Structural and functional analysis of the Na+/H+ exchanger

Abstract

The mammalian NHE (Na+/H+ exchanger) is a ubiquitously expressed integral membrane protein that regulates intracellular pH by removing a proton in exchange for an extracellular sodium ion. Of the nine known isoforms of the mammalian NHEs, the first isoform discovered (NHE1) is the most thoroughly characterized. NHE1 is involved in numerous physiological processes in mammals, including regulation of intracellular pH, cell-volume control, cytoskeletal organization, heart disease and cancer. NHE comprises two domains: an N-terminal membrane domain that functions to transport ions, and a C-terminal cytoplasmic regulatory domain that regulates the activity and mediates cytoskeletal interactions. Although the exact mechanism of transport by NHE1 remains elusive, recent studies have identified amino acid residues that are important for NHE function. In addition, progress has been made regarding the elucidation of the structure of NHEs. Specifically, the structure of a single TM (transmembrane) segment from NHE1 has been solved, and the high-resolution structure of the bacterial Na+/H+ antiporter NhaA has recently been elucidated. In this review we discuss what is known about both functional and structural aspects of NHE1. We relate the known structural data for NHE1 to the NhaA structure, where TM IV of NHE1 shows surprising structural similarity with TM IV of NhaA, despite little primary sequence similarity. Further experiments that will be required to fully understand the mechanism of transport and regulation of the NHE1 protein are discussed.

Full text

Biochem. J. (2007) 401, 623–633 (Printed in Great Britain) doi:10.1042/BJ20061062 623
REVIEW ARTICLE
Structural and functional analysis of the Na+/H+exchanger
Emily R. SLEPKOV, Jan K. RAINEY, Brian D. SYKES and Larry FLIEGEL1
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
The mammalian NHE (Na+/H+exchanger) is a ubiquitously ex-
pressed integral membrane protein that regulates intracellular pH
by removing a proton in exchange for an extracellular sodium
ion. Of the nine known isoforms of the mammalian NHEs, the
first isoform discovered (NHE1) is the most thoroughly character-
ized. NHE1 is involved in numerous physiological processes in
mammals, including regulation of intracellular pH, cell-volume
control, cytoskeletal organization, heart disease and cancer. NHE
comprises two domains: an N-terminal membrane domain that
functions to transport ions, and a C-terminal cytoplasmic regul-
atory domain that regulates the activity and mediates cytoskeletal
interactions. Although the exact mechanism of transport by NHE1
remains elusive, recent studies have identified amino acid residues
that are important for NHE function. In addition, progress has
been made regarding the elucidation of the structure of NHEs.
Specifically, the structure of a single TM (transmembrane) seg-
ment from NHE1 has been solved, and the high-resolution struc-
ture of the bacterial Na+/H+antiporter NhaA has recently been
elucidated. In this review we discuss what is known about both
functional and structural aspects of NHE1. We relate the known
structural data for NHE1 to the NhaA structure, where TM IV
of NHE1 shows surprising structural similarity with TM IV of
NhaA, despite little primary sequence similarity. Further experi-
ments that will be required to fully understand the mechanism of
transport and regulation of the NHE1 protein are discussed.
Key words: cation transport, intracellular pH, membrane protein,
Na+/H+exchanger (NHE), NhaA, structure–function analysis.
INTRODUCTION
The mammalian NHE (Na+/H+exchanger) is an integral mem-
brane protein that functions to exchange one intracellular proton
for one extracellular sodium ion. By its involvement in ion fluxes,
the NHE serves to regulate pHi(intracellular pH) and cell volume,
and to initiate changes in the growth or functional state of cells
[1]. Aside from its physiological role, the NHE serves important
roles in human pathology. Transport by the NHE plays a pivo-
tal role in the damage caused to the human myocardium during
and following a myocardial infarction [2], and it is considered to
represent a key step in the oncogenic transformation of cancerous
cells [3].
To date, the structure of the mammalian NHE remains elusive.
However, a great deal is known about many residues that are
required for the activity and regulation of the exchanger. In addi-
tion, the structure of a peptide of a single TM (transmembrane)
segment from the mammalian NHE has been published recently
[4]. This structure has some similarity to the structure of a func-
tionally important TM segment from the Escherichia coli Na+/H+
antiporter NhaA, suggesting that these two proteins may have
a similar structural architecture, although they share little se-
quence similarity.
MAMMALIAN NHE ISOFORMS
To date, nine isoforms (NHE1–NHE9) have been identified within
the mammalian NHE family [5,6]. The isoforms share approx.
25–70%amino acid identity, with calculated relative molecular
masses ranging from approx. 74000 to 93000 [5,7]. Hydropathy
analysis of the exchangers predicts that they have similar mem-
brane topologies, with an N-terminal membrane domain consist-
ing of 12 predicted TM segments and a more divergent C-terminal
cytoplasmic domain [5]. The NHE1 isoform is the ‘housekeeping’
isoform of the exchanger and is ubiquitously expressed in the
plasma membrane of virtually all tissues. It is the primary NHE
isoform found in the plasma membrane of the myocardium [2].
The NHE2–NHE5 isoforms are also localized to the plasma
membrane, but have more restricted tissue distributions. NHE2
and NHE3 are predominantly located in the apical membrane
of epithelia and are highly expressed in kidney and intestine
[8,9]. NHE4 is most abundant in stomach, but is also expressed
in intestine, kidney, brain, uterus and skeletal muscle [8]. NHE5
is expressed predominantly in brain, but may also be present
at low levels in other non-epithelial tissues, including spleen,
testis and skeletal muscle [10,11]. The isoforms NHE6–NHE9
are ubiquitously expressed and are present in intracellular com-
partments [6]. These organellar membrane NHEs are presumed
to regulate luminal pH and the cation concentration of the intra-
cellular compartments [6]. NHE6 expression is highest in heart,
brain and skeletal muscle and is localized to early recycling
endosomes [6,12]. The NHE7 isoform is localized predominantly
to the trans-Golgi network, and differs from the other NHE
isoforms in that it mediates the influx of either Na+or K+in
exchange for H+[13]. The highest levels of NHE8 expression
are found in skeletal muscle and kidney, and this isoform is
mainly localized to the mid- to trans-Golgi compartments [6].
