From the Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565 Japan
Received for publication, December 30, 2002
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ABSTRACT |
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To identify important amino acid residues
involved in intracellular pH (pHi) sensing of
Na+/H+ exchanger 1, we produced
single-residue substitution mutants in the region of the exchanger
encompassing the putative 11th transmembrane segment (TM11) and its
adjacent intracellular (intracellular loop (IL) 5) and extracellular
loops (extracellular loop 6). Substitution of Arg440 in IL5
with other residues except positively charged Lys caused a large shift
in pHi dependence of
22Na+ uptake to an acidic side, whereas
substitution of Gly455 or Gly456 within the
highly conserved glycine-rich sequence of TM11 shifted pHi dependence to an alkaline side. The
observed alkaline shift was larger with substitution of
Gly455 with residues with increasing sizes, suggesting the
involvement of the steric effect. Interestingly, mutation of
Arg440 (R440D) abolished the ATP depletion-induced acidic
shift in pHi dependence of
22Na+ uptake as well as the cytoplasmic
alkalinization induced by various extracellular stimuli, whereas with
that of Gly455 (G455Q) these functions were preserved.
These mutant exchangers did not alter apparent affinities for
extracellular transport substrates Na+ and H+
and the inhibitor
5-(N-ethyl-N-isopropyl)amiloride. These results suggest that positive charge at Arg440 is required for
normal pHi sensing, whereas mutation-induced perturbation of the TM11 structure may be involved in the effects of
Gly mutations. Thus, both Arg440 in IL5 and Gly residues in
the conserved segment of TM11 appear to constitute important elements
for proper functioning of the putative "pHi
sensor" of Na+/H+ exchanger 1.
The Na+/H+ exchangers
(NHEs)1 are plasma membrane
transporters that regulate pH homeostasis, cell volume, and
transepithelial Na+ absorption (1-4). The activity of
ubiquitous exchanger isoform NHE1 is controlled by various extrinsic
factors including hormones, growth factors, pharmacological agents, and
mechanical stimuli (1-4). These stimuli have been shown to enhance
NHE1 activity by shifting its intracellular pH (pHi) dependence
to an alkaline side. This phenomenon is usually explained by assuming that there exists an allosteric regulatory site for intracellular protons in NHE1 that is distinct from the Na+ or
H+ transport site and that external stimuli increase the
affinity of such H+ modifier site (Ref. 5; see Refs. 1-4
for reviews). Regulation of NHE1 by these stimuli has been reported to
occur through a variety of signaling molecules, i.e.
calcinuerin B homologous protein (6, 7), Ca2+/calmodulin
(8, 9), low molecular mass GTPases Ras and Rho (10-12), p42/44
mitogen-activated protein kinases (13), p90 ribosomal S6 kinase (14),
14-3-3 protein (15), Nck-interacting kinase (16), and
phosphatidylinositol 4,5-bisphosphate (17). However, the
interrelationship of these signaling molecules and the mechanism by
which they modulate the interaction of the putative H+
modifier site with intracellular protons is still unclear.
The NHE family members have similar overall structures, consisting of
an N-terminal transmembrane domain (~500 amino acids) containing 12 membrane spanning segments and a C-terminal cytoplasmic regulatory domain (~300 amino acids) (1-3). We previously showed that deletion of the cytoplasmic domain of NHE1 greatly shifted the
pHi dependence of exchange to an acidic side, with the steep
pHi dependence of exchange being maintained (18). Furthermore,
we have presented evidence that the cytoplasmic domain of NHE1 contains
several subdomains, which increase or decrease the pHi
sensitivity (19). These data may be consistent with the idea that the
putative H+ modifier site exists within the N-terminal
transmembrane domain and that the cytoplasmic domain regulates the
accessibility of regulatory protons to the site. However, the
structural basis for such an idea is largely unknown.
Based on the accessibility of introduced cysteine residues to
sulfhydryl reagents, we have recently presented a new membrane topology
model of NHE1 in which the exchanger consists of 12 transmembrane segments with N- and C-tails located in the cytosol (20), although a
previous study reported the N-tail to be cleaved as a signal peptide
during the exchanger biogenesis (21). In a subsequent cysteine-scanning
mutagenesis study, we found that mutation of Tyr454 or
Arg458 in a new transmembrane domain 11 (TM11) impaired the
plasma membrane expression of NHE1 (22), suggesting the importance of
these residues for proper folding of NHE1. Furthermore, our preliminary experiments suggested that some residues in TM11 regulate pHi sensitivity. In addition, it was noted that TM11 contains a
phylogenetically conserved Gly-rich segment (see Fig. 1B).
These findings prompted us to study the functions of amino acid
residues within TM11 and adjacent intracellular (IL5) and extracellular
loops (EL6).
In this study, we found that mutation of Arg440 within IL5
decreases the pHi sensitivity, whereas mutation of Gly residues within the Gly-rich segment of TM11 increases it, suggesting that the
region encompassing IL5 and TM11 is critical for the regulatory function of NHE1. This is the first identification of important residues involved in pHi sensing within the transmembrane domain of NHE1.
