Cloning and expression of the
Na+/H+
exchanger from Amphiuma RBCs:
resemblance to mammalian NHE1
Lee Anne
McLean1,
Shaheen
Zia2,
Fredric A.
Gorin2, and
Peter M.
Cala1
Departments of 1 Human
Physiology and 2 Neurology, School
of Medicine, University of California, Davis, California
95616
 |
ABSTRACT |
The cDNA
encoding the
Na+/H+
exchanger (NHE) from Amphiuma
erythrocytes was cloned, sequenced, and found to be highly homologous to the human NHE1 isoform (hNHE1), with 79% identity and 89%
similarity at the amino acid level. Sequence comparisons with other
NHEs indicate that the Amphiuma
tridactylum NHE isoform 1 (atNHE1) is
likely to be a phylogenetic progenitor of mammalian NHE1. The atNHE1
protein, when stably transfected into the NHE-deficient AP-1 cell line
(37), demonstrates robust
Na+-dependent proton transport
that is sensitive to amiloride but not to the potent NHE1 inhibitor
HOE-694. Interestingly, chimeric NHE proteins constructed by exchanging
the amino and carboxy termini between atNHE1 and hNHE1 exhibited drug
sensitivities similar to atNHE1. Based on kinetic, sequence, and
functional similarities between atNHE1 and mammalian NHE1, we propose
that the Amphiuma exchanger should
prove to be a valuable model for studying the control of pH and volume
regulation of mammalian NHE1. However, low sensitivity of atNHE1 to the
NHE inhibitor HOE-694 in both native
Amphiuma red blood cells (RBCs) and in
transfected mammalian cells distinguishes this transporter from its
mammalian homologue.
sodium/proton antiport; amiloride; HOE-694; intracellular pH; red
blood cells
 |
INTRODUCTION |
THE UBIQUITOUSLY EXPRESSED
Na+/H+
exchanger (NHE) has been shown to participate in a variety of
physiological processes, including sodium absorption, cell development,
intracellular pH (pHi)
regulation, and cell volume regulation. Abnormalities in NHE function
have been implicated in such pathophysiological conditions as diabetic nephropathy, hypertension, ischemia-reperfusion injury, and
tumor proliferation (24, 36, 41-43). The first demonstration in
mammals of the obligatory counterexchange of equimolar
Na+ for
H+ was performed in 1976 on
isolated brush-border membranes from rat intestine and kidney (30) and
suggested that NHE participates in epithelial
Na+ absorption. During that same
year, Johnson and Epel (26) demonstrated that postfertilization
activation of sea urchin eggs was associated with intracellular
alkalinization mediated by
Na+/H+
exchange. Subsequent studies by Thomas and Aickin directly implicated NHE-dependent regulation of pHi in
snail neurons (44) and mouse soleus muscle (1). The role of the NHE in
the regulation of cell volume was first demonstrated by our laboratory,
in 1980, in studies using red blood cells (RBCs) from the giant
salamander, Amphiuma
tridactylum (9).
These initial studies underscored the broad distribution and
versatility of the NHE, yet they raised questions regarding the molecular equivalence of the proteins performing the various functions. Distinct differences in substrate affinity and inhibitor potency led
many to suspect that the
Na+/H+
exchange functions may be carried out by related rather than identical
proteins. This has since been proven to be the case, with five distinct
mammalian plasma membrane isoforms, NHE1-NHE5 (27, 35, 40, 45,
46), identified thus far, in addition to the cAMP-activated
NHE
isoform from trout RBCs (5). More detailed information concerning the
structural and functional similarities and differences among the
various vertebrate NHE isoforms can be obtained from several excellent,
recently published reviews (31, 33, 50, 52).
The present study is focused on the cloning and functional expression
of the NHE from Amphiuma RBCs. The
Amphiuma exchanger performs the same
housekeeping roles as the mammalian isoform NHE1, functioning in both
volume and pH regulatory capacities (1, 3, 9, 10). Consistent with its
role in mediating volume and pH regulation, the
Amphiuma NHE, like other NHE1
isoforms, is a highly regulated, inducible transporter. Activation by
hyperosmotic shrinkage or intracellular acidification can result in an
increase of NHE1 activity by one to two orders of magnitude, with
restoration to prestimulus levels as volume or pH is regulated. The
NHE-mediated pH and volume regulatory functions in
Amphiuma RBCs are remarkably robust
and are strikingly similar to those of mammalian systems (8-12).
These kinetic and functional similarities between the Amphiuma RBC NHE and mammalian NHE1
led us to postulate that the primary structures of these transport
proteins would be similar and therefore that control mechanisms would
also be comparable for the two transport proteins. On the basis of the
striking conservation of primary structure between the
Amphiuma and human NHE1 isoforms and
the high levels of NHE expression in the
Amphiuma RBCs, we conclude that the
Amphiuma NHE is a useful model in
which to study the biochemical and molecular details of mammalian NHE1
volume and pH-dependent regulation.
 |
MATERIALS AND METHODS |
RNA isolation.
Total intracellular RNA was isolated from
Amphiuma RBCs, heart, lung, liver, and
kidney using a modification of the one-step procedure of Chomczynski
and Sacchi (14). Very high RNase activities in
Amphiuma RBCs necessitated use of a
lysis buffer that contained 14 M guanidinium and urea (Ultraspec,
Biotecx Laboratories). RBCs were separated from plasma using low-speed
centrifugation (1,000 g) and washed
twice in ice-cold 120 mM NaCl before addition of lysis buffer, while
other Amphiuma tissues were
homogenized with a polytron.
Poly(A)+ RNA was isolated from
total cellular RNA following two successive enrichments using
oligo(dT)-cellulose (39).
RNA blot analysis.
Poly(A)+ RNA (5 µg) from each
Amphiuma tissue was size fractionated
on a 1.2% agarose-2.2 M formaldehyde gel with RNA markers ranging from
0.24 to 9.5 kb (Bethesda Research Lab). Fractionated poly(A)+ RNA was transferred to a
nylon membrane by capillary action and cross-linked to the membrane by
ultraviolet light (Stratalinker 1800, Stratagene). Membranes were
prehybridized in 50% formamide, 5× SSPE (1× SSPE is 0.15 M
NaCl, 0.01 M NaHPO4, and 0.001 M
EDTA, pH 7.4), 2× Denhardt's solution, and 0.1%
SDS for 6 h at 42°C, hybridized for 16 h with radiolabeled cDNA
probes (21), and washed in 0.1% SDS and 0.2× SSC (1× SSC
is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) at 60°C.
Radioactive bands on the RNA blots were digitized using a
PhosphorImager with ImageQuant software (Molecular Dynamics). The
scanned autoradiographs were imported directly from ImageQuant into
Adobe Photoshop without altering the relative intensities of the
radioactive bands (29).
Amphiuma NHE cDNAs were amplified by
PCR to generate a gene-specific cDNA library.