The recently identified NHE9 isoform is localized to late recycling
endosomes [6].
NHE1 TOPOLOGY AND TRANSPORT PROPERTIES
The NHE1 isoform is the most well-characterized isoform of the
NHE family. As shown in Figure 1, NHE1 is 815 amino acids in
Abbreviations used: CAII, carbonic anhydrase II; CaM, calmodulin; CHP, calcineurin homologous protein; EL, extracellular loop; ERK1/2, extracellular-
signal-regulated kinase 1/2; ERM, ezrin, radixin and moesin; HSP70, heat-shock protein 70; IL, intracellular loop; MAPK, mitogen-activated protein kinase;
NHE, Na+/H+exchanger; pHi, intracellular pH; PIP2, phosphatidylinositol 4,5-bisphosphate; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase;
TM, transmembrane.
1To whom correspondence should be addressed (email lfliegel@ualberta.ca).
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624 E. R. Slepkov and others
Figure 1 Model of NHE1 showing the membrane and cytoplasmic domains
Upper panel: topology of the membrane domain which functions to transport cations. This
illustration is based on the findings of Wakabayashi et al. [90]. Pink circles, residues implicated
in both ion transport and inhibitor binding; orange circles, residues implicated in ion binding
and transport; yellow circles, residues implicated in inhibitor binding, green circles, residues
implicated in NHE1 folding and targeting to the plasma membrane. Lower panel: representation of
the cytoplasmic domain which functions to regulate the membrane domain through interactions
with signalling molecules.
length, with residues 1–500 comprising the membrane domain and
residues 501–815 comprising the cytoplasmic tail. The membrane
domain of NHE1 is both necessary and sufficient for ion transport,
whereas the cytosolic domain is involved in regulation of the activ-
ity of the exchanger [14]. Ion flux via the exchanger is driven by
the TM Na+gradient and requires no direct metabolic energy
input. NHE1 exhibits a simple Michaelis–Menten dependence on
extracellular Na+, with a reported apparent Kmof 5–50 mM
[15]. Extracellular Li+and H+compete with Na+for binding at
the Na+-binding site, and high extracellular concentrations of K+
inhibit NHE1 [16]. In contrast with the simple Michaelis–Menten
dependence on extracellular Na+, intracellular acidification allo-
sterically increases the activity of NHE, resulting in a rapid in-
crease in pHi.
NHEs are targets for inhibition by the diuretic compound
amiloride and its analogues, and by novel benzoylguanidine
derivatives [17]. Comparisons of the different NHE isoforms show
that they have varying affinities for these inhibitors, with the fol-
lowing order of sensitivity under similar experimental conditions:
NHE1 NHE2 >NHE5 >NHE3 >NHE4 [5,16]. Because NHE1
is the isoform that is most sensitive to inhibition, and is the only
isoform that is present in the plasma membrane of the myo-
cardium, the selective properties of these inhibitors can be ex-
ploited therapeutically.
PHYSIOLOGICAL FUNCTIONS OF NHE1
Na+/H+exchange is critical for a variety of physiological func-
tions. The mammalian NHE protects cells from intracellular
acidification, as shown by the fact that mutant cell lines devoid
of Na+/H+exchange activity are extremely sensitive to acidosis
[18,19]. Because NHE is activated by decreased pHi,when
acidosis occurs it increases NHE1 activity, resulting in the return
of pHito resting values. NHE also serves as a major Na+entry
pathway in many cell types, and as such it regulates both sodium
fluxes and cell volume after osmotic shrinkage [20–22]. Although
the mechanism by which NHE1 regulates cell volume remains
elusive, it is known that the volume- or osmolarity-sensitive site
within NHE1 is located in the N-terminal 566 amino acids of this
protein [23].
In addition to its role in regulating cellular pH and volume,
NHE also initiates shifts in pHithat stimulate changes in the
growth or functional state of cells [18,24]. NHE1 is required
for normal cell growth and proliferation, and cell proliferation is
significantly reduced in cells expressing an inactive NHE1 mutant
[25,26]. NHE1 activity is also important for cell differentiation.
Transcription of NHE1 increases during differentiation in both
human leukaemic cells and P19 embryonal carcinoma cells, and
the P19 cells that have NHE1 inhibited or deleted are markedly
deficient in their ability to differentiate [26–28]. NHE1 also plays
a role in either promoting or inhibiting apoptosis, with its role
varying depending on cell type [29–32].
NHE1 AS A STRUCTURAL ANCHOR
NHE1 acts as a structural anchor that is involved in regulating
cytoskeletal organization. NHE1 is restricted to specialized mem-
brane domains in some cells, such as in the lamellipodia of fibro-
blasts. There, NHE1 associates with the cytoskeleton via a di-
rect interaction with the actin-binding ERM (ezrin, radixin and
moesin) proteins at residues 553–564 in the C-terminal tail of
NHE1 [33,34]. Fibroblasts expressing NHE1 with mutations that
disrupt the interaction show impaired formation of focal adhesions
and actin stress fibres. The cells also have an irregular shape, and,
in wounding assays, migration of the cells is greatly impaired
[33,35,36]. NHE1 therefore has a key role in cell migration
through its interaction with the cytoskeleton and its restricted
location. These observations, plus the fact that NHE1 binds
several other proteins in the cytoplasmic regulatory domain, have
led to the hypothesis that NHE1 can act as a plasma membrane
scaffold that brings together many proteins so they can interact
functionally [37]. For example, in addition to its association
with ERM proteins and the cytoskeleton, NHE1 associates with
signalling molecules such as PIP2(phosphatidylinositol 4,5-
bisphosphate). PIP2binds in the same region as ERM proteins and
ERM proteins require PIP2for F-actin binding. It is hypothesized
that the proximity of PIP2on NHE1 facilitates ERM association
with F-actin [37]. However, further investigation is necessary
to confirm if the binding of NHE1 with a variety of proteins
facilitates their interaction in complicated processes.
