Topological analysis of NHE1, the ubiquitous
Na+/H+
exchanger using chymotryptic cleavage
Lamara D.
Shrode1,
Bing Siang
Gan1,
Sudhir J. A.
D'Souza1,
John
Orlowski2, and
Sergio
Grinstein1
1 Division of Cell Biology,
Hospital for Sick Children, Toronto, Ontario M5G 1X8; and
2 Department of Physiology, McGill
University, Montreal, Quebec, Canada H3G 1Y6
 |
ABSTRACT |
Proteases,
glycosidases, and impermeant biotin derivatives were used in
combination with antibodies to analyze the subcellular distribution and
transmembrane disposition of the
Na+/H+
exchanger NHE1. Both native human NHE1 in platelets and epitope-tagged rat NHE1 transfected into antiport-deficient cells were used for these
studies. The results indicated that
1) the entire population of
exchangers is present on the surface membrane of unstimulated platelets, ruling out regulation by recruitment of internal stores of
NHE1; 2) the putative extracellular
loops near the NH2 terminus are
exposed to the medium and contain all the N- and
O-linked carbohydrates;
3) by contrast, the putative
extracellular loops between transmembrane domains 9-10 and
11-12 are not readily accessible from the outside and may be
folded within the protein, perhaps contributing to an aqueous ion
transport pathway; 4) the extreme COOH terminus of the protein was found to be inaccessible to
extracellular proteases, antibodies, and other impermeant reagents,
consistent with a cytosolic localization; and
5) detachment of ~150 amino acids
from the NH2-terminal end of the
protein had little effect on the transport activity of NHE1.
intracellular pH; sodium/hydrogen antiport; amiloride
 |
INTRODUCTION |
SODIUM/HYDROGEN EXCHANGERS (NHE) are ubiquitous
proteins that play a central role in the regulation of intracellular pH
(pHi) and cell volume and
contribute to epithelial Na+
resorption (11, 13). Under physiological conditions, the antiporters
exchange extracellular Na+ for
intracellular H+ by an
electroneutral process that is allosterically activated by
intracellular acidification (1). In addition, activity is also
increased in many cell types by exposure to growth factors (11) and
with cell shrinkage (20).
Six distinct isoforms of the NHE have been identified by molecular
cloning (14, 23). All isoforms share some basic structural similarities. Analysis of the primary structure predicts the existence of two distinct domains: a transmembrane
NH2-terminal region of ~500
amino acids and a hydrophilic COOH-terminal domain of nearly 300 amino
acids. The hydrophobic
NH2-terminal domain probably spans
the bilayer 10-12 times and is thought to encompass the transport
and amiloride binding sites. The COOH-terminal region is believed to
extend into the cytosol, where it presumably plays a regulatory role
(22, 23). This model was derived exclusively from hydropathy analysis
of the deduced primary sequence, and there is little biochemical
evidence to support the proposed topology. In fact, it is unclear
whether the first transmembrane domain is cleaved after serving as a
signal sequence (22), and conflicting evidence exists regarding the
location of the COOH-terminal domain in different isoforms. Antibodies
raised to the terminal 157 residues of NHE1 reacted with this
antiporter only after permeabilization of the plasmalemma (19),
implying a cytosolic localization. In contrast, monoclonal antibodies
raised to the COOH-terminal residues of NHE3 (amino acids 702-832)
effectively labeled this isoform when added externally to intact cells
or right-side-out vesicles (3). These findings may reflect
structural differences between isoforms that are not readily
apparent from analysis of hydropathy plots. Indeed, the predicted
transmembrane-spanning regions often vary when different programs are
used to analyze the hydropathy (9). Clearly, independent verification
of the structural disposition of the exchangers would be a useful
complement to these theoretical analyses. Using the predicted topology
as a cornerstone, we attempted to further elucidate the
transmembrane structure of NHE1 by utilizing enzymatic cleavage,
impermeant probes, and epitope tagging.
 |
MATERIALS AND METHODS |
Materials.
NaCl, KCl, CaCl2,
MgCl2, sodium pyrophosphate,
trisodium citrate, citric acid,
NaHCO3,
NaH2PO4,
and glucose were purchased from Fisher (Pittsburgh, PA). Nigericin,
monensin, and
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM were purchased from Molecular Probes (Eugene, OR). N-glycosidase F,
O-glycosidase, and neuraminidase as
well as n-octyl glucoside were
purchased from Boehringer Mannheim (Laval, QB, Canada). Sepharose CL-4B
and protein A-Sepharose beads were purchased from Pharmacia Biotech
(Baie d'Urfé, QB, Canada). Diisopropyl fluorophosphate was
purchased from Aldrich (Milwaukee, WI). HOE-694 was a generous gift
from Dr. Wolfgang Scholz from Hoechst (Frankfurt, Germany). All other
reagents were purchased from Sigma (Oakville, ON, Canada).
Antibodies.
Mouse monoclonal antibodies that recognize the influenza virus
hemagglutinin (HA) peptide (YPYDVPDYA) were obtained
from BAbCo (Berkeley, CA). Polyclonal antibodies to the human homologue
of NHE1 were raised by injecting rabbits with a fusion protein
encompassing the COOH-terminal 157 residues (658-815) of the
exchanger and subsequently affinity purified as described (17). Both
anti-rabbit and anti-mouse secondary antibodies were purchased from
Jackson Immunochemicals (West Grove, PA).
Platelet isolation.
