were exposed to 5 ␮g/ml
BFA for 3 h before study. The Vmax of H⫹ efflux for
NHE2 was reduced by 38% from 2,575 ⫾ 78 to 1,598 ⫾
38 ␮M (P ⬍ 0.0001) in the BFA-treated cells, whereas
the transport of NHE1, which has the longest half-life
of the NHEs studied, was unchanged (control: 5,015 ⫾
61 ␮M, BFA treatment: 4,755 ⫾ 76 ␮M, P ⫽ NS, Fig.
7A). To verify that reduction of NHE2 transport was
due to a decrease in surface NHE2, we used cell surface
biotinylation to measure NHE2 surface protein with
and without BFA (Fig. 7B). Densitometric analysis
demonstrated that BFA decreased the amount of the
85-kDa NHE2 protein on the cell surface by ⬃50%.
Exposure of NHE2 to hyperosmolarity for 3 h does not
alter the half-life of plasma membrane NHE2. The
short half-life of NHE2 suggested that physiological
regulation might involve changes in the half-life. Because NHE2 is present in the apical membrane of renal
distal tubule and of duodenal absorptive cells and thus
is exposed to hyperosmolarity, including for prolonged
periods, we determined whether prolonged exposure to
HALF-LIVES OF PLASMA MEMBRANE NA⫹/H⫹ EXCHANGERS NHE1–3
C2045
DISCUSSION
hyperosmolarity altered NHE2 function and plasma
membrane half-life. In these studies, PS120/NHE2
cells were exposed to Na⫹ medium or Na⫹ medium
plus 150 mM mannitol for 3 h, and NHE2 activity
was determined. After 3 h of exposure to hyperosmolarity, NHE2 activity was still inhibited compared
with control (43% inhibition, n ⫽ 3, data not shown).
After 3 h of exposure to hyperosmolarity, half-life of
plasma membrane NHE2 was determined by use of
biotinylation and was studied 0, 2.5, 5, 7, 10, and 19
h later (i.e., plus 3 h), being maintained in the same
hyperosmolar conditions that inhibited NHE2. After
3 h of exposure to hyperosmolarity, the half-life of
plasma membrane NHE2 was not altered (difference
in half-life of plasma membrane p85 exposed to hyperosmolarity vs. time control 1.1 ⫾ 1.2 h, n ⫽ 3, not
significant).
AJP-Cell Physiol • VOL
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Fig. 6. Effect of lysosomal and proteosomal inhibitors on the amount
of internalized NHE2V and NHE3V. PS120 cells were pretreated for
3 h with leupeptin (100 ␮g/ml), chloroquine (50 ␮M), NH4Cl (20 mM),
or lactacystin (50 ␮M). Cells were then biotinylated at 4°C with
NHS-SS-biotin and placed in the continued presence of inhibitor at
37°C for 1 h before being returned to 4°C. Surface biotin was stripped
as described in MATERIALS AND METHODS. Results for NHE2V are
shown in A–C (lysosomal inhibitors) and E (proteosomal inhibitor).
Effect of the lysosomal inhibitor leupeptin on NHE3 is shown in D.
Numbers above blots represent microliters of sample loaded (n ⫽ 3
experiments).
In this study we have characterized the half-life of
plasma membrane NHE1–3 and have demonstrated
that the half-life of plasma membrane NHE2 is relatively short (3 h) in both fibroblasts (PS120) and epithelial (Caco-2) cells compared with that of NHE3 and
NHE1 (14 h and 24 h, respectively). Both 85- and
75-kDa forms of NHE2 had similar plasma membrane
half-lives, suggesting that both forms that reach the
plasma membrane are undergoing similar cellular
trafficking pathways. The short half-life of plasma
membrane NHE2 is supported by the fact that inhibiting both the synthetic pathway and degradation rapidly alters the transport activity of NHE2. Transport
and surface levels of NHE2 are reduced by 3-h treatment with inhibition of the synthetic pathway using
Brefeldin A. This suggests the importance of new synthesis in maintaining plasma membrane of NHE2 over
a short time period. Furthermore, it appears that the
degradation of NHE2 is mediated, at least in part, by
the lysosomes. Treatment with the lysosomal inhibitor
leupeptin (22, 32) and the lysosomotropic agents chloroquine and NH4Cl (29) increased the amount of detectable endocytosed NHE2 protein, presumably by
inhibiting its breakdown in the lysosomal compartment, over a 3-h time period. The rapid degradation of
NHE2 appears to be predominantly by the lysosomes,
because inhibition of the proteosomal pathway did not
affect the amount of endocytosed NHE2 and NHE2
does not appear to be ubiquitinated.
This study demonstrates fundamental differences in
the cellular processing of NHE2 and NHE3. NHE3 is
known to undergo a dynamic process of basal recycling
between the plasma membrane and an endosomal recycling compartment that is PI 3-kinase dependent (11,
19). However, in this study we have demonstrated that
basal NHE2 transport is not affected by the PI 3-kinase inhibitor wortmannin. Furthermore, immunofluorescence studies showed that NHE3 has a discrete
juxtanuclear localization that has previously been
shown to be the recycling endosome on the basis of
colocalization with the transferrin receptor and the
SNARE protein cellubrevin (11, 17). In contrast, NHE2
had a more diffuse cytoplasmic and perinuclear localization. Although the wortmannin data do not rule out
non-PI 3-kinase-dependent cycling for NHE2, as occurs
with part of FGF stimulation of NHE3 in PS120 fibroblasts (17), this, combined with the contrasting subcellular localization with NHE3, suggests a lack of basal
recycling of NHE2.