NHE1 IN DISEASE
NHE1 has been implicated in the physiology of several diseases,
with the majority of research focusing on the role of NHE1
in heart disease and cancer. In the myocardium, under normal
conditions, NHE1 removes excess intracellular acid in exchange
for extracellular sodium. The increased intracellular sodium is
removed by regulatory membrane proteins, including the Na+/K+
ATPas e a n d t he Na+/Ca2+exchanger. Problems arise in the
myocardium with the increased production of protons that occurs
in the human myocardium during and following a myocardial
infarction [38]. The mechanism by which this occurs has been the
subject of much investigation. In the first part of this mechanism,
ischaemia results in increased anaerobic glycolysis, which leads
to a large increase in the production of protons. This serves to
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Structural and functional analysis of the Na+/H+exchanger 625
activate NHE1, which then exchanges intracellular protons for
extracellular sodium, leading to a rapid accumulation of sodium
in the cell [39–41]. Na+/K+ATPase function may be inhibited by
the decrease in high-energy phosphate stores that occurs during
ischaemia. The increased sodium concentration within the cell
drives an increase in calcium within the cell via reversal of the
Na+/Ca2+exchanger. This results in a buildup of calcium in the cell
that triggers various pathways, leading to cell death. It is
known that inhibiting NHE1 can have beneficial effects on the
myocardium during ischaemia and reperfusion, and the use of
NHE1 inhibitors to protect the heart against ischaemic damage has
been well established in animal studies [42–44]. The importance
of NHE1 in ischaemia–reperfusion injury is further supported by
the fact that genetic ablation of NHE1 in mice protects the heart
from ischaemia–reperfusion injury [45].
Despite the promising results generated in animal models,
results from clinical studies of NHE1 inhibition have not been
very positive. Two large studies have failed to find any significant
benefit of an NHE1 inhibitor. Any benefits were restricted to a
subset of patients who underwent coronary artery bypass graft
surgery. Additionally, a small study of 100 patients did reveal that
the NHE1 inhibitor cariporide reduced infarct size and improved
left ventricular function in post-infarction patients undergoing
angioplasty [46–48]. A more recent study [49] showed that cari-
poride administration caused a modest, but significant, reduction
in myocardial infarction after coronary artery bypass graft
surgery; however, in this study a concurrent increase in
cerebrovascular events occurred in high-risk patients, and this
risk outweighed the positive results.
It has long been known that NHE is important for tumour
growth, because tumour cells deficient in Na+/H+exchange activ-
ity either fail to grow tumours or show severely retarded growth
when implanted into immune-deprived mice [50]. It is now evi-
dent that NHE1 causes a reversal of the pH gradient in many types
of transformed and/or malignant cells so that the intracellular
environment is alkaline and the extracellular environment is
acidic [3]. This ‘malignant acidosis’ is considered to represent
a key step in oncogenic transformation and is necessary for
the development and maintenance of a transformed phenotype
[31,51,52]. Whereas NHE1 functions normally to regulate pH
in untransformed cell types, in at least some transformed cells
the tumour microenvironment aberrantly activates the protein.
For example, in breast cancer cells, serum deprivation is a
common tumour microenvironmental condition and this results
in abnormal activation of NHE1 in this cell type. The activation
of NHE1 was shown to be mediated by effects of RhoA and
Rac1, which were specific to tumour cells. In cancer cells, serum
deprivation provoked inhibition of RhoA activity in the leading
edge of pseudopodia, whereas in non-tumour cells this did not
occur [53,54]. This resulted in increased motility and invasion,
characteristics that are required for metastasis to occur [3,53]. A
recent study [55] has also demonstrated that the microenvironment
of breast tumours can activate NHE1 through CD44. This results
in acidification of the extracellular environment and promotes
breast cancer progression. Thus NHE1 inhibitors have a potential
use in treating various types of cancers. To date, NHE1 inhibitors
have been shown to induce apoptosis in leukaemic and breast
cancer cell lines [31,56]. The efficacy of NHE1 inhibitors for
treating cancer currently awaits preclinical and clinical trials.
REGULATION OF NHE1
The NHE1 isoform is highly regulated. Intracellular acidosis is the
major stimulus that regulates NHE1 activity, which is negligible
under normal physiological conditions, but is rapidly activated as
the pHidecreases [57]. This activation exhibits a Hill coefficient of
approx. 3, indicating that more than one proton binds to NHE1
during the transport cycle [58]. Thus it has been suggested that
NHE1 contains a non-transporting H+-binding site, sometimes
referred to as a ‘proton-modifier site’, which allows an allosteric
regulatory mechanism to lead to a greater increase in NHE
activity than would be expected based on pHi[58]. In addition to
responding to intracellular protons, NHE1 is regulated by phos-
phorylation by various kinases and by interactions with other
cellular proteins. NHE1 is also regulated at the transcriptional
level, allowing both mRNA levels and the amount of NHE1
protein produced to be controlled [59–62].
REGULATION BY PHOSPHORYLATION
The distal region of the C-terminal tail of NHE1, which comprises
amino acids ∼700–815, contains a number of serine and threonine
residues that are phosphorylated by protein kinases in response to
sustained acidosis or to hormone and growth-factor stimulation
[63–65]. Phosphorylation of residues in this region moves the set
point of the exchanger, shifting the exchange activity to be more
active at more alkaline pH values. Kinases that phosphorylate
NHE1 and stimulate its activity include: ERK1/2 (extracellular-
signal-regulated kinase 1/2), via the MAPK (mitogen-activated
protein kinase) cascade [66–68]; p90rsk (p90 ribosomal S6 kinase),
a downstream substrate of ERK1/2 [66,69,70]; the Rho-associated
kinase, p160ROCK [71]; NIK (Nck-interacting kinase) [72]; and
CaMKII (Ca2+/calmodulin-dependent kinase II) [73]. NHE1 is
also directly phosphorylated by p38 MAPK [32]. This kinase
inhibits NHE1 activity in response to angiotensin II via inhibition
of ERK1/2 [74], but it may also stimulate NHE1 and induce
alkalinization in an apoptotic pathway [32]. Protein kinases C
and D are also able to regulate the exchanger, but do not appear
to phosphorylate it directly [68,73,75,76]. In addition, NHE1 is
also regulated by dephosphorylation via protein phosphatase 1
[64,77].