Poly(A)+ RNA (2 µg) from
Amphiuma RBCs was annealed with an
oligo(dT)18 primer, and first-strand cDNA was transcribed using Moloney
murine leukemia viral RT (Superscript II, BRL). Resultant cDNA-RNA
hybrids were incubated with RNaseH and diluted 10-fold for subsequent
PCR. A partial NHE cDNA (clone
I) was amplified by PCR using
primers that contained sequences conserved in human, pig, and trout
NHE1 cDNA (Fig. 1, Table
1). Two additional contiguous cDNA
regions (clones
II and
III) were successively generated by PCR to provide the 3' untranslated nucleotide sequence necessary for a primer-specific NHE cDNA library
(clone
IV).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Cloning strategy for isolation of
Amphiuma
tridactylum
Na+/H+
exchanger isoform 1 (atNHE1) cDNA. Open reading frames (open bars) and
untranslated regions (UTR; solid bars) are depicted based on transcript
size. Predicted transmembrane domains (shaded blocks) are labeled and
correspond with other NHEs. Clones
I and
II were obtained by RT-PCR of
poly(A)+ RNA, and
clone
III was obtained from 3'
extension of cDNA of clone
II (3' RACE). Nucleotide
sequence of clone
III was used for generation of a
sequence-specific, primed cDNA library, from which
clone
IV was isolated. Primers used for
RT-PCR are indicated next to arrows. Primer D5 was used to construct
gene-specific cDNA library.
|
|
Nested PCR conditions for clone
I (1077 bp) and
clone II (772 bp).
The first-strand cDNA templates were amplified in the presence of
primers (100 nM), 1 mM Tris · HCl, 0.01% Triton
X-100, 5 mM KCl, 150 mM MgCl2, 20 nM dNTP, and 2.5 units of Taq DNA
polymerase (Promega). The reaction mixture was denatured at 95°C
for 3 min, followed by two cycles of 94°C for 40 s, 60°C for 40 s, and 72°C for 2 min. The double-stranded DNA was then amplified
with the initial primer pair for 20 cycles at 94°C for 40 s,
58°C for 40 s, and 72°C for 2 min, followed by a final 10-min
extension at 72°C. This PCR reaction was diluted 1:25 (vol/vol) and
amplified with a nested, forward primer and the same reverse primer
(Fig. 1, Table 1).
Clone III
was generated by 3' cDNA extension of
clone II.
Rapid amplification of cDNA ends (3' RACE) was accomplished by
ligating adapters AP1 and AP2 (Marathon cDNA amplification kit,
ClonTech) to clone
II cDNA (Fig. 1). Two gene-specific,
nested forward primers, Fd5 and Fd6, were derived from the
clone
II cDNA sequence (Table 1). The
RACE-PCR contained primers (0.2 µM each), 0.2 mM dNTP, 25 mM
N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid; pH
9.3 at 25°C), 50 mM KCl, 2 mM
MgCl2, 1 mM 2-mercaptoethanol, and
2.5 units of TakaRa-Ex-taq DNA polymerase (Oncor). The initial PCR
amplification using Fd5 and AP2 was as follows: 1 cycle at 94°C for
2 min; 5 cycles at 94°C for 30 s and 72°C for 4 min; 5 cycles
at 94°C for 30 s, 65°C for 30 s, and 72°C for 4 min; 15 cycles at 94°C for 40 s, 60°C for 30 s, and 72°C
for 4 min; and a final cycle of 72°C for 10 min. The PCR
amplification using nested primers Fd6 and AP1 was as follows: 1 cycle
at 94°C for 2 min; 25 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 4 min; and a final cycle of 72°C for 10 min.
Generation of an NHE-specific cDNA library.
A primer-specific cDNA library was constructed with 5 µg of
poly(A)+ RNA from
Amphiuma RBCs by using the D5 oligomer
(Table 1, Fig. 1). First-strand cDNA synthesis utilized RNaseH RT
followed by RNaseH digestion, with the second strand synthesized by
Escherichia coli T4 DNA polymerase (Superscript
Choice System, GIBCO BRL). Double-stranded cDNA was ligated to precut
EcoR I adapters, purified by column
fractionation, and cloned into an EcoR
I-digested ZAP Express vector (Stratagene). Approximately 8 × 104 plaques were screened with a
32P-radiolabeled fragment of
clone
III (nucleotides 2276-2579). Three positive clones were identified and cloned into the pBK-CMV phagemid vector (Stratagene). These clones were verified to be overlapping by bidirectional dideoxynucleotide sequencing using an
automated, fluorescent dideoxynucleotide sequencer (Applied Biosystem,
Gene Sequencing Lab, University of California, Davis, CA).
Clone
IV consisted of a 3160-bp insert that
contained the entire Amphiuma
tridactylum NHE isoform 1 (atNHE1)
open reading frame flanked by 5' and 3' untranslated
regions (UTR).
Construction of NHE expression clones.
Expression constructs from clone
IV used PCR to introduce nucleotide
sequences that encoded cMyc and FLAG epitopes at the amino and carboxy
termini, respectively. The cMyc-atNHE1 construct replaced the first
three codons of atNHE1 with nucleotides encoding the cMyc epitope
(Met-Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu). The cMyc-atNHE1-FLAG
construct inserted the FLAG epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) followed by a termination codon at the end of the carboxy terminus of
the cMyc-atNHE1 construct. These epitope-tagged NHE cDNAs, along with
an atNHE1 cDNA containing no epitope tags (atNHE1), were then subcloned
into the pcDNA 3.1 vector (Invitrogen) (25). A schematic diagram
illustrating the construction of the two
human-Amphiuma chimeras is shown in
Fig. 2. Briefly, the atNhC
(Amphiuma amino terminus, human
carboxy terminus) chimera was constructed by replacing the nucleotide
coding region 1525-2469 of atNHE1 with a
Bsg
I/Xho I fragment of hNHE1 that was
generated by partial digestion. Similarly, the hNatC (human amino
terminus, Amphiuma carboxy terminus)
chimera was constructed by replacement of nucleotide coding region
1501-2415 of hNHE1 with a Bsg
I/Xho I fragment of atNHE1. The hNHE1
cDNA was cloned from a human astrocytoma cell line, and its identity was verified by double-stranded, automated nucleotide sequencing as was
done for the atNHE1 and chimeric NHE constructs.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 2.
Construction of chimeric NHEs. Two
Amphiuma-human chimeras were
constructed by interchanging amino-terminal (including all putative
transmembrane domains) and carboxy-terminal domains of
atNHE1 and human NHE1 (hNHE1). A native
Bsg I restriction endonuclease site is
located immediately following 12th transmembrane segment in both atNHE1
(bp 1525) and hNHE1 (bp 1501) sequences. atNhC
(Amphiuma amino terminus, human
carboxy terminus) chimera was constructed by replacement of nucleotide
coding region 1525-2469 of atNHE1 with a
Bsg
I/Xho I fragment generated by partial
digestion of hNHE1 with same restriction endonucleases. hNatC (human
amino terminus, Amphiuma carboxy
terminus) chimera was similarly constructed by replacement of
nucleotide coding region 1501-2415 of hNHE1 with corresponding
Bsg
I/Xho I fragment from atNHE1. AA,
amino acids.
|
|
Nucleotide sequence analysis.
Analyses and comparisons of nucleic acid sequences were carried out
using the Wisconsin package Genetic Computer Group (GCG) software.
Alignments of multiple protein sequences were performed using CLUSTAL W
(version 3.0, NCSA, University of Illinois). Phylogenetic trees were
derived from the aligned sequences using the protein sequence parsimony
method (PROTPARS, PHYLIP, version 3.5c, NCSA, University of Illinois)
(22) and by the neighbor-joining method of Saitou and Nei (38).