Fresh blood from healthy volunteers (80 ml) was collected into 15-ml
conical tubes. Each tube contained 1.5 ml of an acid-citrate-dextrose (ACD) solution containing (in mM) 100 trisodium citrate, 70 citric acid, and 110 dextrose. To prevent platelet activation, blood was
collected by gravity with a 19-gauge needle. After gentle inversion,
blood was sedimented for 15 min at 250 g to separate the plasma. The
platelet-rich plasma from several tubes was combined and sedimented at
2,300 g for 12 min. The supernatant
was removed, and the pellet was gently resuspended in 5 ml of
Ca2+-free Tyrode solution (CFT)
containing (in mM) 137 NaCl, 12 NaHCO3, 5 dextrose, 2.7 KCl, 2 MgCl2, and 0.4 NaH2PO4
(pH 6.5) with 2% EGTA and 0.35% BSA. After a 10-min incubation at
37°C, 357 ml of ACD were added, and the cells were sedimented at
1,000 g for 10 min. The supernatant
was removed, and the pellet was resuspended in 5 ml of CFT with 0.35%
BSA but without EGTA. The cells were further incubated at 37°C for
10 min, and 357 µl of ACD were added. After centrifugation, the
platelets were resuspended in 3 ml of Tyrode solution, consisting of
CFT solution plus 0.5 mM CaCl2. Cells were counted with a Coulter counter and kept at 37°C until used.
Stable transfection and tissue culture.
Complementary DNA of the full-length rat NHE1, previously engineered to
contain a series of unique restriction sites (to facilitate mutagenic
manipulations), was subcloned into a modified eukaryotic expression
vector pCMV (for construction details, see Ref. 15). One copy of an
influenza virus HA peptide (YPYDVPDYA) was appended to the carboxy
cytoplasmic tail by PCR methodologies. This construct was transfected
into AP-1 cells using a
Ca3(PO4)2
precipitation technique (5). AP-1 cells are Chinese hamster ovary cells
devoid of endogenous NHE activity. They were generated earlier by
chemical mutagenesis, followed by selection using the
"H+-suicide" technique (16),
as previously described (18). AP-1 cells and the stable transfectants
derived thereof (AP-1-NHE1-HA) were selected by repeated acid
challenge, as previously described (16), and cultured in a 5%
CO2-humidified environment at
37°C in
-MEM containing 10% fetal bovine serum, 100 U/ml
penicillin, and 100 µg/ml streptomycin.
pHi measurements.
Platelets (5-10 ×107)
were suspended in 1 ml of Tyrode solution and were incubated at
37°C with 50 mM NH4Cl and 1 mM
BCECF-AM. After 18 min, 5 mM EDTA was added, and platelets were
sedimented by centrifugation. The platelets were next resuspended in 2 ml of a Na+-free salt solution
containing (in mM) 140 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 20 HEPES,
titrated to pH 7.3 at 37°C and immediately placed in a disposable
cuvette positioned in the thermally controlled holder of a Hitachi
F-4000 fluorescence spectrometer. After a baseline recording was
obtained, the indicated amounts of
Na+ were added to elicit NHE
activity. Fluorescence was calibrated vs. pH by the addition of
nigericin and monensin (10 mM each), followed by titration of the
extracellular pH by addition of aliquots of concentrated Tris or MES.
Rates of Na+-induced
H+ (equivalent) extrusion were
calculated by linear regression over the first 30 s of pH recovery
after addition of Na+. When
specified, platelets were first incubated with chymotrypsin, washed
once in Tyrode solution, and then loaded with dye as stated above.
For measurement of pHi,
AP-1-NHE1-HA cells, which were plated onto glass coverslips, were
incubated at room temperature for 10 min with 1 mM BCECF-AM in a
NaHEPES-buffered solution (NHB) containing (in mM) 117 NaCl, 25 NaHEPES, 5 glucose, 5 KCl, 2 MgCl2, and 2 CaCl2. After dye loading, the
coverslips were mounted into a coverslip holder that was placed in a
thermally (37°C) controlled Micro-incubator (Medical Systems)
mounted onto the stage of a Nikon TMD-Diaphot microscope attached to an
M series dual-wavelength illumination system (Photon Technology
International). The cells were alternately excited at 440 and 490 nm
while the fluorescence emission was collected at 530 nm. For acid
loading, cells were incubated in NHB with 30 mM
NH4Cl isosmotically replacing NaCl for 10 min at 37°C and then rapidly transferred to
NH4Cl-free, Na+-free solution in which
Na+ was isosmotically replaced by
N-methyl-D-glucammonium
ion (NMDG). At the end of each experiment, the cells were equilibrated
in KCl solutions of varying pH containing (in mM) 140 KCl, 25 NMDG-HEPES, 5 glucose, 2 MgCl2, 2 CaCl2, and 10 mM nigericin. A
calibration curve was thereby constructed, plotting the extracellular
pH, which is assumed to be equal to the
pHi, against the corresponding fluorescence ratio (21). The systematic error that may be introduced by
underestimation of the intracellular
K+ concentration (4) was not
corrected.
Immunofluorescence.
AP-1-NHE1-HA cells plated onto glass coverslips were grown to ~60%
confluence. They were then washed three times with PBS containing (in
mM) 137 NaCl, 7.74 Na2HPO4,
2.26 NaH2PO4,
and 2.7 KCl and fixed for 30 min at room temperature with 4%
paraformaldehyde in PBS. After fixation, the cells were washed three to
four times with PBS and then incubated with 100 mM glycine in PBS for
10 min. The cells were washed again and, except when indicated, were permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature. After blocking with 5% donkey serum for 1 h, the cells
were incubated with mouse anti-HA (1:1,000) antibody for 1 h. After
this period, cells were washed four to five times with PBS and
incubated for 1 h with a donkey anti-mouse antibody conjugated with Cy3
(1:1,000). Cells were washed for the final time and mounted onto glass
slides using Dako fluorescent mounting medium.
Biotinylation, immunoprecipitation, and immunoblotting of NHE1.
Platelets (108) were suspended
in PBS (pH 7.8) with 0.5 mM NHS-LC biotin (Pierce). After a 20-min
incubation in the dark at 0°C, the cells were sedimented and
resuspended in PBS (pH 7.4) with or without 100 U/ml chymotrypsin for
20 min at room temperature. Platelets were then sedimented and
resuspended in 1 ml of immunoprecipitation buffer containing 150 mM
NaCl, 50 mM HEPES, 5 mM EDTA, 3 mM KCl, 10 mM ATP, 25 mM sodium
pyrophosphate, 1 mM o-phenanthroline, 1 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM
benzamidine, 100 µM
N-tosyl-L-phenylalanine
chloromethyl ketone (TPCK), 1 µM pepstatin A, 20 µM leupeptin plus
10 µg/ml aprotinin, and 1% Triton X-100. Triton-insoluble material
was removed by centrifugation of the cell lysate at 20,000 g for 30 min at 4°C. The
Triton-soluble supernatant was then incubated with Sepharose CL-4B for
1 h at 4°C to preclear nonspecific binding to the beads. NHE1 was
immunoprecipitated from the lysates by incubation for 3 h at 4°C
with a 1:50 dilution of the affinity-purified anti-NHE1 antibody.
