(Received for publication, June 26, 1995; and in revised form, August 18, 1995)
From the
The Na/H
exchanger isoforms
NHE1 and NHE3 are regulated differently by various stimuli. Calcium has
been recognized as one of the major second messengers in such exchanger
regulation. We previously proposed that Ca
-induced
activation of NHE1 occurs via displacement of its autoinhibitory domain
from the H
modifier site due to direct binding of
Ca
/calmodulin. To further validate this hypothesis,
the functional role of the cytoplasmic domain was studied in both
wild-type and chimeric exchangers, i.e. NHE1, NHE3, NHE1 with
the cytoplasmic domain of NHE3(N1N3), and NHE3 with the cytoplasmic
domain of NHE1(N3N1). After expression in exchanger-deficient
fibroblasts (PS120), early response (<80 s) to external stimuli was
assessed as
5-(N-ethyl-N-isopropyl)amiloride-sensitive
Na
uptake. Among stimuli tested
(ionomycin,
-thrombin, phorbol ester, hyperosmotic stress, and
platelet-derived growth factor) that are all known to activate NHE1,
only ionomycin and thrombin induced a significant intracellular
Ca
mobilization and early activation of
Na
uptake, implying that Ca
is a main regulator of NHE1 in the early phase of the agonist
response. However, all the stimuli did not activate NHE3 or N1N3. In
contrast, a significant stimulation of
Na
uptake in response to ionomycin and thrombin was observed in N3N1,
accompanied by an alkaline shift of pH
sensitivity
(
0.2 pH units). Deletion of the cytoplasmic calmodulin-binding
domain within N3N1 resulted in a constitutive alkaline shift of
pH
sensitivity and abolished the activation by
ionomycin and thrombin. Together, these data reinforce our concept of
Ca
-induced activation of NHE1. Furthermore, they
provide evidence for a functional interaction of the autoinhibitory
domain of NHE1 with the H
-modifier site of a different
isoform, NHE3.
Calcium ion is an important second messenger in mammalian cells,
regulating various cell functions including muscle contraction,
secretion, cell cycle progression, and a large variety of nerve cell
functions. In many of these processes, elevation of the intracellular
Ca concentration
([Ca
]
) and subsequent
Ca
-dependent activation of a ubiquitous regulator
protein calmodulin (CaM) (
)have been recognized as a major
mechanism of signal transduction in response to hormonal stimulation or
membrane depolarization(1, 2) .
The electroneutral
plasma membrane Na/H
exchanger
isoform 1 (NHE1) has been shown to be one of the targets regulated by
intracellular
Ca
(3, 4, 5, 6, 7, 8) .
NHE1 (9) is a ubiquitous amiloride-sensitive transporter that
regulates pH
and cell
volume(10, 11) , and its structure-function
relationship has been studied
extensively(7, 12, 13, 14, 15, 16, 17) .
We have recently shown that NHE1 is a CaM-binding protein containing
high and low affinity CaM-binding sites in the middle of the
carboxyl-terminal cytoplasmic domain(17) . Based on the
analysis of function of NHE1 mutant molecules that do not bind CaM, we
proposed that Ca
-induced activation of NHE1 occurs
via direct binding of Ca
/CaM to the high affinity
site that has an autoinhibitory function(7) . However, further
experiments were required to unambiguously confirm this hypothesis
because of lack of evidence for the direct effect of
Ca
/CaM on the exchange activity.
When NHE1 is
activated in response to various stimuli such as growth factors,
calcium, and hyperosmotic stress, it is generally accepted that
pH sensitivity of Na
/H
exchange increases without an apparent change in V
(18, 19, 20) . This
is thought to result from increased affinity of the allosteric modifier
site of the exchanger for the intracellular
H
(21) . However, recently cloned other
exchanger isoforms (NHE2, NHE3, and NHE4) (22, 23, 24, 25) differ greatly from
NHE1 in their regulation. Growth factors activate the epithelial
isoforms NHE2 and NHE3 by increasing V
(26, 27) . Phorbol ester
stimulates NHE1 and NHE2 but inhibits NHE3(26, 27) .