Distance matrices were bootstrapped 1,000 times by sampling sites at
random with replacement. The resultant distance matrices were used to
revise the phylogenetic relationships and obtain confidence values
(PAUP program).
Cell culture.
The mutant Chinese hamster ovary (CHO) cell line AP-1, which lacks
endogenous
Na+/H+
exchange activity (37), was used for stable transfection of plasmids
containing Amphiuma, human, and
chimeric NHE cDNA. Cells were maintained in complete
-MEM (Cellgro)
supplemented with 10% fetal bovine serum (HyClone), 100 U/ml
penicillin, and 100 mg/ml streptomycin (Cellgro) at 37°C, 95%
humidity, and 5% CO2. For
measurements of pHi, cells were
grown on glass coverslips coated with rat tail collagen type I
(Collaborative Biomedical Products).
Stable transfection and expression.
AP-1 cells were transfected with NHE cDNA constructs using the calcium
phosphate-DNA coprecipitation method (13, 32). Positive transfectants
were selected for resistance to 400 µg/ml G418 (GIBCO BRL) and
screened for NHE expression by probing immunoblots of whole cell
lysates with a monoclonal antibody (MAb) directed against the carboxy
terminus of the pig NHE1 isoform (MAb 4E9) (19).
Preparation of cell extracts and immunoblot analysis.
Membrane-enriched fractions utilized for epitopic determination were
prepared by washing confluent cells twice with a rinse solution that
contained PBS (145 mM NaCl, 20 mM
KHPO4, adjusted to pH 7.45 with
KOH), 2 mM EDTA, and protease inhibitors [100 µM diisopropyl
fluorophosphate, 100 µM phenylmethylsulfonyl fluoride, 100 µM
N
-p-tosyl-L-lysine
chloromethyl ketone, 100 µM
N-tosyl-L-phenylalanine chloromethyl ketone, and 0.1-1 µg/ml leupeptin, antipain,
bestatin, chymostatin, pepstatin A, phosphoramidon,
4-(2-aminoethyl)benzenesulfonyl fluoride, aprotinin,
4-amidinophenylmethanesulfonyl fluoride, and benzamidine;
Sigma]. The cells were gently scraped in 1-2 ml of rinse
solution, pelleted at 6,000 g for 10 min at 4°C, resuspended in 1 ml of rinse solution, and then lysed
by sonication. Large cytosolic debris was pelleted by centrifugation at
12,000 g for 10 min, and the
supernatant was subjected to 150,000 g
for 45 min at 4°C. The membrane-enriched pellet was solubilized in
200 µl of 50 mM NaCl, 20 mM Tris, and 1% SDS, plus the
aforementioned protease inhibitors. Whole cell lysates were prepared
from confluent cells harvested by gentle pipetting, pelleted by
centrifugation at 4°C, and lysed directly in sample buffer (1%
SDS, 6 M urea, 72 mM
Na2HPO4,
25 mM
NaH2PO4,
0.015% wt/vol bromphenol blue, and 2% vol/vol 2-mercaptoethanol).
Total protein concentrations were determined using a bicinchoninic acid
assay system with BSA as a standard (Pierce).
Protein homogenates were size fractionated through 7.5% SDS-PAGE and
electrotransferred to polyvinylidene difluoride (PVDF) membrane
(Immobilon P, Millipore). Immunoblots were incubated in blocking
buffer (3% dry milk, PBS, and 0.5% Tween-20) for 1 h before
incubation with the primary monoclonal antibodies anti-NHE1 (MAb 4E9),
anti-cMyc, or anti-FLAG (Eastman-Kodak) for 1 h at 22°C. After
several washes with PBS plus 0.5% Tween-20, immunoblots were incubated
with horseradish peroxidase-conjugated goat anti-mouse IgG secondary
antibody (Zymed) for 1 h at 22°C. Blots were developed by enhanced
chemiluminescence detection (SuperSignal, Pierce). Before blotting with
subsequent primary antibodies, blots were incubated in stripping buffer
(62.5 mM Tris at pH 6.8, 2% SDS, and 0.7% 2-mercaptoethanol) at
65°C for 30 min.
Measurement of pHi.
Cells on coverslips were loaded for 30 min with 1 µM
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF)-AM (Molecular Probes) in HEPES-buffered Ringer (HR) at
37°C, in the absence of HCO
3 and
CO2. HR was composed of (in mM) 130 NaCl, 3 KCl, 20 HEPES, 1 MgCl2, 0.5 CaCl2, 10 glucose, and 10 NaOH,
adjusted to pH 7.4 at 37°C with NaOH or HCl. Coverslips were washed
in HR three times, incubated in HR for an additional 30 min at 37°C
and 0% CO2, and then transferred
to polystyrene cuvettes that permitted continuous perfusion of
solution. Cells were maintained at 37°C in the spectrophotometer
(F-2000, Hitachi Instruments), and BCECF fluorescence was measured at
an emission wavelength of 535 nm, using optimized excitation
wavelengths of 507 and 440 nm. Cells were acidified by perfusion with
10 mM NH+4-HR (in mM: 125 NaCl, 3 KCl, 20 HEPES, 1 MgCl2, 0.5 CaCl2, 10 glucose, 10 NaOH, and 10 mM NH4Cl) for 5 min, followed by
washout in NH+4-free and
Na+-free media for 5 min. The
impermeant cation
N-methyl-D-glucamine (NMDG) was used to substitute for
Na+ in the
Na+-free solution (NMDG-HR), which
was prepared by using NMDG-Cl and NMDG-OH in place of NaCl and NaOH,
respectively. Cells were then perfused with HR with or without
amiloride (Sigma) or HOE-694 (Hoechst), and the recovery rate
(
pHi/
t)
was determined during the initial linear portion of recovery from the
acid load. Amiloride was dissolved in DMSO and HOE-694 was dissolved in
distilled H2O to stock
concentrations of 500 mM and 20 mM, respectively, and both were diluted
to final concentrations in HR as indicated.
Intrinsic buffering capacity (mM
H+/
pHi)
was determined over the range of
pHi studied by perfusing cells
with progressively decreasing concentrations of
NH+4-HR (10, 4, 2, 1, 0.5, and 0 mM
NH+4) (6). Calibration of the fluorescence
ratios was performed using high-K+
solutions of known extracellular pH (in mM: 30 KCl, 110 potassium gluconate, 10 HEPES, 1 MgCl2, 0.5 CaCl2, and 10 glucose, adjusted to
pH values of 6.2, 6.6, 7.0, 7.4, and 7.8 at 37°C using NMDG-OH or
HCl) in conjunction with 5 µM nigericin (Sigma), as described by
Boyarsky et al. (6). All solutions were nominally
HCO
3-free to exclude the effects of
HCO
3-dependent mechanisms on pH recovery.
 |
RESULTS |
Cloning Amphiuma NHE cDNA.