Immune complexes were collected by addition of 60 µl of a 50% slurry
of protein A-Sepharose beads to the lysate and incubation for an
additional 1h at 4°C. The beads were then washed five times with
ice-cold immunoprecipitation buffer and finally resuspended in 50 µl
of twice-concentrated Laemmli sample buffer. The samples were resolved
by 10% SDS-PAGE and then transferred to Immobilon. The blots were
incubated with anti-NHE1 antibody (1:5,000), followed by a horseradish
peroxidase-labeled anti-rabbit secondary antibody (1:5,000).
Immunoreactive bands were detected by enhanced chemiluminescence (ECL;
Amersham). For biotinylation experiments, blots were stripped and
reprobed with horseradish peroxidase-labeled avidin (1:2,500) and
detected by ECL.
Deglycosylation of NHE1.
AP-1-NHE1-HA cells grown on 60-mm dishes were incubated in the presence
or absence of chymotrypsin as specified. The cells were next scraped
off the dish with a rubber policeman, sedimented, and subsequently
resuspended in PBS containing 1 mM PMSF, 1 mM TPCK, 1 µM pepstatin A,
20 µM leupeptin, and 10 µg/ml aprotinin. The cells were then
incubated for 30 min at room temperature with 2.5 mM of the protease
inhibitor diisopropyl fluorophosphate. After this incubation, cells
were sedimented, resuspended, and then incubated for 10 min at room
temperature in a hypotonic buffer containing 10 mM HEPES, 18 mM
potassium acetate, 1 mM EDTA, 1 mM PMSF, 1 mM benzamidine, 100 µM
TPCK, 1 µM pepstatin A, 20 µM leupeptin, and 10 µg/ml aprotinin
(pH 7.2). After centrifugation, the cells were resuspended in 25 µl
of enzyme buffer A, which contained
38.7 mM
Na2HPO4,
11.3 mM
NaH2PO4,
1 mM pepstatin A, 20 mM leupeptin, 10 µg/ml aprotinin, 1%
-mercaptoethanol, and 0.5% SDS. After a 10-min incubation at room
temperature, we added 25 µl of enzyme buffer
B, which is identical to buffer
A, except that SDS was replaced with 3%
n-octyl glucoside. As suggested by the
manufacturer of
O-glycosidase,
n-octyl glucoside was added to
minimize the denaturing effect of SDS, by making the ratio of nonionic
to ionic detergents 10:1. After a further 10-min incubation at room
temperature, deglycosylation enzymes were added, and the samples were
incubated for 5 h at 37°C. To terminate the enzymatic reactions at
the end of the 5-h period, 50 µl of twice-concentrated Laemmli sample
buffer were added. After the samples were resolved by 10% SDS-PAGE,
proteins were transferred to Immobilon membranes that were subsequently
blocked by incubation in PBS containing 0.1% Tween 20 and 5% skim
milk for 2 h. The blots were incubated with a monoclonal antibody to HA
(1:5,000), followed by a horseradish peroxidase-conjugated anti-mouse
antibody (1:5,000) and development by ECL.
 |
RESULTS |
Chymotryptic cleavage of human NHE1.
Human platelets were used as a model system to investigate the topology
of NHE1 because they are readily available and express comparatively
large amounts of the exchanger. The degree of exposure of NHE1 to the
extracellular space was probed by using chymotrypsin. This protease
cleaves the peptide bonds on the carboxy side of accessible tyrosine,
tryptophan, and phenylalanine residues. As illustrated in Fig.
2A, human NHE1 has three potential
cleavage sites on the first putative extracellular loop and several
more on the last three loops. Intact human platelets were incubated for
various times with 100 U/ml chymotrypsin. Proteolysis was halted by
washing the cells once in PBS containing the chymotrypsin inhibitor
TPCK. Whole cell lysates were resolved by SDS-PAGE and subjected to
immunoblotting with an antibody to the COOH-terminal 157 residues (Fig.
1A).
Cleavage of NHE1 by extracellular chymotrypsin was apparent at the
earliest time studied, i.e., 5 min. The first detectable cleavage
reduced the apparent molecular mass from ~110 to ~90 kDa. Because a
comparatively low concentration of chymotrypsin produced quantitative
cleavage within 5 min, the susceptible site(s) must be readily
accessible. A second product of NHE1 proteolysis of molecular mass of
~70 kDa became apparent by 10 min. The abundance of this polypeptide
increased over the next 60 min, and that of the 90-kDa polypeptide
intermediate diminished proportionately.

View larger version (88K):
[in this window]
[in a new window]
|
Fig. 1.
Chymotryptic cleavage of human
Na+/H+
exchanger (NHE1). Isolated human platelets were incubated for indicated
times with 100 U/ml chymotrypsin. A:
whole cell lysates from untreated (Cntl) or chymotrypsinized platelets
were resolved by SDS-PAGE, transferred to Immobilon, and immunoblotted
with anti-NHE1 antibody. Arrows indicate wild-type NHE1 and 2 cleavage
products. Blot is representative of 4 similar experiments.
* Position of albumin, which produced variable amounts of
spurious immunostaining. B: cell
lysates from parallel samples were resolved by SDS-PAGE, and gel was
stained with Coomassie blue. Arrow, actin binding protein (ABP).
|
|
The results of Fig. 1A suggest that
NHE1 exposes at least two distinct sites to the extracellular milieu.