Hyperosmolarity stimulates NHE1, NHE2, and NHE4 but inhibits NHE3 (28, 29, 30) . These differences appear to be
attributable to sequence divergence of the cytoplasmic domains of these
NHE isoforms. The amiloride-resistant NHE3 that is expressed in the
apical membrane of epithelial cells in kidney or intestine is the least
related isoform among four mammalian NHEs. The NHE3 cytoplasmic domain
shows very low sequence homology to that of NHE1. Particularly, the
high affinity CaM-binding site sequence of NHE1 is very different from
the corresponding sequence in NHE3. These findings prompted us to study
differences in the Ca
regulation between NHE1 and
NHE3.
In this work, we studied the mechanism of Ca
regulation in the early phase of agonist stimulation in fibroblastic
cells expressing NHE1, NHE3, or their chimeras. Here we show that
Ca
-insensitive NHE3 becomes activatable in response
to an ionomycin- or thrombin-induced increase in
[Ca
]
by replacing the
complete cytoplasmic domain of NHE3 with that of NHE1. Deletion mutant
analysis revealed that this new function conferred by grafting the
cytoplasmic domain of NHE1 was due to the transfer of its
autoinhibitory CaM-binding site.
Figure 1:
Constructs and expression of chimeric
molecules. A, comparison of high affinity CaM binding region A
of rat NHE1 and its corresponding sequence of rat NHE3. Sequences were
aligned as described by Orlowski et al.(43) . B, schematic representation of NHE1/NHE3 chimeric and deletion
constructs. Coding regions for NHE1 and NHE3 are represented by open and closed rectangles, respectively. Numbers show amino acids. C, EIPA concentration dependence of Na
uptake by cells expressing NHE
variants. This experiment were performed by the
Li
-loading method as described previously (12) .
Fig. 2A shows immunoblots of NHE transfectants
probed with a NHE1-specific antibody (RP-cd) and a monoclonal antibody
(P4D5) recognizing the VSVG epitope. In this experiment, we used cells
stably transfected with pNHE3-VSVG and pN1N3-VSVG in place of pNHE3 and
pN1N3 for the availability of the antibody P4D5. Apparent molecular
masses (80 kDa) of NHE3-VSVG and N3N1 were lower than those of
NHE1 and N1N3-VSVG. The results were consistent with the finding that
NHE3 is not glycosylated (13) and the observations obtained
with kidney (36) or intestine (37) brush border
membranes using a NHE3-specific antibody. The expression levels of NHE1
and N3N1 were higher than that of N3N1/
637-656, whereas the
expression level of NHE3-VSVG was higher than that of N1N3-VSVG. These
differences in expression agreed well with those in the V
values for
Na
uptake (25 and 8 nmol/mg/min for pNHE3-VSVG and pN1N3-VSVG,
respectively; see also Fig. 5and Fig. 6). Although
expression of N1N3-VSVG was low, two bands were detected (Fig. 2A). The lower band may represent a
non-glycosylated immature form of N1N3-VSVG.
Figure 2:
Immunoblot analysis of expressed NHE
variants (A) and binding of NHE3 to CaM-Sepharose (B). A, crude membranes from cells expressing NHE
variants (NHE1, N3N1, and N3N1/637-656, 10 µg each; NHE3
and N1N3, 20 µg each) were subjected to immunoblot analysis as
described under ``Experimental Procedures.'' A polyclonal
antibody RP-cd was used for NHE1, N3N1, and N3N1/
637-656,
whereas a monoclonal antibody P5D4 was used for NHE3 and N1N3. In this
experiment, plasmids pNHE3-VSVG and pN1N3-VSVG were used for
availability of the antibody P5D4. B, detergent-solubilized
membrane proteins from cells expressing NHE3 were incubated with
CaM-Sepharose beads in the presence of 0.1 mM CaCl
or 1 mM EGTA as described under ``Experimental
Procedures.'' After brief centrifugation, an aliquot (20 µl
each) of supernatant was subjected to immunoblot
analysis.