An initial NHE cDNA probe was amplified from
Amphiuma RBC
poly(A)+ RNA using oligonucleotide
primers that correspond to highly conserved nucleotide sequences in the
human and pig NHE1 and trout
NHE isoforms
(clone
I; Fig. 1, Table 1). Nucleotide
sequencing of clone
I demonstrated 74 and 60% nucleotide
identity with the hNHE1 (40) and trout
NHE (5) isoforms,
respectively. Radiolabeled clone
I was hybridized with an RNA blot
containing poly(A)+ RNA from
Amphiuma tissues, and a transcript of
~7 kb was detected (Fig. 3). The open
reading frames of other cloned NHE cDNAs are encompassed by <2.5 kb,
so that the putative high-molecular-mass transcript of
Amphiuma NHE indicated the likelihood
of large flanking 5' and 3' UTR. This large transcript size
suggested that an Amphiuma cDNA
library constructed from the polyadenylated 3' terminus might not extend sufficiently upstream to contain the nucleotide sequence complementary to clone
I (Fig. 1). Accordingly,
nested primers and polymerase chain amplification were used to extend
3' from the nucleotide sequence of
clone
I to encompass the entire coding region of the NHE carboxy terminus
(clones
II and
III; Fig. 1).

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 3.
Northern blot of Amphiuma tissue RNA.
Poly(A)+ RNAs (5 µg each) from
Amphiuma tissues were separated on a
1.2% agarose-2.2 M formaldehyde gel.
Lane
1, red blood cells (RBCs);
lane 2, heart; lane
3, lung; lane 4,
liver; lane 5, kidney. cDNA probes
were amplified from atNHE1 cDNA corresponding to 3' UTR
(nucleotides 2416-2579; Fig. 4) and were radiolabeled using random
primers (21). Arrow points to an ~7-kb band on autoradiograph that
corresponds to mean transcript size for atNHE1.
|
|
A gene-specific, unamplified cDNA library was then synthesized using a
primer (D5) corresponding to the proximal 3' UTR of clone
III. The longest clone
(clone IV) isolated from the cDNA library was
3160 bp and was demonstrated by nucleotide sequencing to contain the
entire open reading frame. The nucleotide and predicted amino acid
sequences of clone
IV are depicted in Fig.
4. Successive rehybridization
of the RNA blot with radiolabeled cDNA probes corresponding to the
3' UTR, the carboxy-terminal region, and a transmembrane region
confirmed the presence of 6.8- to 7.1-kb NHE transcripts in
Amphiuma RBCs, heart, lung, liver, and
kidney (Fig. 3).

View larger version (98K):
[in this window]
[in a new window]
|
Fig. 4.
Nucleotide sequence of atNHE1 cDNA and deduced amino acid sequence of
protein. Nucleotides are numbered at
right of sequence relative to putative
translation initiation site. Amino acids are numbered at
left of sequence and are shown by
their single-letter abbreviations. Presumptive TATA boxes are
underlined. * In-frame stop codon.
|
|
Comparisons of atNHE1 with other NHE sequences.
The Amphiuma exchanger exhibits 79%
identity and 89% similarity with the hNHE1 isoform at the amino acid
level (Table 2, see also Fig. 6). The
atNHE1 protein is 80% identical to the other cloned amphibian NHE from
Xenopus
laevis oocyte (Xl-NHE) (7) and 63%
identical to the
NHE (5) isoform isolated from trout RBCs (Table 2).
The Amphiuma NHE is more similar to
the NHE1 isoforms than to the other mammalian isoforms
(NHE2-NHE5), as shown by the phylogenetic tree of aligned,
full-length sequences (Fig. 5). The atNHE1
and hNHE1 amino acid sequences (Fig. 6)
retain a high degree of homology throughout the putative
membrane-spanning domains M2-M12 (87% amino acid identity), as
well as the proximal 200 amino acids of the carboxy terminus (90%
identity). The largest divergence in sequence homology exists at the
initial, amino-terminal 90 amino acids that include the putative M1
transmembrane domain (17% amino acid identity), which may be cleaved
as a signal peptide, and at the last carboxy-terminal 100 amino acids
(52% identity).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
Phylogenetic tree depicting relationship of full-length
atNHE1 to other vertebrate NHE proteins. Alignments of
multiple protein sequences were performed using CLUSTAL W (version
3.0), and phylogenetic trees were derived from aligned sequences using
protein sequence parsimony method (PROTPARS, PHYLIP, version 3.5c).
Branch lengths are proportioned to represent mean number of differences
per amino acid residue. Numbers at nodes represent confidence values
determined by bootstrap analysis of 1,000 replicates (PAUP program).
Xl-NHE, Xenopus laevis NHE isoform.
|
|

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 6.
Comparison of amino acid sequence of atNHE1 with hNHE1. atNHE1 consists
of 813 amino acids with 12 putative transmembrane domains (M1-M12)
analogous to current model of hNHE1 (4, 31, 51). Two predicted N-linked
glycosylation sites are located in 1st extracellular loop, and a
potential signal peptide cleavage site is also indicated. Overall,
there exist 79% amino acid identity and 89% similarity between
Amphiuma and hNHE1 isoforms.
|
|
Expression of Amphiuma NHE in AP-1
cells.
The epitope tags cMyc and FLAG were respectively ligated to the amino
and carboxy termini of the atNHE1 cDNA to investigate the potential
posttranslational processing of the
Amphiuma NHE protein. AP-1 cells were
stably transfected to express the following atNHE1 cDNA constructs:
atNHE1, cMyc-atNHE1, and cMyc-atNHE1-FLAG. Whole cell lysates of the
transfected cells were screened for expression with an anti-NHE1
antibody (MAb 4E9) on immunoblots. Multiple clones, with varying levels
of expression, were found to express atNHE1 for each of the three cDNA
constructs. The retention of the cMyc and FLAG epitope tags was
evaluated using a membrane-enriched protein fraction that was isolated
from nontransfected cells and each of the transfected AP-1 cell lines.
The proteins were size-fractionated by SDS-PAGE, electrotransferred to
PVDF membrane, and successively probed with monoclonal antibodies to
NHE1, cMyc, and FLAG (Fig. 7).
Immunoblots probed with the anti-NHE1 antibody (Fig.
7A) show that atNHE1 is expressed in
each of the transfected AP-1 cell lines, with bands of comparable size
shown for each of the three constructs (atNHE1, cMyc-atNHE1, and
cMyc-atNHE1-FLAG). The anti-NHE1 MAb detects two bands that correspond
with the unmodified protein with a predicted molecular mass of 90.6 kDa
and a posttranslationally modified protein at ~110-115 kDa (Fig.
7A). There are two potential N-glycosylation sites for atNHE1 in the first amino-terminal
extracellular loop (Asn-80 and Asn-84), compared with a single site
that is glycosylated at Asn-75 in hNHE1 (17) (Fig. 6), and preliminary studies from our laboratory indicate that atNHE1 is also N-glycosylated (unpublished observations). Both the cMyc-atNHE1 and cMyc-atNHE1-FLAG transfectants expressed high levels of NHE protein, although expression was still less than that seen in native
Amphiuma RBCs (Fig.
7A, lane
7). The high abundance of NHE
protein in the Amphiuma RBCs is also
demonstrated by comparison with a nontransfected human astrocytoma cell
line (Fig. 7A,
lane
8) that expresses endogenous NHE1
protein.

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 7.