This conclusion rests on the assumption that the platelet membrane
remains intact and impermeant to chymotrypsin during prolonged
incubation with the protease. This was ascertained by analyzing the
polypeptidic pattern of intact platelets after varying times of
incubation with the protease. Whole cell lysates were analyzed by
SDS-PAGE and stained with Coomassie blue (Fig.
1B). Of particular interest are
intracellular proteins known to be susceptible to proteolysis. In
platelet lysates, it is well established that the three heaviest major
polypeptides, with molecular masses of 280, 215, and 200 kDa,
correspond to actin binding protein (ABP), talin, and myosin, respectively (7). It is noteworthy that these proteins remained unaffected throughout the 60-min incubation period used in Fig. 1A. Incipient degradation of these
proteins, especially of ABP, was noted only after 90 min. On the other
hand, when platelets were permeabilized with Triton before exposure to
chymotrypsin, ABP, talin, and myosin were rapidly and completely
degraded (not illustrated). Thus platelets remain intact and
impermeable to chymotrypsin for at least 60 min. This conclusion was
validated during the course of pHi
measurements by the intracellular retention of fluorescent dyes and by
the stability of transmembrane ionic gradients (see below).
Biotinylation of human NHE1.
The previous results suggest that a sizable portion of NHE1 is exposed
to the external medium. This notion was confirmed using an impermeant
biotin derivative capable of reacting covalently with
-amino groups
of lysine residues. Intact platelets were reacted with NHS-LC biotin
and then incubated for 5 min in the presence or absence of
chymotrypsin. As shown in Fig.
2B,
biotinylation did not alter the electrophoretic mobility of NHE1 nor
the ability of chymotrypsin to cleave the exchanger, as revealed by
immunoblotting. The occurrence of biotinylation was verified by
overlaying the same blots with peroxidase-coupled avidin (Fig.
2C). Importantly, although the
presence of covalently bound biotin is readily detectable in the
full-length NHE1, the probe was absent after the initial rapid cleavage
catalyzed by chymotrypsin. Because the antibody used for immunoblotting
detects the COOH-terminal region of the protein, we conclude that not
all of the lysine residues proposed to be exposed to the external
medium are accessible for biotinylation. Our data suggest that only the
lysine residues contained within ~25 kDa of the
NH2-terminal end of the protein
are accessible (see Fig.
2A).

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 2.
Chymotryptic cleavage and biotinylation of human NHE1.
A: diagram of putative topology of
human NHE1 depicting residues that are potential chymotrypsin cleavage
sites (aromatic amino acids; circles) and biotinylation sites (lysines;
triangles). Probable chymotrypsin cleavage sites in first external loop
are marked with an arrowhead. Site of N-linked glycosylation and
potential O-glycosylation sites are also depicted (squares).
B and
C: isolated human platelets were
treated with (+) or without ( ) NHS-LC biotin as described in
METHODS and, when specified, were
subsequently incubated for 5 min with 100 U/ml chymotrypsin. NHE1 was
immunoprecipitated, resolved by SDS-PAGE, and blotted onto Immobilon.
Specificity of precipitation was assessed with preimmune serum (pi).
B: immunoblot using anti-NHE1
antibody. Slight decrease in mobility in biotinylated sample in
rightmost lane was not a reproducible observation.
C: blot in
B was stripped and reprobed by using
horseradish peroxidase-conjugated avidin.
B and
C are representative of 2 separate
experiments.
|
|
Effect of extracellular chymotryptic cleavage on NHE1 function.
Earlier structure-function analysis revealed that NHE activity persists
in mutants lacking most of the cytosolic COOH-terminal tail (22). It
was therefore concluded that the transmembrane, NH2-terminal region is necessary
and sufficient for transport. However, the regions of the
NH2-terminal domain required for
activity were not defined. The finding that the protein can be
proteolytically severed at two sites near the
NH2 terminus enabled us to test the role of this domain in ion exchange. We measured NHE activity in
platelets by recording their ability to recover from an acid load
imposed by prepulsing with NH4Cl.
As shown in Fig.
3A,
control cells recovered very rapidly from the acid load, but only when Na+ was present in the medium.
This recovery was sensitive to both amiloride and HOE-694, which is a
relatively specific inhibitor of NHE1 when used in the low micromolar
range (6). Pretreatment of the platelets with chymotrypsin had no
obvious effect on the ability of the cells to recover from the acid
load nor on the susceptibility of NHE to amiloride or HOE-694 (Fig.
3B). Even after a 60-min treatment
with chymotrypsin, the rate of
Na+-induced alkalinization was
decreased only marginally and was not statistically different from the
control rate (Fig. 3C). Importantly, the Na+-induced extrusion of
H+ (equivalents) was virtually
eliminated by 1 µM HOE-694 in both the control and
protease-treated cells (Fig. 3).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of chymotryptic cleavage on NHE1 activity. Control platelets
(A) or platelets pretreated with 100 U/ml chymotrypsin for 60 min (B)
were loaded with
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF) and acidified by an NH4Cl
prepulse. Cells were then suspended in
Na+-free
K+ medium with or without
amiloride (1 mM) or HOE-694 (1 µM), as indicated, and intracellular
pH (pHi) was monitored
fluorometrically as described in
METHODS. Where indicated by arrow, 28 mM Na+ was added to medium.
C: rate of
Na+-induced alkalinization was
measured in pHi 6.3-6.5
interval in control cells and in cells treated with chymotrypsin for 60 min. Data are means ± SE of no. of experiments indicated.
* Significant difference of P < 0.05 with respect to appropriate control.
|
|
A more detailed comparison of the kinetic properties of NHE in control
and chymotrypsin-treated platelets is presented in Fig.
4. The dependence of the exchanger on
pHi, which is typically sigmoidal
with half-maximal activity near
pHi 6.65, was very similar after
treatment for 60 min with up to 200 U/ml chymotrypsin (Fig. 4A). Because the buffering power was
similar in the two populations of cells and to facilitate comparison,
relative rates of recovery, rather than absolute
H+ (equivalent) fluxes, are
illustrated. Comparable results were obtained in eight control and four
chymotrypsin-treated experiments. Finally, the extracellular
Na+ concentration dependence,
measured at pHi 6.4, was also
indistinguishable before and after chymotryptic treatment (Fig.