Materials--
Biotin maleimide was purchased from Molecular
Probes Inc. MTSET and streptavidin-conjugated agarose were
purchased from Toronto Research Chemicals Inc. and Pierce,
respectively. Amiloride derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was a
gift from the New Drug Research Laboratories of Kanebo, Ltd. (Osaka,
Japan). 22NaCl was purchased from PerkinElmer Life
Sciences. An NHE1-specific polyclonal antibody (RP-cd) was raised as
described previously (8). All other chemicals were of the highest
purity available.
Cells, Culture Conditions, and Stable Expression--
An
Na+/H+ exchanger-deficient cell line (PS120)
(23) and corresponding transfectants were maintained in Dulbecco' s
modified Eagle' s medium (Invitrogen) containing 25 mM
NaHCO3 and supplemented with 7.5% (v/v) fetal calf serum,
penicillin (50 units/ml), and streptomycin (50 µg/ml). The cells were
maintained at 37 °C in the presence of 5% CO2. PS120
cells (5 × 105 cells/100-mm dish) were transfected
with each plasmid construct (20 µg) by the calcium phosphate
co-precipitation technique. Cell populations that stably express mutant
NHE1 were selected by the "H+ killing" procedure as
described (18).
Construction of Na+/H+
Exchanger Mutants--
A plasmid carrying cDNA coding for the
Na+/H+ exchanger (NHE1 human isoform)
containing some unique restriction sites cloned into mammalian
expression vector pECE was described previously (18). A cDNA
construct for Cys-less NHE1 devoid of all endogenous cysteine residues
was also described previously (20). For construction of point mutant
cDNAs, we used a PCR-based strategy as described previously (20),
using two template plasmids coding for the wild-type or Cys-less NHE1.
Briefly, using synthesized sense and antisense primers containing
appropriate mutations, DNA fragments were generated by PCR. These
fragments were digested and inserted into appropriate sites on the
wild-type or Cys-less NHE1 plasmid. The DNA sequences of the PCR
fragments were confirmed with a PerkinElmer Life Sciences model 373S
autosequencer. We used the prefix "cl-" for point mutants produced
from Cys-less NHE1 as a template.
Labeling with Biotin Maleimide--
Biotin maleimide labeling of
NHE1 mutant molecules was carried out as described previously (20).
Briefly, confluent cells cultured on 60-mm dishes were washed twice
with 5 ml of PBSCM (phosphate-buffered saline containing 0.1 mM CaCl2 and 1 mM
MgCl2, pH 7.2) and then incubated in 1 ml of PBSCM
containing 0 or 5 mM MTSET for 30 min at room temperature.
The cells were washed twice with 5 ml of PBSCM and then incubated in 1 ml of PBSCM containing 0.5 mM biotin maleimide (100 mM stock in Me2SO) for 30 min at room
temperature. The cells were washed once with PBSCM containing 1%
2-mercaptoethanol, once with PBSCM, and then collected in
the tube by centrifugation. The cells were solubilized with
1 ml of lysis buffer containing 1% Triton X-100, 150 mM
NaCl, 20 mM Hepes/Tris (pH 7.4), 1 mM
phosphate-buffered saline, and 1 mM benzamidine. After
centrifugation for 10 min at 15,000 rpm, the supernatant was mixed with
streptavidin-agarose beads (30 µl of resin), followed by incubation
for 1 h at 4 °C with mild agitation. The agarose beads were
washed more than five times with 1 ml of lysis buffer, mixed with 50 µl of SDS-PAGE sample buffer containing 3% SDS, and then boiled for
10 min at 100 °C. The proteins were separated on an 8.5% gel by
SDS-PAGE and analyzed by immunoblotting with the NHE1 antibody as
described previously (20). The blots were developed with an ECL
detection system (Amersham Biosciences). When indicated, the cells were
permeabilized with digitonin (50 µg/ml) or streptolysine O
(Sigma) immediately before biotinylation, as described previously
(20).
Measurement of 22Na+
Uptake--
22Na+ uptake activity and its
pHi dependence were measured by the K+/nigericin
pHi clamp method (19). Serum-depleted cells in 24-well dishes
were preincubated for 30 min at 37 °C in Na+-free
choline chloride/KCl medium containing 20 mM Hepes/Tris (pH
7.4), 1.2-140 mM KCl, 2 mM CaCl2,
1 mM MgCl2, 5 mM glucose, and 5 µM nigericin. In some experiments (see Fig. 6), the
preincubation solution contained 2 µg/ml oligomycin and 5 mM 2-deoxyglucose instead of glucose.
22Na+ uptake was started by adding the same
choline chloride/KCl solution containing 22NaCl (37 kBq/ml)
(final concentration, 1 mM), 1 mM ouabain, and 100 µM bumetanide. In some wells, the uptake solution
contained 0.1 mM EIPA. After 1 min, the cells were rapidly
washed four times with ice-cold phosphate-buffered saline to terminate
22Na+ uptake. pHi was calculated from
the imposed [K+] gradient by assuming the equilibrium
[K+]i/[K+]o = [H+]i/[H+]o and an
intracellular [K+] of 120 mM. The data were
normalized as to protein concentration, which was measured by
bicinchoninic assay system (Pierce) using bovine serum albumin as a standard.
Measurement of Changes in pHi--
The changes in
pHi induced by various extracellular agents were measured by
the [14C]benzoic acid equilibration method, as described
previously (18).