Figure 5:
Effect of ionomycin on pH dependence of EIPA-sensitive
Na
uptake by various NHE transfectants.
The rate of
Na
uptake and pH
during uptake were measured with NHE transfectants (A, NHE3; B, N1N3; C, N3N1; D,
N3N1/
637-656) in the absence (
) or presence (
) of
5 µM ionomycin.
Figure 6:
Effect of thrombin on pH dependence of EIPA-sensitive
Na
uptake by various NHE transfectants.
The rate of
Na
uptake and pH
during uptake were measured with NHE transfectants (A, NHE3; B, N1N3; C, N3N1; D,
N3N1/
637-656) in the absence (
) or presence (
) of
2 units/ml thrombin.
CaM is known to bind to
the NHE1 cytoplasmic domain in a Ca-dependent
manner(17) . Consistent with this, we found that the expressed
N3N1 also bound to CaM-Sepharose (data not shown). In addition,
deletion of the high affinity CaM-binding site (amino acids
637-656) from the cytoplasmic domain of NHE1 markedly reduced the
ability of NHE1 and N3N1 to bind to CaM-Sepharose (data not shown, but
see Fig. 6of (17) ). Interestingly, we also found that
the expressed NHE3-VSVG bound to CaM-Sepharose in a
Ca
-dependent manner (Fig. 2B). We
thus conclude that like NHE1, NHE3 is also a CaM-binding protein. In
this paper, however, we did not analyze the precise localization of the
CaM-binding site and its binding affinity in detail.
Figure 3:
Effect of various stimuli on
[Ca]
in cells
expressing NHE variants. A change in
[Ca
]
was measured by
monitoring fura-2 fluorescence in cells expressing NHE1 (A),
NHE3 (B), or N3N1 (C) as described under
``Experimental Procedures.'' At the time shown by arrows, various agents were added into the cuvette at the
following final concentrations: 2 units/ml for thrombin, 0.5 or 5
µM for ionomycin, 200 nM for PMA, and 100 mM for sucrose.
Next, we examined whether various
agents activate the Na/H
exchange in
the early phase (
1 min) of stimulation. In experiments shown in Fig. 4, we acidified cells by a classical
NH
prepulse (5 mM NH
Cl for 30 min). This NH
concentration was chosen to produce a moderate acidification to
``sensitize'' the
Na
uptake
measurement and at the same time to minimize stimuli-induced pH
change that may affect the exchange activity. After the
NH
removal, pH
changed to a
steady level of 6.96 ± 0.13 (n = 17) in
unstimulated cells. Under these conditions, thrombin, PMA, and PDGF-BB
did not produce a significant change in this pH
value,
whereas ionomycin and sucrose produced slight cytoplasmic acidification
in some experiments (up to 0.1 pH unit). The extent of this cytoplasmic
acidification was variable in experiments using different batches of
cells. As shown in Fig. 4, short stimulation with ionomycin and
thrombin significantly stimulated EIPA-sensitive
Na
uptake in NHE1 transfectants, whereas
PMA did not activate it. Although hyperosmolarity (sucrose) only
slightly activated NHE1, the extent of its activation was much less
than those for ionomycin and thrombin. PDGF-BB did not activate
Na
uptake (data not shown). There is thus
a good correlation between Ca
mobilization and early
activation of NHE1 in the responses to various stimuli.
Figure 4:
Effects of various stimuli on the rate of
EIPA-sensitive Na
uptake. Cells were
loaded for 30 min with 5 mM NH
Cl, washed with
nominally Na
-free choline chloride solution, and
placed for 40 s in the same medium.