INTERACTION WITH SIGNALLING MOLECULES
NHE activity is regulated by interaction with a variety of sig-
nalling molecules, and these interactions are shown in Figure 1
(lower panel). To date, three calcium-binding proteins have been
shown to interact with the exchanger: CaM (calmodulin) and CHP
(calcineurin homologous protein) act to stimulate NHE1, whereas
tescalcin inhibits NHE1. CaM binds to the cytoplasmic C-terminal
tail of NHE1 at two sites: a high-affinity site that is located at
amino acids 636–656 (CaM-A; Kd∼20 nM) and a low-affinity
site that is located at amino acids 657–700 (CaM-B; Kd∼350 nM)
[78]. The high-affinity site regulates NHE1 activity by functioning
as an auto-inhibitory domain, and either deletion of this site or
binding of Ca2+/CaM to this site abolishes the inhibitory effect
[79]. A second Ca2+-binding protein that interacts with NHE1
is CHP [80,81]. Endogenous CHP always contains two tightly
bound Ca2+ions when it is associated with NHE1, and it binds
to amino acids 515–530 in the C-terminal cytoplasmic domain
of NHE1 [80,82]. Finally, tescalcin binds to the final 180 amino
acids in the C-terminal tail of NHE1 and functions to inhibit the
activity of the exchanger [83,84].
Other signalling molecules that bind to NHE1 are: CAII
(carbonic anhydrase II), the adaptor protein 14-3-3, PIP2,andthe
mammalian HSP70 (heat-shock protein 70). CAII binds to amino
acids 790–802 at the distal end of the C-terminal tail of NHE1 and
increases the activity of the exchanger [85,86]. Phosphorylation
of NHE1 at a site within amino acids 634–789 causes an increased
interaction between NHE1 and CAII [85,86]. The binding of
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626 E. R. Slepkov and others
14-3-3 protein to NHE1 is also dependent on phosphorylation of
the exchanger, with binding occurring only when Ser703 is phos-
phorylated [87]. In Chinese-hamster fibroblasts, 14-3-3 protein
binding is thought to participate in serum-stimulated exchanger
activation by preventing dephosphorylation of Ser703 and by stabil-
izing an active conformation [87]. The signalling molecule PIP2
binds to NHE1 at two putative PIP2-binding motifs within the
C-terminal domain at residues 513–520 and 556–564, and deletion
of these binding motifs results in reduced transport activity in vivo
[88]. Binding of PIP2to NHE1 is required for optimal activity of
the exchanger, and this interaction, at least partially, accounts
for the ATP dependence of NHE1. Finally, HSP70 binds directly
to the C-terminal regulatory domain of NHE1, an interaction that
is probably involved in the folding and processing of the antiporter
[89].
STRUCTURE OF THE MEMBRANE DOMAIN
Relatively little is known about the structure of NHE1, because of
the inherent difficulties associated with crystallizing membrane
proteins. Thus molecular biology techniques have been used to
gain some understanding of the general structure of NHE1. For
example, the membrane topology of NHE1 shown in Figure 1
(upper panel) was determined experimentally by means of substi-
tuted-cysteine-accessibility analysis [90]. This analysis confirmed
predictions that NHE1 has 12 TM segments, with both the N- and
C-termini located in the cytosol. In addition, it identified three
membrane-associated loop regions, IL2 (intracellular loop 2), IL4
and EL5 (extracellular loop 5) which may be involved in NHE1
function.
The mature form of NHE1 is localized to the plasma membrane
and is glycosylated at both N- and O-linked sites. The N-linked
glycosylation is not necessary for Na+/H+exchange function
and biosynthesis [91,92]. In addition, it is known that NHE1
forms homodimers in intact cells, and that dimer formation is not
required for Na+/H+exchange activity [93–95]. Although it was
originally thought that the membrane domain of NHE1 alone is
sufficient to allow for dimerization, a recent study showed that
the proximal C-termini (amino acids 503–580) have a strong pro-
pensity to interact directly with each other, suggesting that the two
C-termini of the NHE1 dimer may also interact with each other
[94,95]. Finally, structural information has been deduced about
the C-terminal cytoplasmic domain of NHE1 using CD spectro-
scopy. This method revealed that the cytoplasmic tail of NHE1
is 35%α-helix, 17%β-turn and 48%random coil, and that
the structure of the cytoplasmic tail is more compact at regions
proximal to the membrane domain, whereas regions distal to the
membrane domain are more flexible and display calcium-depend-
ent conformational changes [83,96].
We recently published the first high-resolution structure of a TM
segment of the human Na+/H+exchanger [4]. A TM IV peptide
was expressed and purified, and its structure was determined in
a membrane-mimetic environment (Figure 2). From this NMR
structure it is clear that TM IV does not, as a whole, assume a
single conformation. Rather, sections within TM IV have distinct
structural characteristics. Specifically, TM IV is composed of
three sections of four to nine residues that converge structurally
and only one region is α-helical: Asp159–Leu163 form a series of
β-turns; Leu165–Pro168 has an extended structure; and Ile169 –Pro176
form an α-helix. As the structure of the peptide was determined
in a membrane-mimetic solvent in the absence of the balance
of the protein, these three structured regions rotated quite freely
with respect to each other at swivel points that are located at
Phe164 and Pro168/Ile169 (Figure 2). It must be presumed that in
the entire NHE1 protein the interaction of TM IV with other TM
Figure 2 Structure of a TM IV peptide in a membrane -mimetic environment
Convergent stretches (grey) of residues Asp159–Leu163 ,Leu
165–Pro168 , and Ile169–Phe176 in
relation to pivot residues Phe176 and Pro168/Ile169 are shown [137]. The flexible N- and C-termini
are represented by dashed lines. Note that only the proline side chains are indicated. Reproduced
from [4] with permission.
segments would restrict the rotation about these swivel points.
Aside from this TM segment, no other information has been pub-
lished regarding the structure of NHE1. However, in contrast with
results from TM IV, we have recently determined TM VII is a
kinked α-helix [96b].