Statistics--
The data for the pH dependence of
22Na+ uptake were simulated by fitting the
values to the sigmoidal dose-response equation, the rate of
EIPA-sensitive 22Na+ uptake = Vmax/(1 + 10^(log(pK Precise Topology of the Putative 11th Transmembrane Domain and
Neighboring Loops of NHE1--
Based on the results of cysteine
accessibility analysis, we previously presented a novel topology model
of NHE1, as shown in Fig. 1A
(20). Here, to obtain more precise topological information of the
regions surrounding TM11, we introduced single Cys residues into these
regions of Cys-less NHE1 and expressed mutants in the exchanger-deficient cells PS120. All of the mutant exchangers except
cl-K438C, cl-R440C, cl-I451C, cl-G456C, and cl-R458C were well
expressed in PS120 cells (Fig.
2A; also see Fig. 7 of Ref. 20). On SDS-PAGE, we observed two forms of exchanger protein with high
(~110 kDa) and low (~90 kDa) molecular masses, which are
thought to be a mature protein with N- and
O-linked glycosylation and an immature protein containing
only N-linked high mannose oligosaccharide, respectively
(24). The cells were then incubated with 0.5 mM biotin
maleimide after preincubation with or without a membrane-impermeable
MTSET, and biotinylated proteins were subsequently recovered with
streptavidin-agarose, followed by immunoblot analysis with an anti-NHE1
to visualize biotinylated exchangers (Fig. 2B).
Mutant exchangers cl-L468C, cl-L469C, cl-D470C, cl-K471C, cl-K472C,
cl-H473C, cl-F474C, cl-P475C, and cl-M476C with high molecular mass
were efficiently labeled with biotin maleimide (Fig. 2B; not
shown for cl-K471C, but see Ref. 20). cl-E151C, used as a positive
control, was also labeled, whereas Cys-less NHE1 was not (data not
shown; see Ref. 20). The biotinylation of mutant exchangers was
significantly inhibited by pretreatment with membrane-impermeable MTSET
(Fig. 2B), suggesting that these residues face the outside. On the other hand, cl-G466C, cl-D478C, and Cys477* (native
cysteine) were not labeled with biotin maleimide, and cl-Y467C was only
faintly labeled (Fig. 2B). In contrast, cl-N437C, cl-I441C,
cl-V442C, cl-K443C, cl-K447C, and cl-D448C were labeled with biotin
maleimide only when the cells were permeabilized with digitonin (Fig.
2C; data not shown for some mutants) or streptolysine O (see
Ref. 20), suggesting that these positions face the inside. Some Cys
mutants, i.e. cl-F439C, cl-L444C cl-T445C, cl-P446C, and
cl-Q449C, were only faintly labeled even when cells were permeabilized with digitonin (Fig. 2C; data not shown for cl-T445C and
cl-P446C). We found that introduced cysteines at Phe450,
Ile452, Ala453, Tyr454,
Gly455, Leu457, Gly459,
Ala460, Ile461, Ala462,
Phe463, Ser464, and Leu465 were not
labeled with biotin maleimide in either intact or permeabilized cells
(data not shown; see Ref. 20). Based on these results, we propose that:
(i) the putative TM11 consists of 18 residues from Gln449
to Tyr467, (ii) the putative TM12 starts from
Cys477, (iii) the region from Leu468 to
Met476 forms the extracellular loop (EL6), (iv) the region
from Asn437 to Asp448 forms part of the
intracellular loop (IL5), and (v) some residues in IL5 may not be
completely exposed to the cytosol. These topological data would be
useful for the functional study of the exchanger in which residues in
TM11 and its surrounding regions are mutated (see below).
Identification of Residues Modifying pHi Sensitivity of
NHE1--
We introduced single Cys residues into the region covering
most of IL5 and TM11 in the wild-type NHE1. In the wild-type
background, R440C was well expressed in PS120 cells, but K438C, I451C,
Y454C, and R458C were not. All Cys mutants expressed well in cells
exhibited high EIPA-sensitive 22Na+ uptake
activity when cells were acidified. We found that substitution of
Arg440 with Cys greatly shifted the pHi dependence
of 22Na+ uptake to an acidic side with the
pK of less than 6.2 (Fig.
3A). We could not determine
the precise pK value for this mutant because of incomplete
attainment of the maximal activity. On the other hand, mutations at
other neighboring residues in IL5 did not significantly affect the
pHi dependence (Fig. 3B). Intriguingly, unlike the
Arg440 mutation, mutations of Gly455 and
Gly456 in TM11 significantly shifted the pHi
dependence of 22Na+ uptake to an alkaline side,
respectively (Fig. 3C). The pK values increased
by ~0.3 pH units with these mutations. When these experiments were
repeated more than three times, the pK values of mutant
exchangers other than R440C, G455C, and G456C were in the range of
6.5-6.8, which is not very different from that of the wild-type NHE1
(Fig. 3D). Thus, we identified Arg440 in IL5 and
Gly455/Gly456 in TM11 as residues greatly
influencing the pHi sensitivity of NHE1.
We substituted Arg440 and Gly455 with other
residues and analyzed the functional consequences of these mutations.