4B).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of chymotrypsin on pHi and
external Na+ dependence of NHE1.
Platelets were incubated without (control) or with 200 U/ml
chymotrypsin for 20 min and loaded with BCECF for measurement of
pHi.
A: platelets were acidified to varying
degrees, and Na+-induced rate of
recovery was determined as in Fig. 3. To facilitate comparison, data
were normalized to rate of alkalinization recorded at pH 6.3, and
normalized initial rate of pH change is plotted against
pHi. Absolute rate of
alkalinization measured at pHi 6.3 was 0.91 ± 0.18 pH units/min (n = 7) for controls and 0.69 ± 0.22 pH units/min
(n = 4) for chymotrypsin-treated
samples. No significant difference (P > 0.05) in absolute rate of recovery was observed between control or
chymotrypsin-treated cells at any
pHi.
B: platelets were acidified to pH 6.4 and recovery was measured with addition of varying
Na+ concentrations. Initial rate
of recovery from acidification is plotted against
Na+ concentration. Data are means ± SE of no. of experiments indicated. No significant difference
(P > 0.05) in
Na+ dependence was observed
between control or chymotrypsin-treated cells at any extracellular
Na+ concentration.
|
|
Expression and characterization of epitope-tagged NHE1.
Although the predicted topology for all the NHE isoforms suggests that
the COOH-terminal tail is located intracellularly, a recent report (3)
suggests that the extreme COOH terminus of NHE3 might in fact be
extracellular. On the other hand, antibodies to the putative cytosolic
domain of NHE1 reacted with the exchanger only after permeabilization
of the cell membrane (19), implying that the epitopes are
intracellular. However, these experiments were performed using
antibodies raised to a fusion protein encompassing the COOH-terminal
157 amino acids, and the precise antigenic determinants are not known.
It is therefore conceivable that although the immunoreactive part of
the tail is indeed located intracellularly, the extreme COOH terminus
is exposed extracellularly, by virtue of an additional transmembrane
crossing.
A similar caveat applies to our experiments in platelets, which used a
polyclonal antibody to the COOH-terminal 157 residues. An extracellular
COOH-terminal fragment could conceivably have been cleaved by
chymotrypsin, remaining undetected if it did not contain antigenic
determinants or was too small to be resolved by the PAGE system used.
To establish more precisely the location of the COOH terminus of NHE1,
we constructed a vector to express full-length NHE1 tagged at
its COOH terminus with an influenza virus HA peptide (Fig.
5A).
This epitope-tagged exchanger was stably transfected into NHE-deficient
AP-1 cells, yielding AP-1-NHE1-HA cells. To verify expression of the
HA-tagged NHE1, the transfectants were lysed, resolved by SDS-PAGE, and
immunoblotted with a monoclonal anti-HA antibody. As illustrated in
Fig. 5B, two immunoreactive bands were
detected in AP-1-NHE1-HA cells: a major, diffuse band of ~100 kDa,
corresponding to mature, tagged NHE1, and a sharper, smaller band of
~80 kDa, which probably represents incompletely glycosylated NHE1
trapped in the secretory pathway. A similar immature form was reported
earlier after heterologous expression of NHE1 (6). Importantly, no
immunoreactive bands were detected in untransfected AP-1 cells (Fig.
5B).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
Epitope-tagged rat NHE1: heterologous expression in AP-1 cells.
A: predicted topology of rat NHE1.
Position of hemagglutinin (HA) tag is indicated. Potential chymotrypsin
cleavage sites (circles) and glycosylation sites (squares) are
depicted. Probable chymotrypsin cleavage sites are marked with an
arrowhead. B: AP-1 cells that were
stably transfected with HA-tagged NHE1 (AP-1-NHE1-HA) and their
untransfected counterparts (AP-1) were lysed, resolved by SDS-PAGE,
transferred to Immobilon, and immunoblotted with an anti-HA antibody.
Representative of 3 similar experiments.
|
|
To use the tagged constructs for assessment of the topology of NHE1, it
was important to ascertain that introduction of the epitope did not
alter the properties of the exchanger. This was evaluated functionally.
AP-1-NHE1-HA cells as well as the parental AP-1 cells were acid loaded
as pHi was recorded
fluorometrically (Fig. 6). The
transfectants recovered from the acidosis on reintroduction of
Na+ to the medium, whereas the
untransfected parental cells remained acidic despite the addition of
Na+ (Fig.
6A). These findings imply that at
least a fraction of the tagged NHE-1 reaches the plasmalemma, in which
it effectively catalyzes
Na+/H+
exchange. In fact, the rate of exchange was greater in AP-1-NHE1-HA cells than in wild-type Chinese hamster ovary cells (WT5) expressing the endogenous antiporter. As shown in Fig. 6,
B and
C, the activity observed in the
transfectants bears all the hallmarks of NHE1: the extrusion of
H+ was found to be
Na+ dependent and sensitive to
micromolar doses of ethylisopropyl amiloride and HOE-694.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 6.
Functional assessment of NHE1-HA expression in AP-1 cells.
A: AP-1 or AP-1-NHE1-HA cells were
loaded with BCECF and acidified by an
NH4Cl prepulse. Tail end of
prepulse is illustrated. pHi was
measured fluorometrically in
Na+-free
[N-methyl-D-glucammonium
ion (NMDG) medium] or
Na+-rich medium, as indicated by
bars. B: AP-1-NHE1-HA cells were BCECF
loaded and acidified as in A. Recovery
from acidification was monitored in
Na+-rich medium in presence or
absence of 1 µM ethylisopropyl amiloride (EIPA), as indicated.
C: accumulated data of rates of
Na+-induced recovery from an
acidification in wild-type Chinese hamster ovary cells (WT), AP-1
cells, or AP-1-NHE1-HA cells. Presence or absence of
Na+, HOE-694 (HOE), and EIPA is as
indicated. Data are means ± SE of no. of experiments indicated.