RESIDUES INVOLVED IN NHE1 FUNCTION
Although the exact mechanisms of transport and inhibitor binding
by NHE1 are not known, specific residues within the membrane
domain of NHE1 have been implicated as being important for ion
binding and transport. The location of these residues is highlighted
in Figure 1 (upper panel), and Table 1 summarizes the effects of
these mutations.
TM IV
Numerous residues in TM IV have been implicated in NHE1
function. For example, a Phe165 →Tyr mutation in TM IV of
the hamster NHE1 sequence (corresponding to human Phe161)
causes both an increase in resistance to inhibitors and a decrease
in Vmax for Na+[97]. In addition, a Leu167 →Phe mutation
(corresponding to human Leu163) causes increased inhibitor resi-
stance with no effect on Na+transport. In 1997, Counillon et al.
[98] used random mutagenesis and found that a Gly174 →Ser
mutation in TM IV causes a modest increase in resistance to
amiloride with no effect on Na+transport. They also made an
NHE1 mutant with a Leu163 →Phe/Gly174 →Ser double mutation,
and this mutant possessed a strongly reduced affinity for HOE
694 [(3-methylsulfonyl-4-piperidinobenzoyl)guanidine methane-
sulfonate] and a 2-fold decrease in sodium affinity. A Phe162 →Ser
substitution in TM IV has been found to cause a dramatic decrease
in affinity for cariporide and a 10-fold decrease in Na+affinity
[99].
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Table 1 Amino acids known to be involved in NHE1 structure and function
Summary of residues in the membrane domain of NHE1 required for ion transport, inhibitor binding and/or expression and targeting of the exchanger. Amino acid numbering corresponds to the human
NHE1. For mutants that were studied in other mammalian models, the species used and the corresponding amino acid number are indicated in parentheses. The location of the residue within the topology
of the exchanger, the specific mutations studied and the effects of the mutations are indicated. EIPA, 5-(
N
-ethyl)-
N
-isopropylamiloride; HOE 694, (3-methylsulfonyl-4-piperidinobenzoyl)guanidine
methanesulfonate; MPA, 5-(
N
-methyl)-
N
-propylamiloride; MTSET, 2-(trimethylammonium)ethyl methanethiosulfonate bromide.
Amino acid Location Mutation Effect of mutation Reference
Gly148 (rat: Gly152) EL2 Gly →Ala Increase in
K
ifor EIPA [7]
Pro153/Pro154 (rat: Pro157 /Pro158) EL2 Pro →Ser/Pro →Phe Increase in
K
ifor EIPA [7]
Decrease in transport
Phe161 (hamster: Phe165)TMIVPhe→Tyr Increase in
K
ifor amiloride and MPA [97]
Decrease in transport
Phe161 TM IV Phe →Cys Lines ion-transport pore [4]
Phe162 TM IV Phe →Ser 1500-fold increase in
K
ifor cariporide [99]
Increase in
K
mfor Na+
Leu163 (hamster: Leu167)TMIVLeu→Phe, Ala, Arg, Trp Increase in
K
ifor amiloride, MPA and HOE 694 [97]
Leu →Tyr Eliminates Na+/H+transport
Pro167 TM IV Pro →Gly, Ala, Cys Decreased Na+/H+transport, expression [100]
and plasma membrane targeting
Pro168 TM IV Pro →Gly, Ala, Cys Decreased Na+/H+transport [100]
Gly174 TM IV Gly →Ser,Asp Increase in
K
ifor amiloride and HOE 694 [98]
Leu163/Gly174 TM IV Leu →Phe/Gly →Ser Increase in
K
ifor HOE 694 [98]
Increase in
K
mfor Na+
Arg180 IL2 Arg →Cys MTSET treatment decreases activity [90]
Gln181 IL2 Gln →Cys MTSET treatment decreases activity [90]
Glu262 TM VII Glu →Gln Eliminates Na+/H+transport [102]
Glu →Asp Restores partial Na+/H+transport
Increases
K
mfor Li+
Asp267 TM VII Asp →Asn Eliminates Na+/H+transport [102]
Asp →Glu Restores Na+/H+transport
His349 TM IX His →Gly, Leu Increase in
K
ifor amiloride [103]
His →Tyr, Phe Decrease in
K
ifor amiloride
Glu346 (rat: Glu350)TMIXGlu→Asp Increase in
K
ifor EIPA and HOE 694 [7,105]
Decrease in transport
Increase in
K
mfor Na+
Glu →Asn, Gln Increase in
K
ifor EIPA
Decrease in transport
Gly352 (rat: Gly356)TMIXGly→Ala, Ser, Asp Increase in
K
ifor EIPA [7]
Decrease in transport
Glu391 EL5 Glu →Gln Decreased Na+/H+transport [102]
Glu →Asp Restores Na+/H+transport
Arg440 IL5 Arg →Cys, Lys, His, Asp, Glu, Leu Shifts pHidependence to acidic side [107]
Tyr454 TM XI Tyr →Cys Retained in endoplasmic reticulum [106]
Gly455 TM XI Gly →Cys, Gln, Thr, Val Shift pHidependence to alkaline side [107]
Gly →Ala, Asp, Asn, Ser No effect on pHidependence
Gly456 TM XI Gly →Cys Shifts pHidependence to alkaline side [107]
Arg458 TM XI Tyr →Cys Retained in endoplasmic reticulum [106]
We have been investigating the importance of residues in TM
IV of NHE1 for several years, and we have found numerous resi-
dues in this TM segment that are required for normal Na+/H+
exchange activity. For example, we found that both Pro167 and
Pro168 are required for normal NHE1 activity, whereas Pro178 is
not [100]. In addition, mutation of Pro167 affects the expression
and membrane targeting of NHE1 [100]. Thus both Pro167 and
Pro168 are critical for normal NHE1 function, and may be required
to directly interact with transported cations, or to allow TM IV to
assume a unique structure or undergo a conformational change
that is required for NHE1 activity. Mutation of these prolines
may also affect the structure or folding of NHE1 that may cause
aberrant targeting of the protein.