Immunoblot analysis of atNHE1 expressed in AP-1 cells. Crude membrane
protein (~100 µg) from each of following cell extracts was resolved
by 7.5% SDS-PAGE: nontransfected AP-1 cells
(lane
1), cMyc-atNHE1 transfected AP-1
clone G6 (lane
2), cMyc-atNHE1-FLAG transfected
AP-1 clone C5 (lane
3), and untagged atNHE1 transfected
AP-1 clone A4 (lane
4). Positive controls for cMyc and
FLAG antibodies were run in lanes
5 and
6, respectively. For comparison, 50 µg of membrane-enriched protein from wild-type
Amphiuma RBCs
(lane
7) and from nontransfected mammalian
cells expressing endogenous NHE1 (U251 astrocytoma;
lane
8) were run on a separate
immunoblot. A: immunoblots were probed
with anti-NHE1 monoclonal antibody (MAb 4E9).
Lane
4 (untagged atNHE1-transfected AP-1
cells) is shown with an increased exposure time due to somewhat lower
levels of expression relative to other constructs.
B: immunoblot was stripped and
reprobed with an anti-cMyc antibody. KCC2-1CT cell line overexpresses a
cMyc epitope-tagged
K+-Cl
cotransporter, and a membrane-enriched protein fraction (1 µg) was
included as a positive control (lane
6).
C: same blot was restripped and
reprobed with an anti-FLAG antibody, with a bacterial alkaline
phosphatase-FLAG fusion protein (0.5 µg) serving as a positive
control (lane
5). Marker sizes are in kDa.
|
|
The immunoblot in Fig. 7A was stripped
and reprobed with an anti-cMyc antibody (Fig.
7B), using a membrane-enriched
fraction from the KCC2-1CT cell line
(lane
6) that expressed a cMyc-tagged K+-Cl
cotransporter for a positive control. Neither of the epitope-tagged AP-1 transfectants expressed a detectable cMyc epitope (Fig.
7B), but a strong signal was
detected from the expressed cMyc-tagged K+-Cl
cotransporter protein. Prolonged exposure of this immunoblot beyond the
linear range of enhanced chemiluminescence detection did not reveal any
additional signal from the AP-1 transfectants (data not shown). These
data suggest that a region of the amino terminus containing the cMyc
epitope tag is cleaved from the mature protein, consistent with the
hypothesis that part of the amino terminus functions as a signal
peptide sequence (17). Reprobing of the same blot with the anti-FLAG
antibody demonstrated retention of the carboxy-terminal epitope in the
AP-1 transfectants (Fig. 7C). There
was some nonspecific binding of the anti-FLAG antibody to other AP-1
cell proteins, but the unique NHE proteins detected by the anti-NHE1
antibody were also clearly observed using the anti-FLAG antibody
(lane
3, Fig.
7C).
Expression of hNHE1 and chimeric NHEs in AP-1 cells.
cDNA encoding the full-length sequence of hNHE1 and two chimeric
Amphiuma-human exchangers
(atNhC and hNatC) were stably transfected into AP-1 cells.
The atNhC chimera consists of the amino-terminal domain of the
Amphiuma exchanger, including the
entire transmembrane-spanning region M1-M12, and the
carboxy-terminal domain of the hNHE1 protein, whereas the hNatC chimera
contains the amino-terminal and transmembrane domains from human and
the carboxy-terminal region of
Amphiuma (see Fig. 2). As was observed
with the atNHE1 constructs, multiple clones for each cDNA construct
were found to express an NHE1 protein when probed by immunoblot
analysis. Several clones from each cDNA construct were tested for
function by monitoring recovery of
pHi following acidification using
the fluorescent ratio dye BCECF, and all these clones exhibited
significant NHE activity. A single clone from each of the
NHE-transfected AP-1 cells was selected for more detailed analysis.
Functional characterization of Amphiuma,
human, and chimeric NHE transfectants.
Na+/H+
exchange activity was evaluated by measuring the maximal
pHi/
t
following an acid load, as described in Measurement of
pHi. Cells were acidified to a
mean pHi of 6.95 ± 0.06, with no significant difference between the various cell lines. The rate of
H+ efflux (mM
H+/min) was calculated by
multiplying the rate of recovery by the intrinsic buffering capacity
(mM
H+/
pHi),
which was empirically derived from separate experiments. The buffer
capacity was linearly dependent on
pHi, with a similar trend for both
the transfected and nontransfected AP-1 cells (data not shown).
The typical recovery from intracellular acidification, under nominally
HCO
3-free conditions for transfected and control AP-1 cells is shown in Fig. 8.
Nontransfected AP-1 cells (Fig. 8A)
exhibited modest Na+-independent
alkalinization following the acid load (0.12 mM
H+/min), but this recovery was
insensitive to the NHE1 inhibitors amiloride and HOE-694. Furthermore,
the inhibitors bafilomycin (V-type
H+-ATPase inhibitor) or DIDS
(anion exchange inhibitor) did not affect the rate of recovery (data
not shown). In contrast, each of the NHE-transfected AP-1 cells (Fig.
8B) exhibited rapid recovery of
pHi in response to acidification
following the reintroduction of
Na+ to the perfusate. As shown in
Table 3, the atNHE1-transfected AP-1 cells
displayed the most robust pH recovery response, with an average
H+ flux rate of 6.58 ± 0.60 mM/min, >50 times that of nontransfected AP-1 cells and nearly double
that of the hNHE1 transfectants.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 8.
Recovery of intracellular pH
(pHi) in nontransfected and
transfected AP-1 cells following acidification. Cells on coverslips
were acidified using a 10 mM NH+4 prepulse,
and pHi was monitored by following
BCECF fluorescence. A: nontransfected
AP-1 cells display modest alkalinization following an acid load that is
independent of extracellular Na+
and not inhibited by NHE1 inhibitors (Inh) amiloride (Amil) or HOE-694
(HOE). B: AP-1 cells transfected with
cDNA encoding atNHE1, hNHE1, atNhC chimeric NHE, or hNatC chimeric NHE
rapidly recover from acidification on reintroduction of
Na+ to perfusion medium.
Representative of at least 3 experiments each.
|
|
View this table:
[in this window]
[in a new window]
|
Table 3.
Comparison of maximum JH+ and inhibition
constants for transfected and nontransfected AP-1 cells following
acidification
|
|
Inhibition of pH recovery by amiloride for each of the NHE
transfectants was dose dependent, whereas nontransfected AP-1 cells were unaffected (Fig.
9A, Table
3). Values of IC50 for amiloride were similar for the atNHE1 transfectant and the two chimeric proteins,
with values ranging from 2.6 to 6.1 µM. These values are similar to
the 1.6 µM reported for rat NHE1 expressed in AP-1 cells (34) and the
3 µM reported for hNHE1 expressed in the NHE-deficient Chinese
hamster lung fibroblast (PS120) cell line (18). Interestingly, the
calculated IC50 for our
hNHE1-transfected AP-1 cells was 24 µM, nearly an order of magnitude
higher than that reported elsewhere. This disparity, however, primarily
reflects the different experimental conditions between our studies
(external Na+ concentration 140 mM) and those of others (external
Na+ concentration ~0 mM). Using
the 22Na uptake protocol of
Orlowski and Kandasamy (34), in nominally Na+-free medium, we measured
amiloride IC50 values of 4.7 ± 0.5 µM for hNHE1-transfected AP-1 cells and 1.3 ± 0.2 µM for
atNHE1-transfected AP-1 cells (data not shown).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 9.