Last 3 columns were compared with respective control (AP-1-NHE1 cells
with Na+) and significance of
difference is noted. ** P < 0.01; *** P < 0.001.
|
|
The expression of NHE1-HA was further evaluated by immunofluorescence.
In accordance with the immunoblotting data, AP-1 cells failed to react
with the HA-specific antibody (Fig. 7,
left). In contrast, those cells
stably transfected with NHE1-HA demonstrated a diffuse,
membrane-associated labeling (Fig. 7,
middle). In some cells, perinuclear
intracellular staining was observed, consistent with accumulation of
some of the immature NHE1 in the endoplasmic reticulum, likely due to
overexpression (see inset, Fig. 7,
middle). Importantly, immunolabeling
was observed only after the AP-1-NHE1-HA cells had been permeabilized
with Triton-X-100 (Fig. 7, middle) but not in fixed, unpermeabilized cells (Fig. 7,
right). This indicates that the HA
epitope is intracellular and therefore implies that the COOH-terminal
end of NHE1 is located intracellularly as well.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 7.
Detection of NHE1-HA in intact and permeabilized AP-1 cells by
immunofluorescence. AP-1 (left) or
AP-1-NHE1-HA cells (middle and
right) were fixed with 4%
paraformaldehyde and treated with
(left and
middle) or without
(right) 0.1% Triton X-100 in PBS
for 20 min. Samples were then probed with a monoclonal anti-HA
antibody, followed by a Cy3-labeled goat anti-mouse secondary antibody.
Inset to
middle illustrates perinuclear,
probably endoplasmic reticular staining.
|
|
Chymotryptic cleavage of rat NHE1.
Treatment of the platelet exchanger with chymotrypsin and use of the
polyclonal antibody indicated that cleavage occurred near the
NH2-terminal domain of NHE1 but
could not rule out the occurrence of COOH-terminal cleavage. This
possibility was considered experimentally using the heterologously
expressed rat NHE1, which was epitope tagged at its COOH terminus.
AP-1-NHE1-HA cells were exposed to chymotrypsin for various times, and
whole cell lysates were resolved by SDS-PAGE, followed by
immunoblotting with a monoclonal antibody to HA. Within 5 min,
chymotrypsin cleaved NHE1-HA, yielding an immunoreactive fragment of
apparent molecular mass of 64 kDa (Fig. 8).
The immunoreactivity appeared to increase in the 80-kDa region,
suggesting an alternate cleavage site. However, the appearance of this
product was obscured by the presence of the immature form of NHE1 in
the transfectants. No further cleavage products were noted for up to 20 min (not illustrated). These results imply that chymotrypsin cleaves
only at sites located in the
NH2-terminal half of the protein
and are consistent with the notion that the COOH terminus is localized
entirely in the cytosolic environment.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 8.
Chymotryptic cleavage of HA-tagged rat NHE1: effect on glycosylation
sites. AP-1-NHE1-HA cells were incubated with (+) or without ( )
100 U/ml chymotrypsin for 10 min. Cell lysates were then prepared and
treated with indicated glycosidases for 5 h, as described in
METHODS. Samples were then resolved by
SDS-PAGE, transferred to Immobilon, and immunoblotted with anti-HA
antibody. Treatment with chymotrypsin,
N-glycosidase F
(N-Gly), neuraminidase (Neura), or
O-glycosidase
(O-Gly) is indicated. Representative
of 3 experiments.
|
|
Localization of glycosylation sites in rat NHE1.
The human NHE1 was shown earlier to exhibit both N- and O-linked
carbohydrates (6). The sequence of the rat homologue (Fig. 5A) similarly predicts several
potential O-linked glycosylation sites and a potential
N-linked glycosylation site. As shown in Fig. 8, incubation
of lysates, obtained from AP-1-NHE1-HA cells, with
N-glycosidase F increased
the mobility of the mature rat NHE1 from an apparent molecular mass of
~100 to ~83 kDa. The mobility was similarly increased by a combined
treatment with neuraminidase and
O-glycosidase. These observations
suggest that, like the human NHE1, the rat homologue is glycosylated on
both asparagine and serine residues. It is noteworthy that the minor
band of ~75 kDa was virtually unaffected by the glycosidases,
consistent with the notion that it is a poorly glycosylated immature
form of NHE1.
Having established that proteolysis occurs only near the
NH2 terminus, we proceeded to
define the position of the cleavage sites with respect to the sites of
glycosylation. Cells were initially treated with chymotrypsin, then
lysed, and subjected to deglycosylation. As shown in Fig. 8, the
mobility of the chymotryptic products was similar, ~65 kDa, whether
the samples had been treated with or without
N-glycosidase F or with the
combination of neuraminidase and
O-glycosidase. These findings imply
that the carbohydrate moieties had been removed by the protease
chymotrypsin. The increased sharpness of the bands obtained after
proteolysis is consistent with this conclusion.
 |
DISCUSSION |
The experiments described above provide evidence to complement our
understanding of the structure of NHE1 predicted by analysis of
hydropathy.
COOH terminus.
Earlier biochemical studies suggested that most of the COOH-terminal
domain of NHE1 (~300 residues) is located intracellularly. The
evidence supporting this notion includes the following:
1) the finding that antibodies
raised to the terminal 157 amino acids react only after cells are
permeabilized (19); 2) the
observation that all the known phosphorylation sites are within the
COOH-terminal domain (22); and 3)
the report that calmodulin can bind to two sites within this domain
(2). On the other hand, a more recent result indicates that at least
part of the COOH-terminal region of NHE3 appears to be exposed
extracellularly (3).
Our data using both native and epitope-tagged NHE1 indicate that the
extreme COOH terminus of this protein lies within the cell, since it
was not accessible to external antibodies or to an impermeant biotin
derivative. Moreover, none of the hydrophobic residues that are
substrates for chymotryptic cleavage in the COOH-terminal tail are
exposed to the protease when it is added to intact cells. Thus our
findings are most easily reconciled with the original model, reproduced
in Fig. 5A, in which the COOH-terminal hydrophilic domain lies entirely within the cytosol. The discrepancy between our findings and those of Biemesderfer et al. (3) may reflect
differences between isoforms. However, preliminary experiments in our
laboratory indicate that, as in the case of NHE1, an epitope tag placed
at the COOH terminus of NHE3 is accessible only after permeabilization
of the cells and is resistant to proteolytic treatment in intact but
not in permeabilized cells (unpublished observations).