We also used the substituted-cysteine-accessibility method
[101] to examine both functional and structural aspects of TM
IV in NHE1. We found that TM IV is exceptionally sensitive to
mutation, with each of the 23 single-cysteine mutations resulting
in a significant decrease in exchanger activity [4]. In addition, we
examined the sensitivity of the active single-cysteine mutants to
thiol-reactive reagents and found that the mutant Phe161 →Cys
was significantly inhibited by this treatment [4]. Therefore, in
addition to being extremely sensitive to mutation, TM IV, and
specifically Phe161, line the ion-transport pore of NHE1.
The loop regions at either end of TM IV also contain residues
that are important for NHE1 function. Mutation of three residues
in the second exomembrane loop at the N-terminal end of TM
IV (EL2) affects both the drug sensitivity and the activity of the
exchanger [7]. IL2, at the C-terminal end of TM IV, also contains
residues that may line the ion-transport pore, because treating the
mutants Arg180 →Cys or Gln181 →Cys at IL2 with membrane-
impermeant thiol reagents severely inhibits transport [90].
TM VII
TM VII is clearly involved in the ion-binding and transport
capabilities of NHE1, because both Glu262 and Asp267 are essential
for NHE1 activity [102]. Both of the mutations Glu262 →Gln and
Asp267 →Asn abolished Na+/H+exchange activity, whereas the
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628 E. R. Slepkov and others
conservative mutations Glu262 →Asp and Asp267 →Glu, which
retain the acidic side chain, restored Na+/H+exchange activity.
In addition, the mutant Glu262 →Asp had a lower affinity for Li+
than the wild-type exchanger. Because Li+has a smaller ionic
radius than Na+, this decreased affinity may be due to the shorter
side-chain length of the aspartate residue reducing the ability of
the exchanger to co-ordinate the smaller Li+ion. Thus TM VII is
involved in the cation-exchange mechanism of NHE1, and Glu262
is a likely target for directly interacting with transported cations.
TM IX
The importance of TM IX for NHE1 activity was first demon-
strated when it was determined that mutating His349 affects the
amiloride sensitivity of NHE1 [103]. Moreover, chimaeric NHE
proteins made by interchanging a 66-amino-acid segment con-
taining TM IX and its adjacent loops from NHE1 and NHE3
displayed reciprocal alterations in their sensitivities to inhibitors,
but retained normal Na+transport properties [104]. Recently, two
residues within TM IX of rat NHE1, Glu350 and Gly356 (corres-
ponding to Glu346 and Gly352 in human NHE1), were identified as
major determinants of drug sensitivity [7]. In addition, mutation
of Glu346 also affects the Na+affinity of NHE1 [105].
TM XI
A number of residues in TM XI may be involved in either ion
transport or proper targeting to the plasma membrane, as shown
by the fact that mutation of these residues alters NHE1 function
[106]. Specifically, two mutants in TM XI, Tyr454 →Cys and
Arg458 →Cys, are retained in the endoplasmic reticulum [106]. In
addition, the mutations Gly455 →Cys and Gly456 →Cys in TM XI
shift the pHidependence of the exchanger to a more alkaline value
(pK∼7), whereas the mutation Arg440 →Cys in IL5 at the N-
terminal end of TM XI shifts the pHidependence to a more acidic
value (pK<6.2) [107]. The shift in pKobserved with mutation
of Gly455 was dependent on the size of the substituted side-chain
residue, implying that these mutations may perturb the structure
of TM XI, thereby indirectly affecting the H+-modifier site. In the
case of Arg440, mutation to a lysine residue had a more modest
effect on the pKthan other mutations, indicating that a positive
charge at this site may be important for normal pHisensitivity.
Further experiments measuring 22Na+efflux from cells in which
the Na+/H+exchanger was functioning in the reverse mode also
support the conclusion that both IL5 and TM XI play a crucial
role in the proper functioning of the H+-modifier site [107].
Other proximal membrane regions involved in NHE1 function
The first extracellular loop (EL1), the membrane-associated seg-
ment (EL5) and the proximal region of the cytoplasmic domain
all contain residues that are involved in NHE1 function. EL1 is
involved in the differences in volume sensitivity between isoforms
of the Na+/H+exchanger. Specifically, amino acids 41–53 are res-
ponsible for inhibiting hyperosmolarity-induced activation of
NHE2, resulting in the lack of RVI (regulatory volume increase)
that is evident in this isoform [108]. However, the N-terminal
region (amino acids 1–95) of NHE1 do not appear to be involved
in the volume-sensing mechanism of this isoform [108].
The possible functional involvement of the membrane-asso-
ciated segment within NHE1 is of interest, because this segment
contains several polar amino acids and is reminiscent of the
selectivity filter of potassium channels [102]. It has been demon-
strated that Glu391, located in the membrane-associated segment,
Figure 3 Structure of the
E. coli
Na+/H+antiporter NhaA
Ribbon representation of NhaA viewed in parallel with the membrane. The 12 TM segments
are labelled with Roman numerals. TM segments IV and XI have a helix-extended chain–helix
conformation. The cytoplasmic (upper) and periplasmic (lower) faces of the membrane are
indicated by broken lines. TM segments are colour coded as follows: I, pink; II, cyan; III, blue;
IV, red; V, grey; VI, green; VII, yellow; VIII, orange; IX, green; X, pale yellow; XI, brown; XII cyan.
This Figure is adapted from [112] with permission from
Nature
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Ltd. (http://www.nature.com/).
is important for activity [102]. A Glu391 →Gln mutation resulted
in a partial reduction in activity, and a Glu391 →Asp mutation,
an alternative acidic residue, restored Na+/H+exchanger activity.
Thus the membrane-associated segment plays a role in the ion-
binding and transport properties of NHE1.
A highly conserved histidine-rich sequence of amino acids in
the proximal region of the cytoplasmic domain, H540YGHHH545,
is also involved in NHE1 function. Mutation of this sequence to
H540HHHHH545 has no effect on the activation of NHE1 by pro-
tons, but did cause a decrease in the maximal velocity of the ex-
changer [109]. Thus this conserved sequence is involved in NHE1
function, but is not involved in proton sensing.
STRUCTURE OF PROKARYOTIC Na+/H+ANTIPORTERS
NHEs are ubiquitous throughout the Animal Kingdom. In bac-
teria, Na+/H+exchange serves a role in osmotic regulation and
removes an internal sodium ion in exchange for external H+.