Na
uptake was then measured for 40 s in the same medium containing 1
mM
NaCl and 1 mM ouabain. Various agents
described in the figure were continuously present for total 80 s in the
choline chloride solution at the following concentrations: 2 units/ml
for thrombin, 5 µM for ionomycin, 200 nM for PMA,
and 100 mM for sucrose. Percentage activation of the
EIPA-sensitive
Na
uptake activity was
plotted in the ordinate. In the presence of thrombin, PMA, or
sucrose, EIPA-insensitive
Na
uptake was
less than 15% of total
Na
uptake in all
transfectants, but it was slightly higher in the presence of ionomycin
(less than 25% of total
Na
uptake). The
latter result may indicate the presence of
Ca
-dependent
Na
uptake
activity (probably Na
/Ca
exchange)
in this cell line. Data are means ± S.D. of at least three
independent experiments.
In sharp
contrast to NHE1, the epithelial isoform NHE3 was not activated by
ionomycin, thrombin, PMA, and sucrose (Fig. 4). PDGF-BB also was
not effective (data not shown). A chimera N1N3 also did not respond to
these agents. Interestingly, the reciprocal chimera N3N1 was markedly
activated by ionomycin and thrombin. However, these activating effects
of ionomycin and thrombin were abolished in cells expressing the
deletion mutant of N3N1 (N3N1/637-656) lacking the high
affinity CaM-binding site (Fig. 4).
We measured pH dependence of EIPA-sensitive
Na
uptake by NHE variants. In cells expressing NHE3 and N1N3, short
stimulation with ionomycin and thrombin did not affect the pH
dependence curve ( Fig. 5and Fig. 6). We found that
thrombin did not induce a detectable increase in the V
value of NHE3 exchange activity (Fig. 6A). This
finding is inconsistent with the recent reports (26, 27) that growth factors activate NHE3 by
increasing its V
value. This discrepancy is
likely to be due to a difference in exposure time to growth factors.
Our exposure time (80 s) is much shorter than those used in these
previous studies. In contrast to NHE3 and N1N3, in cells expressing
N3N1, these agents clearly shifted the pH
dependence curve
to an alkaline side by about 0.2 pH unit without a change in the
maximal activity (V
) ( Fig. 5and Fig. 6). The extent of this alkaline shift was almost the same
as that in NHE1 (see Fig. 2of (7) ). Such a shift in
pH
dependence, however, did not occur in a deletion mutant
of N3N1 (N3N1/
637-656) lacking the high affinity CaM-binding
site. Thus, the cytoplasmic domain determines the notable difference in
the response to ionomycin or thrombin between NHE1 and NHE3. In
addition, the high affinity CaM-binding site of NHE1 is required for
this response.
It is important to note that unstimulated cells
expressing NHE1, NHE3, and their chimeras exhibited markedly different
pH dependences of
Na
uptake
activity. Fig. 7A shows pH
dependence
curves normalized by the V
value of each NHE
variant. NHE3 exhibited pH
dependence with a very high
pK value (
7.1). In contrast, N3N1 had pH
dependence with a relatively acidic pK value (
6.6).
This pK value is slightly lower than that of NHE1 (pK =
6.75)(7, 15) . However, N1N3
exhibited pH
dependence with an intermediate pK value (
6.9). Thus, the order of pH
sensitivity
was NHE3 > N1N3 > NHE1 > N3N1. Therefore, pH
sensitivity appears to be determined by a complex mechanism
involving the interaction between the transmembrane and cytoplasmic
domains of NHE isoforms. In addition, we found that deletion of the
high affinity CaM-binding site induced a constitutive alkaline shift of
pH
dependence in N3N1 (Fig. 7B).
Figure 7:
The pH dependence of
EIPA-sensitive
Na
uptake in unstimulated
NHE transfectants. Comparison of pH
dependences of
Na
uptake by cells expressing NHE3, N3N1,
and N1N3 (A) or N3N1 and N3N1/
637-656 (B).
The rate of EIPA-sensitive
Na
uptake was
normalized by the maximal rate (V
) (taken as
100%) of
Na
uptake in each experiment.
Data were taken from two independent
experiments.