NH2 terminus.
As reported for the human NHE1 (6), we found that the rat homologue of
the exchanger contains both N- and O-linked carbohydrates (Fig. 8). In
the human NHE1, mutagenesis studies found that N75 is the only
potential N-glycosylation site utilized, implying that the first
putative loop is indeed extracellular. These observations could not be
confirmed by epitope tagging of the extreme
NH2 terminus, possibly because
this portion of the protein is cleaved cotranslationally as part of a
signal sequence.
As an alternative approach, we used chymotryptic treatment to verify
the transmembrane disposition of NHE1. In platelets, the protease
cleaved the exchanger quantitatively under conditions in which
intracellular proteins remained unaffected. These findings indicate
that the entire population of NHE1 is present on the surface membrane
of unstimulated platelets. This rules out recruitment of intracellular
exchangers as a viable mechanism of stimulation, as found to be the
case for H+ pumps and glucose
transporters in other tissues (e.g., Ref. 12).
As reported earlier (6), two immunoreactive bands can be observed in
transfected cells. The smaller of these polypeptides was proposed to be
an intermediate form in the biosynthesis of NHE1 (6). Our observations
in the AP-1-NHE1-HA cell line, which are stably transfected with rat
NHE1, support this notion in that 1)
the smaller polypeptide was insensitive to chymotrypsin, suggesting an
intracellular location, probably within the secretory pathway, and
2) the putative precursor was
insensitive to both N- and
O-glycosidase, indicating that it
contains little, if any, carbohydrate. Accordingly, unlike the mature
NHE1, this species migrates as a comparatively sharp band on SDS-PAGE.
Because the biosynthetic intermediate was not detected in either native
platelets (this report) or salivary acinar cells (17), it is
conceivably a result of overexpression in heterologous transfectants
and may be of little physiological importance.
We detected two distinct chymotryptic cleavage sites in human NHE1 and
at least one site in rat NHE1. A second site may exist in rat NHE1, but
the product of its cleavage is obscured by the presence of the
~80-kDa immature species in the transfectants. The precise location
of the cleavage sites is difficult to establish based solely on the
molecular mass of the products, because the contribution of the
carbohydrate to the electrophoretic mobility of the protein is
uncertain and because the sites of O-linked glycosylation are not
defined. We therefore proceeded to deglycosylate NHE1 after
proteolysis. As shown in Fig. 8, the ~65-kDa fragment yielded by
chymotrypsin treatment did not contain detectable N- or O-linked
carbohydrate. On the basis of the amino acid composition of the rat
NHE1, we can estimate that cleavage occurred at most ~150 residues
from the NH2 terminus, i.e., in
the first or second putative extracellular loops (see arrowheads in
Fig. 5A). The portion cleaved off
includes the sites of attachment of both the N- and O-linked sugars:
these probably include N76 and one or more of the serine residues
contained between residues 35 and 66 in the first external loop.
Biotinylation experiments using an impermeant succinimidyl derivative
indicated that at least one lysine residue in human NHE1 is readily
exposed extracellularly. These sites were found to be near the
NH2 terminus, inasmuch as they
were cleaved by short incubation with chymotrypsin (Fig.
2C). There are 10 lysine residues in
the predicted extracellular loops of the human NHE1, 2 on the first
loop and the remainder on the fifth and sixth loops (Fig.
2A). We favor the interpretation
that only one or both of the lysine residues in the first loop are
accessible from the outside. This would imply that the putative
extracellular loops between transmembrane domains 9-10 and
11-12, despite their size and hydrophilicity, are not
fully exposed to the outside medium. In accordance with this
interpretation, chymotrypsin failed to cleave in this region, despite
the presence of multiple tyrosine and phenylalanine residues that are
theoretical substrate sites for the protease (see Figs.
2C and
5A). Hence the regions between transmembrane domains 9-10 and 11-12 may lie along the
membrane surface or be folded within the protein, perhaps contributing to the aqueous path for ion translocation. In addition, it is possible
that these two loops are involved in dimerization of NHE1 (8), which
may involve intermolecular disulfide linkage (9).
Functional consequences of cleavage.
Extensive chymotryptic cleavage of human NHE1, which reduced the size
of the polypeptide containing the antigenic determinants to ~70 kDa,
had little effect on ion exchange activity. Neither the rate nor the
apparent affinity of the exchanger for external Na+ or internal
H+ were measurably affected.
Therefore structural integrity is not essential for NHE1 activity. It
is noteworthy, however, that because our antibodies could not detect
the NH2-terminal region of the protein, the cleaved fragments may have remained associated with the
membrane and the COOH-terminal portion of the protein after proteolysis, possibly continuing to contribute to the functional activity. Resolution of these alternatives will require development of
antibodies to the first external loop or epitope tagging of this
region.
 |
ACKNOWLEDGEMENTS |
We thank Dr. G. Goss for his contribution to preliminary
experiments that facilitated this work.
 |
FOOTNOTES |
This work was supported by grants from the Medical Research Council
(MRC) of Canada (to S. Grinstein and J. Orlowski). L. D. Shrode is the
recipient of a postdoctoral fellowship from the Arthritis Society of
Canada. B. S. Gan is supported by grants from the Plastic Surgery
Educational Foundation, Plastic Surgery Research Fund, and Toronto
Hospital Dept. of Surgery. S. J. A. D'Souza is the recipient of a
postdoctoral fellowship from the MRC. S. Grinstein is cross-appointed
to the Dept. of Biochemistry, University of Toronto, and is an
International Scholar of the Howard Hughes Medical Institute, an MRC
Distinguished Scientist, and current holder of the Pitblado Chair in
Cell Biology.
Address for reprint requests: S. Grinstein, Div. of Cell Biology, 555 University Ave., Toronto, Ontario, Canada M5G 1X8.