E. coli has two antiporters, NhaA and NhaB. They exchange
Na+or Li+for H+. NhaA is indispensable for transport and is
electrogenic with a stoichiometry of 2H+/Na+[110]. It is the
most well studied of the prokaryotic Na+/H+exchangers and
has been well characterized by site-specific mutagenesis, and has
also been overexpressed, purified and crystallized. Despite their
similarity in function with the mammalian NHEs, NhaA, NhaB
and other Na+/H+exchangers, such as those found in yeast, do
not share a large amount of similarity in their primary sequence.
It may be that some critical amino acids involved in transport
are conserved in their position and in their function in cation
co-ordination [111]; however, this has yet to be determined.
Crystal structure of the Na+/H+antiporter from
E. coli
Recently, the crystal structure of the Na+/H+antiporter, NhaA,
from E. coli has been solved. Although NhaA shares little
sequence homology with NHE1, these proteins share a similar
basic topology with 12 membrane-spanning segments and both
the C- and N-termini in the cytoplasm. Figure 3 shows the 3.45 Å
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Structural and functional analysis of the Na+/H+exchanger 629
Figure 4 Proposed mechanism of transport by the
E. coli
Na+/H+antiporter NhaA
The TM IV–TM XI assembly and its interaction with TM IX is shown. (A) Acidic pH-locked conformation. TM IX is bent, and the conformation of the TM IV–TM XI assembly only partly exposes the
Na+-binding site. (B) Alkaline pH causes a conformational change in helix IX that results in a reorientation of the TM IV–TM XI assembly. This exposes the Na+-binding site (yellow circle) to
the cytoplasmic funnel (red broken lines and red circle) and blocks it from the periplasm (orange line). (C)Na
+binding causes the cation-loaded binding site to be exposed to the periplasm. Upon
release of the cation, key aspartic acid residues are protonated, shifting NhaA back into the cytoplasm-exposed conformation in (B). This Figure is adapted from [112] with permission from
Nature
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2005 Macmillan Magazines Ltd. (http://www.nature.com/).
resolution structure of NhaA in an acid-locked conformation. This
structure reveals a negatively charged funnel that opens to the
cytoplasm and ends in the middle of the membrane at the putative
ion-binding site [112]. Of the 12 TM segments, ten span the
bilayer as α-helices, whereas two (TMs IV and XI) are composed
of a short helix, an extended polypeptide chain and a short helix
(Figure 3). It should also be noted that, despite weak sequence
similarity, TM segments III, IV and V form a bundle with strong
structural similarity to the bundle formed by TM segments X, XI
and XII. These bundles are in the opposite orientation relative to
the membrane and bring TM segments IV and XI into close prox-
imity at the ion-binding site while providing a balanced electro-
static environment. Extended (also referred to as ‘unwound’)
segments in pairs of TM segments at active sites have also been
observed in the SERCA1a (sarcoplasmic/endoplasmic reticulum
Ca2+-ATPase 1a); [113] and in a bacterial homologue of Na+/Cl−-
dependent neurotransmitter transporters (LeuTAA) [114]. In each
case, the extended portions of otherwise helical TM segments
are in close proximity to each other and form ion- and substrate-
binding sites.
The mechanism of NhaA regulation is thought to be dependent
on TM IX, which is known to contribute to the ‘pH sensor’ and
undergo a pH-induced conformational change. In the structure,
TM IX is a distorted helix that is in contact with the TM IV–TM
XI assembly at the centre of the membrane. Thus it is thought that
at alkaline pH the conformation of TM IX changes, resulting in
a reorientation of the TM IV–TM XI assembly that would fully
expose the Na+-binding site to the cytoplasm. Subsequent binding
of Na+would then trigger another small movement of the TM
IV–TM XI assembly, thereby exposing the cation-loaded binding
site to the periplasm. Figure 4 shows this proposed mechanism
of pH regulation and ion translocation. This mechanism, which
requires only a small conformational change, is consistent with
the extremely high catalytic activity of NhaA.
STRUCTURAL SIMILARITIES BETWEEN NHE1 AND NHAA
It has been proposed that the three-dimensional architecture of
NhaA may be similar to that of the mammalian NHEs, despite the
fact that these proteins share little sequence similarity in align-
ments of primary structure and have different transport stoi-
chiometry [111,115,116]. In a number of membrane proteins,
intramolecular structural homology has been observed within
three-dimensional structures for regions having limited primary
sequence similarity [112,114,117–122], including the case of
NhaA, as described above [112,119,120]. Despite differences in
transport mechanism, such as differing stoichiometery, the dis-
tantly related proteins NHE1 and NhaA do share a number of simi-
lar characteristics. In each of these proteins the pH regulatory site
is different from the active site, a loop participates in the pH
response and each protein exists as an oligomer within the mem-
brane [95,115,123,124].
In the NMR structure of TM IV from NHE1 (Figure 2), an ex-
tended structure in the middle of the segment can be observed
which is reminiscent of those seen in the ion-/substrate-binding
sites of NhaA, SERCA1a [113] and LeuTAA [114]. Although it
is certainly a possibility that an isolated TM peptide in mem-
brane-mimetic conditions will assume a non-physiologically rel-
evant structure, a number of isolated TM segments under such
conditions have been shown to be both functional and properly
structured where a structure of the full-length protein exists [125–
132]. Beyond the general motif of paired TM segments in close
proximity with extended regions interrupting structure, direct
comparison of the crystal structures of NhaA, SERCA1a and
LeuTAA shows that neither TM IV or TM XI of NhaA has strong
structural similarity with M4 or M6 of SERCA1a or to TM I
or TM VI of LeuTAA (results not shown). At best, a five-residue
stretch of M4 of the extended portion of SERCA1a has 0.98 Å
CαRMSD (root mean square deviation) overlap with TM IV
of NhaA. Comparison of TM IV of NHE1 with TM IV and
TM XI of NhaA demonstrates strong structural similarity, parti-
cularly between the pair of TM IV segments.