Fig. 4 shows the pHi dependence
of 22Na+ uptake in cells expressing R440D or
R440K. Amino acid substitution of Arg440 with Asp greatly
shifted the pHi dependence to an acidic side. In the
inset to Fig. 4, we present the ratios between the uptake
activities of various substitution mutants of Arg440 at
pHi 6.8 and 5.4, in place of the respective pHi dependences. The ratio decreased significantly when Arg440
was substituted with Lys, His, Asp, Glu, or Leu, indicating the acidic
shift in the pHi dependence. It is notable that the effect of
the Arg440 to Lys substitution on the pHi
dependence was modest (pK = ~6.4) compared with the
substitution with other residues (Fig. 4). Thus, the positive
charge of Arg440 appears to be important for the normal
pHi sensitivity.
Fig. 5 (A and B)
shows the results of amino acid substitution at Gly455.
Substitution of Gly455 to Gln, Thr, or Val significantly
shifted the pHi dependence of 22Na+
uptake to an alkaline side, whereas substitution to Ala, Asp, Asn, or
Ser did not shift or only modestly shifted. We plotted the
pK values against the surface area or volume of substituted amino acid side chains. The data suggest that the pK increases when
Gly455 is substituted with bulky residues (Fig.
5C).
Mutations of Arg440 and Gly455 Do Not
Change the Apparent Affinities for Extracellular Na+,
H+, and EIPA--
We analyzed several other kinetic
parameters of Na+/H+ exchange in cells
expressing R440D and G455Q that exhibited acidic and alkaline
pHi dependence of 22Na+ uptake,
respectively. Fig. 6 (A-C)
shows the extracellular Na+ concentration dependence of
EIPA-sensitive 22Na+ uptake. The
Km values for Na+ estimated from double
reciprocal plots (Fig. 6, A-C, insets) were
similar for the wild-type NHE1 (9.22 ± 1.04 mM),
R440D (9.30 ± 0.90 mM), and G455Q (14.08 ± 1.75 mM). The dependences of 22Na+
uptake on extracellular pH (pHo) and on the EIPA
concentration were also similar for the wild-type NHE1, R440D, and
G455Q (pK for pHo, 7.29 ± 0.03, 7.50 ± 0.08, and 7.38 ± 0.02; IC50 for EIPA, 106.3 ± 7.3, 91.2 ± 0.1, and 85.0 ± 8.0 nM,
respectively). The data suggest that mutations of Arg440
and Gly455 only affect the pHi dependence of
exchange.
Effects of Mutations of Arg440 and Gly455
on Some NHE Regulatory Functions--
Cellular ATP depletion is known
to inhibit the exchange activity by reducing the pHi
sensitivity and/or maximal activity (18, 25-28). Fig.
7 (A and B) shows
the effect of ATP depletion on the pHi dependence of
22Na+ uptake in cells expressing R440D or
G455Q. Interestingly, ATP depletion did not change the pHi
sensitivity in cells expressing R440D (Fig. 7A), although it
caused ~50% reduction of Vmax (data not
shown). In contrast, ATP depletion greatly shifted the pHi
dependence of cells expressing G455Q to an acidic side without a change
in Vmax. However, it is notable that even under
ATP depletion G455Q exhibited more alkaline pHi dependence of
22Na+ uptake (pK, ~6.4) compared
with the wild type (pK, ~6.0).
Finally, we measured the changes in pHi caused by
various extracellular stimuli. In cells expressing the wild-type NHE1
and G455Q, we detected cytoplasmic alkalinization induced by thrombin,
platelet-derived growth factor BB, hyperosmotic stress (sucrose),
phorbol 12-myristate 13-acetate, lysophosphatidic acid, and the
recently described activator Li+ (29), indicating that
all of these stimuli are capable of activating exchangers (Fig.
8). Of note, the extent of
cytoplasmic alkalinization caused by hyperosmotic stress or
Li+ was reduced in cells expressing G455Q. In contrast,
such alkalinization by extracellular stimuli was not observed in cells
expressing R440D. Lack of alkalinization was presumably due to the
acidic shift of the pHi dependence of activity of
R440D.
In this study, after obtaining precise information on the topology
of the portion of NHE1 encompassing TM11 and its surrounding regions,
we performed scanning mutagenesis analysis of these regions to search
for residues possibly involved in the regulation of pHi
dependence of Na+/H+ exchange. We found that
mutation at Arg440 of IL5 of NHE1 greatly shifted
pHi dependence of EIPA-sensitive 22Na+
uptake to an acidic side, whereas mutation at Gly455 or
Gly456 of TM11 shifted it to an alkaline side. Mutation of
many other residues in IL5 and TM11, in which it did not impair
exchanger expression in the plasma membrane, did not alter the
pHi dependence. Furthermore, these arginine and
glycine mutations did not alter the apparent affinities of NHE1 for
external transport substrates Na+ and H+ and
the inhibitor EIPA. We observed similar opposite shifts of the
pHi dependence with these NHE1 mutants when we
measured the reverse mode of Na+/H+ exchange,
i.e. EIPA-sensitive 22Na+ efflux
from 22Na-loaded
cells,2 supporting the
possibility that the mutations alter the interaction of intracellular
protons with the H+ modifier site rather than the transport
site. It thus appears likely that these single mutations exert rather
specific effects on the pHi sensor of NHE1.