Received 4 November 1997; accepted in final form 15 April 1998.
 |
REFERENCES |
1.
Aronson, P. S.,
J. Nee,
and
M. A. Suhm.
Modifier role of internal Na+ inactivating the Na+/H+ exchanger in renal microvillus membrane vesicles.
Nature
299:
161-163,
1982[Medline].
2.
Bertrand, B.,
S. Wakabayashi,
T. Ikeda,
J. Pouyssegur,
and
M. Shigekawa.
The Na+/H+exchanger isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins. Identification and characterization of calmodulin-binding sites.
J. Biol. Chem.
269:
13703-13709,
1994[Abstract/Free Full Text].
3.
Biemesderfer, D.,
P. A. Rutherford,
B. DeGray,
and
P. S. Aronson.
Immunocyochemical studies of NHE3: evidence that an epitope within the C-terminal domain is exoplasmic (Abstract).
Mol. Biol. Cell
6:
H43,
1995.
4.
Boyarsky, G.,
C. Hanssen,
and
L. A. Clyne.
Inadequacy of high K+/nigericin for calibrating BCECF. II. Intracellular pH dependence of the correction.
Am. J. Physiol.
271 (Cell Physiol. 40):
C1131-C1145,
1996[Abstract/Free Full Text].
5.
Chen, C.,
and
H. Okayama.
High-efficiencey transformation of mammalian cells by plasmid DNA.
Mol. Cell. Biol.
7:
2745-2752,
1987[Medline].
6.
Counillon, L.,
J. Pouysségur,
and
R. A. F. Reithmeier.
The Na+/H+ exchanger NHE1 possesses N- and O-linked glycosylation restricted to the first N-terminal extracellular domain.
Biochemistry
33:
10463-10469,
1994[Medline].
7.
Crawford, N. K,
S. Authi,
and
N. Hack.
Isolation and characterization of platelet membranes prepared by free flow electrophoresis.
Methods Enzymol.
215:
5-20,
1992[Medline].
8.
Fafournoux, P.,
J. Noel,
and
J. Pouyssegur.
Evidence that Na+/H+ exchanger isoforms NHE1 and NHE3 exist as stable dimers in membranes with a high degree of specificity for homodimers.
J. Biol. Chem.
269:
2589-3596,
1994[Abstract/Free Full Text].
9.
Fliegel, L.,
and
P. Dibrov.
The Na+/H+ Exchanger (1st ed.). Austin, TX: Landes, 1997, p. 1-20.
10.
Grinstein, S.,
and
A. Rothstein.
Mechanisms of regulation of the Na+/H+ exchanger.
J. Membr. Biol.
90:
1-12,
1986[Medline].
11.
Grinstein, S.,
D. Rotin,
and
M. J. Mason.
Na+/H+ exchange and growth factor-induced cytosolic pH changes. Role in cellular proliferation.
Biochim. Biophys. Acta
988:
73-97,
1989[Medline].
12.
Kandror, K. V.,
and
P. F. Pilch.
Compartmentalization of protein traffic in insulin-sensitive cells.
Am. J. Physiol.
271 (Endocrinol. Metab. 34):
E1-E14,
1996[Abstract/Free Full Text].
13.
Noël, J.,
and
J. Pouysségur.
Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms.
Am. J. Physiol.
268 (Cell Physiol. 37):
C283-C296,
1995[Abstract/Free Full Text].
14.
Orlowski, J.,
and
S. Grinstein.
Na+/H+ exchangers of mammalian cells.
J. Biol. Chem.
272:
22373-22376,
1997[Free Full Text].
15.
Orlowski, J.,
and
R. A. Kandasamy.
Delineation of transmembrane domains of the Na+/H+ exchanger that confer sensitivity to pharmacological antagonists.
J. Biol. Chem.
271:
19922-19927,
1996[Abstract/Free Full Text].
16.
Pouyssegur, J.,
C. Sardet,
A. Franchi,
G. L'Allemain,
and
S. Paris.
A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH.
Proc. Natl. Acad. Sci. USA
81:
4833-4837,
1984[Abstract].
17.
Robertson, M. A.,
M. Woodside,
J. K. Foskett,
J. Orlowski,
and
S. Grinstein.
Muscarinic agonists induce phosporylation-independent activation of the NHE1 isoform of the Na+/H+ antiporter in salivary acinar cells.
J. Biol. Chem.
272:
287-294,
1997[Abstract/Free Full Text].
18.
Rotin, D.,
and
S. Grinstein.
Impaired cell volume regulation in Na+-H+ exchange-deficient mutants.
Am. J. Physiol.
257 (Cell Physiol. 26):
C1148-C1156,
1989.
19.
Sardet, C.,
L. Counillon,
A. Franchi,
and
J. Pouysségur.
Growth factors induce phosphorylation of the Na+/H+ antiporter, a glycoprotein of 110 kD.
Science
247:
723-726,
1990[Medline].
20.
Shrode, L. D.,
J. D. Klein,
W. C. O'Neill,
and
R. W. Putnam.
Shrinkage-induced activation of Na+/H+ exchange in primary rat astrocytes: role of myosin light chain kinase.
Am. J. Physiol.
269 (Cell Physiol. 38):
C257-C266,
1995[Abstract/Free Full Text].
21.
Thomas, J. A.,
R. N. Buchsbaum,
A. Zimniak,
and
E. Racker.
Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.
Biochem. J.
18:
2210-2218,
1979.
22.
Wakabayashi, S.,
P. Fafournoux,
C. Sardet,
and
J. Pouysségur.
The Na+/H+ antiporter cytoplasmic domain mediates growth factor signals and controls "H+-sensing."
Proc. Natl. Acad. Sci. USA
89:
2424-2428,
1992[Abstract].
23.
Wakabayashi, S.,
M. Shigekawa,
and
J. Pouysségur.
Molecular physiology of vertebrate Na+/H+ exchangers.
Physiol. Rev.
77:
51-74,
1997[Abstract/Free Full Text].
Am J Physiol Cell Physiol 275(2):C431-C439
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society