Specifically, when aligned as shown in Figure 5, 14 residues
of the TM IV segments of each exchanger are structurally
homologous (Figure 5; see also detailed superimpositions in
the Supplementary Figures, http://www.BiochemJ.org/bj/401/
bj4010623add.htm). Most notably, the extended segment in NHE1
at residues 165–168 superimposes extremely well on to the
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630 E. R. Slepkov and others
Figure 5 Structural similarity between TM IV segments of NHE1 and NhaA
Upper panel: sequence alignment of TM IVs of NHE1 and NhaA with arbitrary coloration. Lower
panel: representative NHE1 TM IV structure [4] (arbitrary orientation at swivel point Phe164)
shown alongside the TM IV segment of NhaA [112], with colouring as indicated in the sequence
alignment. Despite little sequence similarity, alignment of the TM IV segments of NHE1 and
NhaA over the residues illustrated allows structural superimposition at the 14 pairs of residues
indicated by arrows. Differences in structure between the Leu163–Phe164 swivel point of NHE1
and the crystal structure of inactive NhaA at Ile128–Pro129 mean that the entire segment does
not superimpose well. However, Asp159–Phe162 of NHE1 shows extremely similar structure to
Ile121–Trp126 of NhaA, and a subset of the NMR structures with the appropriate Pro168/Ile169 swivel
point orientation gives excellent superimposition of Leu165–Gly174 of NHE1 on Ale130 –Gly139 of
NhaA.
extended region of NhaA at residues 130–133. There would
be little chance of predicting this structural similarity based on
primary sequence, since in NHE1 this region has the sequence
Leu-Leu-Pro-Pro versus Ala-Ala-Thr-Asp in NhaA. As would be
expected, the helical segment 169–174 of NHE1 shows a good
superimposition on to the helical segment TM IVc of NhaA, but
the flexibility we observed in the NMR structures at the swivel
point between Pro168 and Ile169 (Figure 2) means that only a subset
of the TM IV structures of NHE1 superimposes well over both the
extended segment and the C-terminal helical region IVc of NhaA.
An example of another structure showing good superimposition
over this entire region is presented in Figure 5. Finally, the turn
structure observed over Asp159–Phe162 of NHE1 superimposes
exceptionally well on to Glu124–Ala127 of NhaA. This covers the
C-terminus of the TM IVp helix, which extends from Ile121–Trp126
in the X-ray structure of the pH-inactivated form of NhaA, and the
initiation of the β-bend observed at Ala127–Ala130 .TMIVofNHE1
also shows a lesser structural similarity to TM XI of NhaA (results
not shown), which would be expected given the quasi-symmetrical
symmetry between TM IV and XI in the NhaA crystal structure
[112,119,120]. Finally, TM IV of NHE1 does not superimpose
well on to the M4 segment of SERCA1a, even though the M4
segment shows some structural similarity to TM IV of NhaA.
Therefore, despite extremely low amino acid sequence similarity,
theNMRstructureofTMIVofNHE1overresiduesAsp
159–Gly174
shows strong structural similarity to the TM IV segment of NhaA
over residues Glu124 –Gly139, with the exception of the swivel point
region Leu163–Phe164 .
Because biochemical studies have shown that TM IV in NHE1
lines the ion-transport pore and contains numerous residues that
are important for NHE1 function [4], it is reasonable to assume
that this TM segment is likely to play a central role in the mech-
anism of NHE1. As shown in Figure 4, conformational change of
TM IV in NhaA about the extended non-helical region is proposed
to play a major role in exposing the Na+-binding site in the
active versus inactive protein. Given the structural similarity of
the extended segment of TM IV in NHE1, alongside its inherent
flexibility, as exhibited by the pair of swivel points immediately
N- and C-terminal to the extended segment, it is very possible that
TM IV of NHE1 is involved in a similar mechanistically important
conformational change. Furthermore, despite an apparent lack of
sequence similarity, it is possible that further structural similarity
exists and that these proteins share a similar tertiary fold. However,
a definitive analysis of the comparative structure of NhaA and
NHE1 will await the resolution of the structure of the entire
membrane domain of NHE1.
CONCLUSIONS AND FUTURE DIRECTIONS
In recent years, there have been many advances made in our under-
standing of the function and regulation of the mammalian NHE.
Several regions of the exchanger have been shown to be involved
in ion-exchange activity, inhibitor binding, interaction with sig-
nalling molecules and regulation by phosphorylation. However,
many unanswered questions still remain. For example, although
it is evident that TM IV lines the ion-transport pore of NHE1
and contains many residues that are important for NHE1 function
and inhibitor binding, less is known about other TM segments.
Thus further mutagenesis studies are required to identify pore-
lining and functionally important residues in other TM segments
of NHE1. In addition, once further pore-lining residues are
identified in TM segments other than TM IV, site-directed chemi-
cal cross-linking experiments may be of use to elucidate structural
information about NHE1. This technique has been used to probe
the three-dimensional structures of many polytopic membrane
proteins and can be used to develop a model for the arrangement
of TM segments [133–136]. Finally, although some structural
information can be deduced about NHE1 based on the NMR
structure of TM IV and similarities between NHE1 and NhaA,
a major goal in the field of NHE research is the elucidation of
high-resolution structural information about NHE1. One way to
accomplish this is to determine the structure of TM segment
peptides. It may also be possible to express and purify larger sec-
tions of the NHE1 protein that encompass several TM segments.
The structures of larger sections of the NHE1 protein would
be especially useful for understanding results from biochemical
studies, because interactions between the TM segments may limit
rotation within the TM segments, allowing the segments to adopt
the conformation that they have in full-length NHE1. Finally,
we await the structure of full-length NHE1, which will allow us
to understand at a molecular level how this protein binds and
transports cations and interacts with inhibitors.
E.R.S. was supported by an AHFMR (Alberta Heritage Foundation for Medical Research)
Studentship. J.K.R. is supported by a Fellowship from the CIHR (Canadian Institutes of
Health Research) Strategic Training Program in Membrane Proteins and Cardiovascular
Disease. B. D.S. receives support as a Canada Research Chair in Structural Biology. L. F. is
supported by an AHFMR Senior Scientist Award. Research by L. F. in this area is supported
by the CIHR.
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Received 13 July 2006/28 September 2006; accepted 30 October 2006
Published on the Internet 12 January 2007, doi:10.1042/BJ20061062
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