In addition to the effect on pHi dependence of basal
Na+/H+ exchange, mutation of Arg440
abolished the ATP depletion-induced acidic shift of pHi dependence of exchange activity as well as the cytoplasmic
alkalinization in response to various extracellular stimuli. We
previously reported (18) that deletion of the entire C-terminal
cytoplasmic domain of NHE1 causes a large acidic shift of pHi
dependence of basal exchange activity and the disappearance of growth
factor-induced cytoplasmic alkalinization. The same deletion was also
found to abolish the ATP depletion-induced acidic shift of pHi dependence of exchange activity. We subsequently provided evidence that
these effects occur because of deletion of the N-terminal portion of
the cytoplasmic domain of NHE1 named subdomain I (amino acids 503-600)
(19). Thus, the effects caused by deletion of subdomain I and single
mutation of Arg440, which is localized in the internal loop
(IL5) connecting TMs 10 and 11, are very similar except that in the
latter the Vmax of exchange activity is somewhat
reduced by ATP depletion.
Recently, we have reported that the calcineurin B homologous protein
CHP is an essential co-factor for the expression of high physiological levels of exchange activity in many plasma membrane NHE
isoforms (6). We showed that CHP is tightly bound to a short
juxtamembrane segment of the exchanger (amino acids 510-530 of
subdomain I in the case of NHE1) (6). Interestingly, in addition to the
large reduction of Vmax, CHP binding-defective NHE1 mutants exhibited a marked shift of the pHi dependence to
an acidic side.3 Thus, the
mutation at Arg440, deletion of subdomain I, and the CHP
binding-defective mutation all caused the same effect, i.e.
a marked shift of the pHi dependence to the acidic side,
suggesting that the region of IL5 containing Arg440 and the
CHP-subdomain I complex are both involved in the preservation of
physiological pHi sensitivity of the exchanger. In this study,
we observed that pHi dependence of exchange activity did not
change extensively by substitution of Arg440 with similarly
charged but somewhat more bulky Lys residue, unlike substitutions with
other residues (Cys, His, Asp, Glu, and Leu). This suggests that the
positive charge of Arg440 is important. An attractive
hypothesis would be that Arg440 interacts with negatively
charged residue(s) localized in the CHP-subdomain I complex and that
its substitution with other residues or cell ATP depletion results in
the disruption of such interaction, thereby altering pHi
sensitivity of the exchanger. Consistent with this hypothesis, there is
a cluster of acidic amino acid residues on the side opposite to one
where CHP binds to NHE1 in a predicted model of CHP
structure.2
As opposed to the mutation of Arg440 in IL5, mutation of
Gly455 or Gly456 in the glycine-rich region
within TM11 shifted the pHi dependence of basal exchange
activity to an alkaline side. Furthermore, in the Gly455
mutant, ATP depletion was able to induce a large acidic shift of the
pHi dependence without a change in Vmax,
a pattern mimicking that of the wild-type NHE1, although the
pHi dependence itself was shifted to the alkaline side under
these conditions (Fig. 7B). In the same mutant, we observed
normal cytoplasmic alkalinization in response to growth factors (Fig.
8). Thus, the underlying mechanisms for the effects of these Gly
mutations and that of the Arg440 mutation in IL5 are likely
to be different.
The glycine-rich region in TM11 of NHE1 is remarkably conserved in
different types of NHEs from bacteria to mammals (Fig. 1B),
suggesting its importance for the NHE function. Glycine has been
suggested to serve as a molecular hinge permitting the occurrence of
conformational changes of intramembrane helices in some proteins (30,
31) or as a notch for orienting multiple helices at the point of
closest packing between membrane helices in several polytopic membrane
proteins (32). In this study, we observed a clear tendency showing that
substitution of Gly455 with more bulky residues are more
effective in causing an alkaline shift of pHi dependence of
exchange activity (Fig. 5C), which indicates that small
residues are required at the position of Gly455 for normal
pHi sensitivity. This appears to be in line with the fact that
some isoforms such as NHEs 4, 6, 7, and 8 have small residues such as
alanine and serine at this position (Fig. 1B). We previously
found that single cysteine substitutions at Tyr454 and
Arg458, both being close to Gly455 or
Gly456 in TM11, cause the retention of NHE1 protein in the
endoplasmic reticulum (22), suggesting that these mutations impair
proper protein folding. On the other hand, mutation of
Gly455 neither prevented the plasma membrane expression of
NHE1 protein nor appeared to alter other transport functions of the
exchanger significantly. Based on all these pieces of information, we
speculate that a relatively mild change in helix packing interaction of TM 11 with other transmembrane helice(s) was induced by the mutation at
Gly455 or Gly456, which indirectly modified the
conformation and function of the putative H+-regulatory
site(s). It is noteworthy that in Escherichia coli antiporter NhaA distantly related to mammalian NHEs, mutation of
Gly338 in the glycine-rich region of TM11 was reported to
render the antiporter active in the wide range of pH, i.e.
practically independent of the pH level between pH 6 and 9 (33).
Although the present and previous results suggest important roles of
IL5, TM11, and the subdomain I-CHP complex in the modulation of
pHi sensing in NHE1, little is known about structures of the
ion transport sites and the putative pH sensor and their interaction.