In this work, we studied the mechanism of Ca regulation of Na
/H
exchange in
fibroblastic cells expressing NHE1, NHE3, and their chimeras. We can
summarize our observations as follows. First, ionomycin and thrombin
but not PMA, hyperosmolarity, and PDGF-BB induced intracellular
Ca
mobilization as well as activation of NHE1 in the
early phase of stimulation. It thus appears that intracellular
Ca
is a main regulator in the early phase of NHE1
activation. Second, ionomycin and thrombin activated neither NHE3 nor a
chimera N1N3, but they activated another chimera N3N1. Thus, the
cytoplasmic domain determines the difference in the
Ca
-induced response between NHE1 and NHE3. Such
regulatory role of the cytoplasmic domain is in accordance with a
recent report that NHE1 becomes activable in response to cAMP by
replacing its cytoplasmic domain with that of trout
NHE(38) . Third, deletion of the high affinity CaM-binding
site from N3N1 induced a constitutive alkaline shift of pH
dependence of
Na
uptake and
abolished the ionomycin- or thrombin-induced activation of
Na
uptake by this chimera. Thus, the
CaM-binding site in the NHE1 cytoplasmic domain is also able to exert
an inhibitory effect on the H
modifier site of NHE3.
All these data are consistent with our previous hypothesis (7) that early Ca
-induced activation of NHE1
occurs via displacement by Ca
/CaM of the
autoinhibitory CaM-binding domain from the H
modifier
site.
Regulation of NHE1 is more complex in the late phase of
stimulation (5-30 min). In the late phase, thrombin, PMA,
PDGF-BB, and hyperosmolarity all activate NHE1 and induce cytoplasmic
alkalinization. The role of Ca in NHE1 regulation in
the late phase is not clear at present. After thrombin stimulation,
[Ca
]
declined to base line in a
relatively short time (see Fig. 3). In addition, there was no
significant Ca
mobilization induced by PMA, PDGF-BB,
or hyperosmolarity under the conditions used in this study (see Fig. 3and ``Results''). On the other hand, previous
studies showed that intracellular Ca
depletion caused
by extracellular or intracellular addition of calcium chelators reduced
the extent of cytoplasmic alkalinization in response to long
stimulation by growth factors such as thrombin and
PDGF-BB(6, 39, 40, 41) .
Furthermore, we previously showed that deletion or point mutation of
CaM-binding domain in NHE1 reduced cytoplasmic alkalinization by
50% in response to long stimulation with thrombin and
80% in
response to hyperosmolarity(17) . These latter results raise
the possibility that slight elevation of
[Ca
]
in subplasmalemma space
not detectable by ordinary fluorescence
[Ca
]
measurements may partially
be involved in the NHE1 regulation even in the relatively long time
range. In our previous paper(17) , we presented evidence
suggesting that another mechanism not involving
Ca
/CaM or direct phosphorylation of NHE1 but
involving unknown regulatory factor(s) that also activates NHE1 in the
late phase of growth factor stimulation.
As described above,
pH sensitivity of chimera N3N1 is up-regulated by
Ca
-mobilizing agents. Thus, the lack of
Ca
response in NHE3 or N1N3 is attributable to the
structure of the NHE3 cytoplasmic domain. We found that NHE3 binds
Ca
/CaM, although at present we do not know the
precise localization of its binding site (see Fig. 2B).
However, the CaM-binding site in NHE3 does not appear to function as a
Ca
/CaM-sensitive autoinhibitory domain as in NHE1,
because NHE3 does not respond to Ca
at least in the
conventional way it was tested. Comparison of the CaM-binding site
sequence of rat NHE1 with the corresponding sequence of rat NHE3
reveals that in rat NHE3, 27 intervening amino acids are inserted
between Ser-605 and Tyr-606, whereas 9 amino acids are conserved
between NHE1 and NHE3 (see Fig. 1A). It is thus
possible that insertion of the intervening sequence in this region of
NHE3 resulted in disruption of the original autoinhibitory CaM-binding
domain during the evolutional process. Recently, CaM antagonist W13 was
reported to stimulate the NHE3 activity(42) . At present, it is
not clear how CaM-binding site(s) of NHE3 is related with the data
obtained with this drug.