Both NHE2 and NHE3 are regulated in part by a
change in their Vmax (21). One of the mechanisms of
NHE3 regulation is changes in the amount of total
NHE3 protein on the plasma membrane vs. the intracellular compartment, and this often involves changes
in the rate of endocytosis and exocytosis. Similarly for
NHE2, there is a large amount of the 75-kDa form
(80%) of NHE2 within the cell, whereas 36% of the
85-kDa form is present in the intracellular compartment (4). Given the short plasma membrane half-life of
C2046
HALF-LIVES OF PLASMA MEMBRANE NA⫹/H⫹ EXCHANGERS NHE1–3
NHE2, this intracellular population may represent
protein associated with the biosynthetic pathway.
However, it remains to be determined whether trafficking of a subpopulation of intracellular NHE2 plays a
role in the regulated state in either a PI 3-kinasedependent or -independent manner.
Proteins with a short half-life are much more effectively regulated by changing rates of protein synthesis
and degradation than are proteins with longer halflives. Because NHE3 is known to undergo rapid internalization and recycling back to the plasma membrane,
the long half-life of plasma membrane NHE3 suggests
that this isoform avoids targeting to the late endosomes and lysosomes. This is demonstrated in the
present study by the lack of effect of the lysosomal
inhibitor leupeptin on the amount of endocytosed
NHE3. The long half-lives of NHE1 and NHE3 suggest
that regulation of basal levels of degradation/synthesis
is not an efficient mechanism for rapid modulation of
levels of NHE1 and NHE3 on the plasma membrane.
In contrast, the short half-life of NHE2 suggests that
changing synthesis or degradation rates can much
more effectively regulate the amount of this isoform on
the plasma membrane.
One possible function of NHE2, given its short
plasma membrane half-life, is to respond to stimuli
through a change in its synthesis and degradation.
Other transport proteins that have rapid plasma membrane turnover include the ENaC and the type II
AJP-Cell Physiol • VOL
Na⫹/Pi cotransporter. For both of these proteins, degradation plays an important role in regulating the
transport rate. In HEK-293 cells, ENaC is ubiquitinated by the binding of Nedd4 to the COOH termini of
the ␣- and ␥-subunits, rapidly targeting the constitutively active channel for proteosomal and lysosomal
degradation. In the condition Liddle’s syndrome,
Nedd4 cannot bind to the channel subunits; thus degradation is slowed and hypertension results because of
increased surface expression of ENaC and increased
absorption of Na⫹ (1, 33). In the case of the Na/Pi
cotransporter, in rat renal brush border and in OK
cells, this transporter is broken down under basal
conditions by the lysosomal pathway, and upon stimulation with PTH the rate of degradation is substantially increased, leading to an inhibition of Na⫹/Pi
cotransport activity (27, 28). By analogy, the short
half-life of NHE2 could be important in maintaining
basal levels of this transporter and also for altering
transporter levels in the regulated state.
The findings in this study may give insight into the
relative specialized cellular roles of NHE2 and NHE3.
Although both are located on the apical surface of renal
and intestinal epithelial cells, in many species their
relative roles have not yet been fully defined. Knockout
studies in mice suggest that NHE3 is the only isoform
carrying out intestinal basal Na⫹/H⫹ exchange,
whereas the role of NHE2 is not well understood (7,
31). On the other hand, NHE2 appears to be important
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Fig. 7. Effect of Brefeldin A (BFA) on NHE2V and NHE1V Na⫹/H⫹ exchange rate and amount of NHE2 on the plasma membrane. A:
PS120/NHE2V and PS120/NHE1V cells were grown to subconfluence on glass coverslips. The rate of Na⫹/H⫹ exchange was measured by
determining the rate of pHi recovery after an acute NH4Cl-induced acid load using BCECF. H⫹ efflux rates, equivalent to Na⫹/H⫹ exchange,
were plotted against intracellular [H⫹]. Na⫹/H⫹ efflux rates were calculated at various pHi, lines were fit to the data by an allosteric model,
and kinetic parameters (Vmax and K⬘[H⫹]i), were estimated. Cells were exposed to vehicle (squares) or 5 ␮g/ml BFA (circles) for 3 h at 37°C
before measurement of Na⫹/H⫹ exchange rate (n ⫽ 3 experiments). B: PS120/NHE2V cells were exposed to 5 ␮g/ml BFA or vehicle for 3 h.
Cells were then biotinylated with NHS-SS-biotin as described in MATERIALS AND METHODS. Equal volumes of the surface fraction were
analyzed by SDS-PAGE and subjected to Western analysis. Numbers above blots represent microliters of sample loaded (n ⫽ 3 experiments).
HALF-LIVES OF PLASMA MEMBRANE NA⫹/H⫹ EXCHANGERS NHE1–3
M. E. Cavet was supported in part by a Wellcome International
Traveling 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-26525, PO1-DK44484, and RO1-DK-51116 and by The Hopkins Center for Epithelial
Disorders.
Present address of M. E. Cavet: Center for Cardiovascular Research, University of Rochester, Box 679, 601 Elmwood Ave., Rochester, NY 14618.
Present address of F. S. de Medina: Department of Pharmacology,
University of Granada, Campus de Cartuja s/n, 18071 Granada,
Spain.
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