In our previous study of membrane topology of NHE1, we found re-entrant
loop-like structures between TMs 4 and 5, between TMs 8 and 9, and
between TMs 9 and 10 (20). In addition, both TM4 and TM9 have been
shown to be important for the interaction of NHE1 with amiloride
analogue inhibitors that compete with the transport substrate
Na+ (34-38). Furthermore, other studies identified a few
polar amino acid residues required for ion exchange within
transmembrane segments in the mammalian NHE1 (38, 39) as well as in the
Na+/H+ antiporters from E. coli (40,
41) and Vibrio alginolyticus (42). Much further study is
required for the elucidation of the structural aspect of
Na+/H+ exchange.
In summary, we have presented evidence that Arg440 in IL5
and Gly455 or Gly456 in TM11 are important
residues that oppositely regulate the pHi sensitivity of NHE1.
This is the first identification of critical residues involved in
pHi sensing within the N-terminal transmembrane domain of NHE1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pHi)n)) (Vmax, the maximal
rate of 22Na+ uptake; pK, pHi giving
half-maximal 22Na+ uptake; n, Hill
coefficient), with the aid of a simulation program included in the
graphing software Graphpad Prizm (Microsoft Corp.). The
concentration dependences of extracellular Na+, pH, and
EIPA for 22Na+ uptake were also fitted to
steady-state kinetic equations with the aid of the same simulation
program. Kinetic parameters are expressed as the best fitted values
with standard errors, whereas other data are expressed as the
means ± S.D. for at least three determinations.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Topology of NHE1 and sequence alignment of
TM11. A, left side, a topological model of
NHE1 based on our previous cysteine accessibility analysis (20).
Right side, precise topologies of IL5, TMs 11 and 12, and
EL6 revealed in this study. The positions examined for cysteine
accessibility are represented by a single-letter amino acid code.
Black and gray circles represent residues
accessible to external and internal SH probes, respectively, whereas
white circles represent residues not or only faintly labeled
by biotin maleimide (see Fig. 2). White squares represent
residues (Lys438, Arg440, Ile451,
Gly456, and Arg458) not studied here because
cells expressing mutants could not be obtained. B, sequence
alignment of TM11 and its neighboring regions in the exchangers from
different species. GenBankTM accession numbers for these
proteins are as follows: human NHE1, P19634; rat NHE2, NP_036785; rat
NHE3, NP_036786; rat NHE4, P26434; human NHE5, NP_004585; human NHE6,
NP_006350; human NHE7, NP_115980; mouse NHE8, NP_683731; trout NHE,
A46188; green crab NHE, AAC26968; Caenorhabditis elegans
NHE, NP_509830; Saccharomyces cerevisiae NHX1, NP_010744;
cynechobacterium (Synechocystis sp.) Syn-NhaP, NP_441245;
Pseudomonas aeruginosa NhaP, NP_252576; and Bacillus
subtilis NhaG, BAA89487.
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Fig. 2.
Biotinylation of neighboring regions of
TM11. Cells expressing single cysteine mutants of Cys-less NHE1
were treated with or without 5 mM MTSET. Then cells were
incubated with 0.5 mM biotin maleimide, solubilized with
lysis buffer, and treated with streptavidin-agarose as described under
"Experimental Procedures." A total cell lysate (20 µg)
(A) or the proteins recovered with streptavidin-agarose
(B and C) were separated by SDS-PAGE, and the
proteins were visualized by immunoblot analysis with an NHE1 antibody.
In C, cells were permeabilized with digitonin before
biotinylation also as described under "Experimental
Procedures."
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[in a new window]
Fig. 3.
pHi dependence of
exchange activity in cells expressing various NHE1 cysteine
mutants. A-C, pHi dependence of
22Na+ uptake was measured in cells expressing
the wild-type or cysteine mutant exchangers in the presence or absence
of 0.1 mM EIPA. pHi was clamped at various values
with K+/nigericin. The wild-type or mutant exchangers
exhibited high 22Na+ uptake activities (15-50
nmol/mg/min at pHi 5.4). The data were fitted to the sigmoidal
dose dependence equation as described under "Experimental
Procedures." In B and C, the data were
normalized to the maximal uptake activity. D, pK
values obtained by curve fitting. The bars represent the
standard errors given by the curve fitting program, whereas the
numbers represent Hill coefficients.
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[in a new window]
Fig. 4.
Effect of amino acid substitution at
Arg440 on pHi dependence of exchange activity.
pHi dependences of EIPA-sensitive
22Na+ uptake were compared among cells
expressing the wild-type NHE1, R440K, and R440D. Inset,
ratios between activities of various substitution mutants of
Arg440 at pHi 6.8 and 5.4 are
presented. The cells expressing these exchangers exhibited high
22Na+ uptake activities (20-45 nmol/mg/min at
pHi = 5.4). The data are the means ± S.D.
of three determinations.
View larger version (24K):
[in a new window]
Fig. 5.
Effect of amino acid substitution
at Gly455 on pHi dependence of exchange
activity. A, pHi dependence of
22Na+ uptake was measured in cells expressing
the wild-type or cysteine mutant exchangers in the presence or absence
of 0.1 mM EIPA. The wild-type or mutant exchangers
exhibited high 22Na+ uptake activities (20-45
nmol/mg/min at pHi = 5.4). B,
pK values were obtained by curve fitting as described under
"Experimental Procedures." The bars represent the
standard errors given by the curve fitting program, whereas the numbers
represent Hill coefficients. C, pK values were
plotted against the surface area (43) or volume (44) of substituted
amino acid side chains.