Dose response curves for inhibition of
Amphiuma, human, and chimeric NHEs
expressed in AP-1 cells. Maximal rates of
H+ efflux
(JH+;
mM/min) following an acid load were determined for atNHE1-, hNHE1-,
atNhC-, and hNatC-transfected AP-1 cells over increasing concentrations
of amiloride and HOE-694 (see MATERIALS AND
METHODS for details). Data were normalized as a
percentage of maximal
JH+
in absence of inhibitor and represent means ± SE of at least 3 experiments each.
|
|
Recent studies have shown that HOE-694 is more selective and more
potent than amiloride for inhibiting the NHE1 isoform (18, 34).
Counillon and co-workers (18) reported an
IC50 of 0.16 µM when using
HOE-694 to inhibit PS120 cells that were stably transfected with hNHE1,
compared with an IC50 of 3 µM
for amiloride. In the present study, HOE-694 inhibited activity of the
hNHE1 transfectant to a greater degree than amiloride, with a
calculated IC50 of 1.2 µM (Fig.
9B, Table 3). However, HOE-694 only
modestly inhibited the atNHE1 transfectants and the two
chimeric NHE transfectants, with calculated
IC50 values of >200 µM (Fig.
9B, Table 3). Due to precipitation of
HOE-694 from the HR solution at concentrations greater than 200 µM,
we were not able to obtain a full dose response curve for this
compound. As with the atNHE1-transfected AP-1 cells, HOE-694
sensitivity of native atNHE1 in intact
Amphiuma RBCs was also minimal (data
not shown).
 |
DISCUSSION |
Our laboratory has previously shown that the NHE expressed in the RBCs
of the giant salamander, A.
tridactylum, is especially active in
both pH and volume regulation (9-11). We have measured amiloride-sensitive Na+ uptake
rates of >30
mM · min
1 · kg
dry cell solid
1 in
Amphiuma RBCs maximally stimulated by
the addition of the phosphatase inhibitor calyculin A (1 µM;
unpublished observations). Consistent with the high level of functional
NHE activity, these RBCs express exceptionally large quantities of the
NHE protein (Fig. 7A). In this
study, the Amphiuma NHE (atNHE1) was
cloned and sequenced to ascertain its similarity to the mammalian NHE1 isoforms and to provide the foundation for developing sequence-specific antibodies for use in studies of atNHE1 control. When the kinetic, functional, and structural similarities between atNHE1 and mammalian NHE1 are considered, together with the low tonic activity of the Amphiuma NHE in
Amphiuma RBCs, the
Amphiuma NHE is a valuable model for
studying the control of pH and volume regulation by mammalian NHE1.
Patterns of transcript and protein expression.
In Amphiuma, the atNHE1 isoform
isolated from the RBCs was detected in all other tissues examined,
including heart, lung, kidney, and liver (Fig. 3). The transcript size
for atNHE1 (~7 kb) is larger than those reported for the human and
rat NHE1 isoforms, 5.1 kb (23) and 4.8 kb (35), respectively. The
transcript size of the recently cloned Xl-NHE isoform from
X.
laevis oocytes (7) has not been
described, but a recent report describes a 6-kb mRNA for a reptilian
NHE homologue (23). Our cloning strategy was directed toward obtaining
and expressing the coding region of the cDNA, and therefore we did not
sequence the entire 7-kb transcript once the complete open reading
frame was identified. However, 3' RACE did indicate that the
3' UTR exceeded 1.1 kb. The significance of these large UTRs for
some NHE transcripts is unknown, but regions within the 3' UTR
have been shown to regulate transcript stability and intracellular
localization in some genes.
The open reading frame of the atNHE1 transcript translates to a protein
consisting of 813 amino acids with a predicted core molecular mass of
90.6 kDa (Fig. 4). Kyte and Doolittle hydropathy plots
predict an approximately 500-amino acid amino-terminal region that
contains 12 putative transmembrane domains followed by a large
carboxy-terminal, cytoplasmic tail of ~300 amino acids (Fig. 6) (28).
This secondary structure prediction is consistent with that predicted
for the other members of the NHE supergene family (50, 52). The
homologies between the Amphiuma,
mammalian, and Xenopus NHE1 proteins
exceed those observed between NHE1 and the other mammalian isoforms
NHE2-NHE5 (Table 2). Figure 5 depicts the phylogenetic
relationship between atNHE1 and 12 other NHE proteins. Multiple protein
alignments using CLUSTAL W were separately determined for the
full-length NHE sequences, the conserved M2-M12 transmembrane
regions, and the cytoplasmic carboxy-terminal regions. The phylogenetic
relationships among the NHE proteins were the same for these
comparisons and reflect the sequence conservation throughout the entire
NHE sequence. These analyses also indicate that the amphibian NHE
proteins, notably atNHE1, are likely to be progenitors of the mammalian NHE1.
Structure and function comparisons.
The putative transmembrane domains M2-M12 are highly conserved
among all of the various NHE isoforms, with the greatest divergence seen in membrane-spanning region M1 (50). Although this M1 domain is
conserved among the different mammalian NHE1 isoforms, comparison of
atNHE1 with hNHE1 reveals only 19% identity in this region. It has
been proposed that this first putative membrane-spanning domain may
function as a signal peptide that is cleaved from the membrane-associated NHE protein (17). As predicted for other NHE
isoforms, the atNHE1 contains a potential peptide cleavage sequence
immediately following the M1 transmembrane domain (47). In this study,
AP-1 cells were transfected with atNHE1 cDNA constructs that contained
an amino-terminal cMyc epitope tag (cMyc-atNHE1 and cMyc-atNHE1-FLAG).
These transfected cells expressed functional atNHE1 protein, with an
approximate molecular mass of 110-115 kDa, that was recognized by
a monoclonal anti-NHE1 antibody (MAb 4E9; Fig.
7A) but not recognized by a
monoclonal anti-cMyc antibody (Fig.
7B). Conceivably, amino-terminal
glycosylation could interfere with recognition of the cMyc epitope,
thus resulting in lack of signal at 110-115 kDa. However, in
addition to the 110- to 115-kDa glycosylated protein, the anti-NHE1
antibody also recognized an 85- to 90-kDa unglycosylated core protein
(Fig. 7A). The failure of the cMyc
antibody to detect this core atNHE1 protein supports the signal peptide
cleavage theory and indicates that the cMyc epitope is cleaved before
glycosylation. The fact that the mature atNHE1 protein is glycosylated
implies that the proposed signal peptide would have to be cleaved
proximal to amino acid 80 or 84, the two potential N-glycosylation
sites present in atNHE1 (Fig. 6).
The membrane-spanning regions M2-M12 share 95-100% homology
between atNHE1 and hNHE1, with four of the transmembrane domains being
identical (Fig. 6). The proposed ion transport region of the exchanger,
spanning transmembrane domains M6 and M7 (also referred to as M5a and
M5b), is highly conserved among all NHE isoforms (20, 31, 50) and is
identical for the atNHE1 and hNHE1 isoforms with the
exception of a single amino acid (Ser-232 of hNHE1 replaced by Ala in
atNHE1). The putative amiloride binding site in transmembrane domain M4
for atNHE1 and hNHE1 also differs by only one amino acid (Val-160 of
hNHE1 replaced by Thr in atNHE1) (15, 50, 51).