The pH sensitivity of chimera
N3N1 as well as NHE1 is constitutively alkaline-shifted by deletion of
the CaM-binding site. This finding suggests that the autoinhibitory
CaM-binding domain of NHE1 functionally interacts with an
``acceptor'' site(s) somewhere in the NHE sequences. This
putative acceptor site(s), which could also be involved in the
H
binding to the exchanger, is most probably located
in the NH
-terminal highly conserved regions of NHE1 and
NHE3. Alternatively, it may be located in the homologous NH terminus of
the cytoplasmic domain that was important for the maintenance of
pH
sensitivity in NHE1 (14) . (
)According to our recent study, the cytoplasmic tail (amino
acids 659-815) of NHE1 is not involved in the Ca
regulation of NHE1 (7) .
Identification of
this acceptor site(s) would lead to a better understanding of the
molecular entity of the H
-modifier site.
In
unstimulated cells expressing NHE1, NHE3, and their chimeras, we
observed marked differences in the pH dependence of
Na
uptake activity (Fig. 7A). The pH
sensitivity of NHE3 is
much higher than that of NHE1. This was confirmed by our finding that
cells expressing NHE3 possess a much higher resting pH
as
compared to cells expressing NHE1, (
)which is consistent
with previous results(27) . This is also consistent with the
recent observation (26) obtained from analysis of pH
recovery rate, but not consistent with other
reports(28, 43) . AP-1 cells were used in the latter
studies, while PS120 cells were used in this study. Thus, there may be
cell specific effects that influence the pH
sensitivity. It
is important to note that pH
dependence was dramatically
shifted to an acidic side, when the cytoplasmic domain of NHE3 was
replaced by that of NHE1 to form N3N1 (Fig. 7A).
Conversely, pH
dependence was significantly shifted to an
alkaline side, when the cytoplasmic domain of NHE1 was replaced by that
of NHE3 to form N1N3. Direction of the pH shift caused by COOH termini
swaps can be explained by incorporation or removal of the
autoinhibitory domain of NHE1. However, a large acidic pH shift
observed in N3N1 is not explained only by incorporation of the
inhibitory domain of NHE1. We found that in cells expressing NHE3-VSVG
lacking the last COOH-terminal 47 amino acids of NHE3, pH
dependence was shifted to a much more acidic side (>0.4 pH
unit) as compared to that in intact NHE3.
Therefore, the
short COOH-terminal tail may function as an activator domain in NHE3.
Thus, pH
sensitivity appears to be determined by a global
mechanism involving interaction between the transmembrane domain and
multiple cytoplasmic subdomains. More precise identification of the
critical region of NHE3 would facilitate understanding of how pH
dependence of Na
/H
exchange is
determined by the interaction between the transmembrane and cytoplasmic
domains of NHE isoforms.
In summary, the present data indicate that
the presence or absence of the autoinhibitory CaM binding region
produces the remarkable difference in the Ca response
between NHE1 and NHE3. Activation of CaM-dependent protein kinase II
has been reported to inhibit Na
/H
exchange in kidney and intestine brush border membranes which
express NHE3(44, 45, 46) . Inhibition of the
apical exchanger by CaM-dependent protein kinase II was also reported
in proximal tubule cell line LLC-PK1(47) . Lack of early
Ca
activation in NHE3 would facilitate this
CaM-dependent protein kinase II-mediated inhibition of the apical
exchanger which lead to inhibition of Na
reabsorption.
On the other hand, activation of NHE1 by Ca
/CaM
binding would play an important role in a rapid and fine pH
regulation in various cells when
[Ca
]
increases. Thus, the
difference of Ca
response appears to reflect distinct
physiological requirements of these two exchangers.