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[in a new window]
Fig. 6.
Comparison of kinetic properties of wild-type
and mutant exchangers. A-C, EIPA-sensitive
22Na+ uptake in cells expressing the wild-type
NHE1, R440D, or G455Q was measured as a function of Na+
concentration. Insets, double reciprocal plots of exchange
activity versus Na+ concentration. D
and E, EIPA-sensitive 22Na+ uptake
in cells expressing the wild-type NHE1, R440D, or G455Q was measured as
a function of extracellular pH or EIPA concentration.
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[in a new window]
Fig. 7.
Effect of ATP depletion on pHi
dependence of exchange activity in cells expressing R440D or
G455Q. A and B, pHi
dependence of EIPA-sensitive 22Na+ uptake was
measured under control (open circles) or ATP-depleted
conditions (closed circles) in cells expressing R440D or
G455Q, respectively. In each panel, exchange activities are normalized
to the ones measured at pHi of 5.4.
View larger version (20K):
[in a new window]
Fig. 8.
Extracellular stimuli-induced changes in
pHi in cells expressing the wild-type and
mutant exchangers. Changes in pHi were measured as
described under "Experimental Procedures" using the
[14C]benzoic acid equilibration method. The cells
expressing the wild type, G455Q, or R440D were stimulated for 15 min at
37 °C with 2 units/ml thrombin, 10 ng/ml platelet-derived growth
factor (PDGF) BB, 200 mM sucrose, 1 µM phorbol 12-myristate 13-acetate (PMA), 10 µg/ml lysophosphatidic acid, or 140 mM
Li+. For stimulation with Li+, the cells were
transferred from Hepes-buffered Dulbecco' s modified Eagle' s medium
to medium containing 140 mM LiCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2.
and 20 mM Hepes/Tris (pH 7.0).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by Grant-in-Aid on Priority Areas 13142210 and Grant-in-Aid for Scientific Research 14580664 from the Ministry of Education, Science, and Culture of Japan, by a grant from the Organization of Pharmaceutical Safety and Research of Japan (Promotion of Fundamental Studies in Health Science), and by Health and Labour Sciences research grants, and Research on Advanced Medical Technology Grant nano-001.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.
To whom correspondence should be addressed: Dept. of Molecular
Physiology, National Cardiovascular Center Research Institute, Fujishirodai 5-7-1, Suita, Osaka 565-8565, Japan. Tel.:
81-6-6833-5012; Fax: 81-6-6872-7485; E-mail: wak@ri.ncvc.go.jp.
Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M213243200
2 S. Wakabayashi, unpublished observations.
3 T. Pang, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: NHE, Na+/H+ exchanger; pHi, intracellular pH; TM, transmembrane domain; EIPA, 5-(N-ethyl-N-isopropyl)amiloride; IL, intracellular loop; EL, extracellular loop; CHP, calcineurin B homologous protein; MTSET, 2-trimethylammoniumethyl methanethiosulfonate.
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REFERENCES |
---|
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---|
1. |
Wakabayashi, S.,
Shigekawa, M.,
and Pouysségur, J.
(1997)
Physiol. Rev.
77,
51-74 |
2. |
Orlowski, J.,
and Grinstein, S.
(1997)
J. Biol. Chem.
272,
22373-22376 |
3. |
Counillon, L.,
and Pouysségur, J.
(2000)
J. Biol. Chem.
275,
1-4 |
4. | Grinstein, S., Rotin, D., and Mason, M. J. (1989) Biochim. Biophys. Acta 988, 73-97[Medline] [Order article via Infotrieve] |
5. | Aronson, P, S., Nee, J., and Suhm, M. A. (1982) Nature 299, 161-163[Medline] [Order article via Infotrieve] |
6. |
Lin, X.,
and Barber, D. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12631-12636 |
7. |
Pang, T.,
Su, X.,
Wakabayashi, S.,
and Shigekawa, M.
(2001)
J. Biol. Chem.
276,
17367-17372 |
8. |
Bertrand, B.,
Wakabayashi, S.,
Ikeda, T.,
Pouysségur, J.,
and Shigekawa, M.
(1994)
J. Biol. Chem.
269,
13703-13709 |
9. |
Wakabayashi, S.,
Bertrand, B.,
Ikeda, T.,
Pouysségur, J.,
and Shigekawa, M.
(1994)
J. Biol. Chem.
269,
13710-13715 |
10. |
Voyno-Yasenetskaya, T.,
Conklin, B. R.,
Gilbert, R. L.,
Hooley, R.,
Bourne, H. R.,
and Barber, D. L.
(1994)
J. Biol. Chem.
269,
4721-4724 |
11. |
Dhanasekaran, N.,
Prasad, M. V.,
Wadsworth, S. J.,
Dermott, J. M.,
and Van Rossum, G.
(1994)
J. Biol. Chem.
269,
11802-11806 |
12. |
Hooley, R., Yu, C.-Y.,
Symons, M.,
and Barber, D. L.
(1996)
J. Biol. Chem.
271,
6152-6158 |
13. |
Bianchini, L.,
L'Allemain, G.,
and Pouysségur, J.