The carboxy-terminal domain of atNHE1, like the amino terminus (from M2
through M12), maintains a high degree of homology with hNHE1 and is
90% identical over the proximal 200 amino acids (Fig. 6), with the
greatest similarity in regions of the carboxy terminus believed to
regulate NHE activity. For example, the high-affinity Ca2+/calmodulin binding region
[amino acids 636-656, hNHE1 (2)] is identical in
atNHE1 and hNHE1, with the exception of Thr-645 (atNHE1) being replaced
by an asparagine in the hNHE1 protein (Asn-637). The region between
amino acids 567-635 (hNHE1) is believed to interact with the
H+-sensing region of the
transmembrane domain and to be crucial for stimulation by phorbol
esters, thrombin, platelet-derived growth factor, and okadaic acid (48,
49). A comparison between hNHE1 and atNHE1 reveals only two dissimilar
amino acids over this region (Leu-614 and Asp-626 of hNHE1 replaced by
Lys and Val, respectively, in atNHE1).
The hNHE1 and atNHE1 proteins differ most notably over the terminal 100 amino acids (52% amino acid identity). Both hNHE1 and atNHE1 contain
putative phosphorylation consensus sites in this region; however, no
common sites are shared between the two transporters. Wakabayashi and
co-workers (49) report that deletion of the last 117 carboxy-terminal
amino acids does not interfere with NHE activation by growth factors,
phorbol esters, or phosphatase inhibitors, nor does such modification
result in an apparent alteration in pH set point. In fact, deletion of
these amino acids resulted in a nearly threefold increase in exchanger
activity. In the present study, we found that the
Amphiuma NHE-transfected AP-1 cells
exhibited activity nearly twice as high as that of the hNHE1
transfectants (Table 3). Interestingly, in response to intracellular
acidification, the atNhC chimeric NHE
(Amphiuma amino terminus, human
carboxy terminus) displayed exchange activity similar to the hNHE1,
whereas the hNatC chimera (human amino terminus,
Amphiuma carboxy terminus) was more
similar to the Amphiuma exchanger.
This implies that the carboxy terminus, notably the terminal ~100
amino acids, plays some role in regulating exchanger activity in
response to acidification. More specifically, the deletion data of
Wakabayashi et al. (49) and our chimera data are
consistent with the notion that the 117 terminal amino acids in the
hNHE1 might serve to suppress exchanger activity in response to changes
in pHi. The fact that the
phosphorylation sites in this carboxy-terminal region differ between
the Amphiuma and human transporters
suggests that a phosphorylation-dependent process could be involved in
suppressing H+-induced exchanger
activity. In addition to pH regulation, preliminary studies indicate
that atNHE1 expressed in AP-1 cells also functions in a volume
regulatory capacity, displaying amiloride-sensitive Na+ uptake in response to cell
shrinkage (unpublished observations).
Sensitivity to amiloride and HOE-694.
The highly similar sequences of atNHE1 and hNHE1 lead us to anticipate
similar responses to known NHE1 antagonists. Specifically, amino acids
proposed to be essential for amiloride sensitivity in hNHE1
[Leu-163 and Gly-174 in the M4 transmembrane domain (16) and
His-349 in the M9 domain (51)] are retained in the atNHE1 sequence. Although amiloride sensitivities for atNHE1 and the two
chimeric proteins were similar to what has been reported for mammalian
NHE1 (Fig. 9A, Table 3), the measured
IC50 for our hNHE1 transfectant
was nearly an order of magnitude higher. This discrepancy between our
amiloride IC50 values and those
reported by others is essentially due to differences in experimental
conditions. All previous reports of antagonist sensitivities have been
performed under nominally Na+-free
conditions (16, 18, 34), whereas the experiments in this study were
performed under physiological conditions of 140 mM external
Na+. Because amiloride displays
competitive behavior with Na+,
potency of amiloride inhibition should be an inverse function of
external Na+ concentration.
Indeed,
22Na+
uptake studies performed in our laboratory under nominally
Na+-free conditions resulted in
amiloride IC50 values of 4.7 µM
for the hNHE1 transfectants and 1.3 µM for the atNHE1-transfected AP-1 cells. Thus it appears that there is an approximately fivefold increase in the amiloride IC50
under conditions of physiological external
Na+ compared with that in
nominally Na+-free medium.
Although results with amiloride are readily explained on the basis of
experimental conditions, the striking difference in response of the
human and Amphiuma NHE isoforms to
HOE-694 was unanticipated. We confirmed earlier studies demonstrating
that HOE-694 was found to be a more potent inhibitor of the hNHE1
isoform than was amiloride (18), with an
IC50 of 1.2 µM (Fig. 9, Table 3). In contrast, the apparent IC50
for HOE-694 inhibition of Amphiuma NHE
in AP-1 cells exceeded 200 µM (Fig.
9B, Table 3), more closely resembling
the IC50 of 650 µM determined
for inhibition of the mammalian NHE3 isoform (18). In addition, both of
the Amphiuma-human chimeras exhibited
an insensitivity to HOE-694 that was similar to the native
Amphiuma exchanger.
A recent study by Orlowski and Kandasamy (34) utilized AP-1 cells
transfected with cDNAs encoding chimeric NHE proteins that contained
different transmembrane domains from mammalian NHE1 and NHE3. These
investigators exploited the differential sensitivities displayed by the
NHE1 (high-affinity) and NHE3 (low-affinity) isoforms to various NHE
antagonists, including amiloride and HOE-694. They demonstrated that
replacement of a 66-amino acid segment, spanning transmembrane domain
M9, from NHE1 with the homologous segment of NHE3 resulted in a
chimeric protein with drug sensitivities that approached that of NHE3.
The converse was also true; replacement of this region of NHE3 with M9
from NHE1 resulted in drug sensitivities that were more similar to
NHE1. These investigators concluded that the region between
transmembrane domains M8 and M10 appears to be an important site of
interaction with the HOE-694 compound. However, this region between M8
and M10 is identical in the atNHE1 and mammalian NHE1 isoforms (Fig.
6). Because both of our chimeric proteins were insensitive to HOE-694,
this suggests that additional interactions between the amino terminus
(including all transmembrane segments) and the carboxy terminus are
contributing to specificity of hNHE1 for HOE-694. The variance in
HOE-694 sensitivities between the native RBC atNHE1 and mammalian NHE1
proteins could be attributed to species-specific differences in
membrane-associated accessory proteins or signal transduction pathways
expressed in the two different cell types. However, when expressed in
the mammalian AP-1 cell line, the
Amphiuma transporter retains the same
response to both HOE-694 and amiloride as in the native red blood cell, suggesting that species-specific differences are unlikely to be involved. Furthermore, the fact that both the atNHE1 protein and the
NHE proteins in the study of Orlowski and Kandasamy (34) were expressed
in the same AP-1 cell line provides additional evidence that the
differences in HOE-694 sensitivity are not due to interactions with
accessory proteins.