(1997)
J. Biol. Chem.
272,
271-279 |
14. |
Takahashi, E.,
Abe, J.-I.,
Gallis, B.,
Aebersold, R.,
Spring, D. J.,
Krebs, E. G.,
and Berk, B. C.
(1999)
J. Biol. Chem.
274,
20206-20214 |
15. |
Lehoux, S.,
Abe, J. I.,
Florian, J. A.,
and Berk, B. C.
(2001)
J. Biol. Chem.
276,
15794-15800 |
16. |
Yan, W.,
Nehrke, K.,
Choi, J.,
and Barber, D. L.
(2001)
J. Biol. Chem.
276,
31349-31356 |
17. |
Aharonovitz, O.,
Zaun, H. C.,
Balla, T.,
York, J. D.,
Orlowski, J.,
and Grinstein, S.
(2000)
J. Cell Biol.
150,
213-224 |
18. | Wakabayashi, S., Fafournoux, P., Sardet, C., and Pouysségur, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2424-2428[Abstract] |
19. | Ikeda, T., Schmitt, B., Pouysségur, J., Wakabayashi, S., and Shigekawa, M. (1997) J. Biochem. (Tokyo) 121, 295-303[Abstract] |
20. |
Wakabayashi, S.,
Pang, T.,
Su, X.,
and Shigekawa, M.
(2000)
J. Biol. Chem.
275,
7942-7949 |
21. |
Miyazaki, E.,
Sakaguchi, M.,
Wakabayashi, S.,
Shigekawa, M.,
and Mihara, K.
(2001)
J. Biol. Chem.
276,
49221-49227 |
22. | Wakabayashi, S., Pang, T., Su, X., and Shigekawa, M. (2000) FEBS Lett. 487, 257-261[CrossRef][Medline] [Order article via Infotrieve] |
23. | Pouysségur, J., Sardet, C., Franchi, A., L'Allemain, G., and Paris, S. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4833-4837[Abstract] |
24. | Counillon, L., Pouysségur, J., and Reithmeier, R. A. F. (1994) Biochemistry 33, 10463-10469[Medline] [Order article via Infotrieve] |
25. |
Cassel, D.,
Katz, M.,
and Rotman, M.
(1986)
J. Biol. Chem.
261,
5460-5466 |
26. |
Orlowski, J.
(1993)
J. Biol. Chem.
268,
16369-16377 |
27. | Yun, C. H. C., Tsé, C. M., and Donowitz, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10723-10727[Abstract] |
28. |
Goss, G. G.,
Woodside, M.,
Wakabayashi, S.,
Pouysségur, J.,
Waddell, T.,
Downey, G. P.,
and Grinstein, S.
(1994)
J. Biol. Chem.
269,
8741-8748 |
29. | Kobayashi, Y., Pang, T., Iwamoto, T., Wakabayashi, S., and Shigekawa, M. (2000) Pfluegers Arch. Eur. J. Physiol. 439, 455-462[CrossRef][Medline] [Order article via Infotrieve] |
30. | Youxing, J., Lee, A., Chen, J., Cadene, M., Chait, B. T., and MacKinnon, R. (2002) Nature 417, 523-526[CrossRef][Medline] [Order article via Infotrieve] |
31. | Toyoshima, C., and Nomura, H. (2002) Nature 418, 605-611[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Javadpour, M. M.,
Eilers, M.,
Groesbeek, M.,
and Smith, S. O.
(1999)
Biophys. J.
77,
1609-1618 |
33. |
Rimon, A.,
Gerchman, Y.,
Kariv, Z.,
and Padan, E.
(1998)
J. Biol. Chem.
273,
26470-26476 |
34. |
Wang, D.,
Balkovetz, D. F.,
and Warnock, D. G.
(1995)
Am. J. Physiol.
269,
C392-C402 |
35. | Counillon, L., Franchi, A., and Pouysségur, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4508-4512[Abstract] |
36. | Counillon, L., Noël, J., Reithmeier, R. A. F., and Pouysségur, J. (1997) Biochemistry 36, 2951-2959[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Orlowski, J.,
and Kandasamy, R. A.
(1996)
J. Biol. Chem.
271,
19922-19927 |
38. |
Khadilkar, A.,
Iannuzzi, P.,
and Orlowski, J.
(2001)
J. Biol. Chem.
276,
43792-43800 |
39. |
Fafournoux, P.,
Noel, J.,
and Pouysségur, J.
(1994)
J. Biol. Chem.
269,
2589-2596 |
40. | Noumi, T., Inoue, H., Sakurai, T., Tsuchiya, T., and Kanazawa, H. (1997) J. Biochem. (Tokyo) 121, 661-670[Abstract] |
41. | Inoue, H., Noumi, T., Tsuchiya, T., and Kanazawa, H. (1995) FEBS Lett. 363, 264-268[CrossRef][Medline] [Order article via Infotrieve] |
42. | Nakamura, T., Komano, Y., and Unemoto, T. (1995) Biochim. Biophys. Acta 1230, 170-176[Medline] [Order article via Infotrieve] |
43. | Chothia, C. (1976) J. Mol. Biol. 105, 1-14[Medline] [Order article via Infotrieve] |
44. | Zamyatnin, A. A. (1972) Prog. Biophys. Mol. Biol. 24, 107-123[CrossRef][Medline] [Order article via Infotrieve] |