A final interesting observation from our current study is the fact that
the resting pHi of the
atNHE1-transfected AP-1 cells (pHi
7.52 ± 0.01, 37°C) was considerably higher than that measured in intact Amphiuma RBCs
(pHi 7.0 ± 0.1, 22°C)
(11). Additional experiments were conducted on atNHE1-transfected AP-1
cells at room temperature to determine whether this was the cause of
the discrepancy in resting pHi.
Although decreasing the temperature did result in lower values of
resting pHi
(pHi 7.38 ± 0.09, 22°C), along with decreased activity in response to intracellular
acidification, it could not account for the entire difference in
steady-state pHi (data not shown).
This suggests that the steady-state
pHi set point of atNHE1 in these
two systems is regulated by additional factors besides the tertiary
structure of the core protein. Such factors could include interaction
with ancillary regulatory proteins or intracellular differences in
tonic kinase or phosphatase activity.
In conclusion, the high degree of similarity between hNHE1 and atNHE1
is reassuring, given the close correspondence in kinetics and
regulation of the human and Amphiuma
antiporters. The fact that the putative regulatory regions are nearly
identical for atNHE1 and hNHE1 suggests that the
Amphiuma RBC transport protein will
continue to be a useful model for the study of mammalian NHE1 function
and regulation. These structure-function observations are significant,
since the Amphiuma RBC appears to be
one of the most abundant sources of NHE1 protein yet described (Fig.
7A). Furthermore, due to its
abundance and low tonic activity, it will be possible to utilize this
system to address questions that are difficult to address in mammalian
cells that have a relative paucity of antiporter protein and high
levels of tonic NHE activity.
 |
ACKNOWLEDGEMENTS |
L. A. McLean and S. Zia contributed equally to this paper.
 |
FOOTNOTES |
We thank Chris Amolo and Jane Roscoe for preparation of the hNHE1 and
chimeric NHE cDNA constructs, Dr. Bradley Shaffer (Dept. of Evolution
and Ecology, University of California, Davis, CA) for assistance with
phylogenetic tree analyses, and Dr. Nanna Jorgensen, Elizabeth Nguyen,
and Jelena Burress for technical assistance. We are grateful to Dr.
Sergio Grinstein (Hospital for Sick Children, Toronto, ON, Canada) for
the AP-1 cell line, Dr. Dan Biemesderfer (Yale University, New Haven,
CT) for the anti-NHE1 antibody (MAb 4E9), and Dr. John Payne for the
anti-cMyc antibody and cell lysates from the KCC2-1CT cell line, which
express a cMyc-tagged
K+-Cl
cotransporter. We also thank Dr. Hans-J. Lang of Hoechst for providing
the HOE-694 compound.
This research was supported by National Heart, Lung, and Blood
Institute (NHLBI) Grant HL-21179 (to P. M. Cala). L. A. McLean was
supported by NHLBI Predoctoral Training Grant HL-07682 (to J. Longhurst).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. M. Cala,
Dept. of Human Physiology, School of Medicine, University of
California, 1 Shields Ave., Davis, CA 95616 (E-mail:
pmcala{at}hph.ucdavis.edu).
Received 10 April 1998; accepted in final form 27 January 1999.
 |
REFERENCES |
1.
Aickin, C. C.,
and
R. C. Thomas.
An investigation of the ionic mechanism of intracellular pH regulation in mouse soleus muscle fibers.
J. Physiol. (Lond.)
273:
295-316,
1977[Medline].
2.
Bertrand, B.,
S. Wakabayashi,
T. Ikeda,
J. Pouyssegur,
and
M. Shigekawa.
The Na+/H+ exchanger isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins. Identification and characterization of calmodulin-binding sites.
J. Biol. Chem.
269:
13703-13709,
1994[Abstract/Free Full Text].
3.
Bianchini, L.,
A. Kapus,
G. Lukacs,
S. Wasan,
S. Wakabayashi,
J. Pouyssegur,
F. H. Yu,
J. Orlowski,
and
S. Grinstein.
Responsiveness of mutants of NHE1 isoform of Na+/H+ antiport to osmotic stress.
Am. J. Physiol.
269 (Cell Physiol. 38):
C998-C1007,
1995[Abstract/Free Full Text].
4.
Bianchini, L.,
and
J. Pouyssegur.
Molecular structure and regulation of vertebrate Na+/H+ exchangers.
J. Exp. Biol.
196:
337-345,
1994[Abstract/Free Full Text].
5.
Borgese, F.,
C. Sardet,
M. Cappadoro,
J. Pouyssegur,
and
R. Motais.
Cloning and expression of a cAMP-activated Na+/H+ exchanger: evidence that the cytoplasmic domain mediates hormonal regulation.
Proc. Natl. Acad. Sci. USA
89:
6765-6769,
1992[Abstract].
6.
Boyarsky, G.,
M. Ganz,
R. Sterzel,
and
W. Boron.
pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO
3.
Am. J. Physiol.
255 (Cell Physiol. 24):
C844-C856,
1988[Abstract/Free Full Text].
7.
Busch, S.
Cloning and sequencing of the cDNA encoding for a Na+/H+ exchanger from Xenopus laevis oocytes (X1-NHE).
Biochim. Biophys. Acta
1325:
13-16,
1997[Medline].
8.
Cala, P. M.
Cell volume regulation by Amphiuma red blood cells. The role of Ca+2 as a modulator of alkali metal/H+ exchange.
J. Gen. Physiol.
82:
761-784,
1983[Abstract].
9.
Cala, P. M.
Volume regulation by Amphiuma red blood cells: the membrane potential and its implications regarding the nature of the ion flux pathways.
J. Gen. Physiol.
76:
683-708,
1980[Abstract/Free Full Text].
10.
Cala, P. M.,
S. E. Anderson,
and
E. J. Cragoe.
Na/H exchange-dependent cell volume and pH regulation and disturbances.
Comp. Biochem. Physiol. A Physiol.
90:
551-555,
1988.
11.
Cala, P. M.,
and
H. M. Maldonado.
pH regulatory Na/H exchange by Amphiuma red blood cells.
J. Gen. Physiol.
103:
1035-1053,
1994[Abstract].
12.
Cala, P. M.,
H. Maldonado,
and
S. E. Anderson.
Cell volume and pH regulation by the Amphiuma red blood cell: a model for hypoxia-induced cell injury.
Comp. Biochem. Physiol. A Physiol.
102:
603-608,
1992.
13.
Chen, C.,
and
H. Okayama.
High-efficiency transformation of mammalian cells by plasmid DNA.
Mol. Cell. Biol.
7:
2745-2752,
1987[Medline].
14.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
15.
Counillon, L.,
A. Franchi,
and
J. Pouyssegur.
A point mutation of the Na+/H+ exchanger gene (NHE1) and amplification of the mutated allele confer amiloride resistance upon chronic acidosis.
Proc. Natl. Acad. Sci. USA
90:
4508-4512,
1993[Abstract].
16.
Counillon, L.,
J. Noel,
R. A. Reithmeier,
and
J. Pouyssegur.
Random mutagenesis reveals a novel site involved in inhibitor interaction within the fourth transmembrane segment of the Na+/H+ exchanger-1.
Biochemistry
36:
2951-2959,
1997[Medline].
17.
Counillon, L.,
J. Pouyssegur,
and
R. A. F. Reithmeier.
The Na+/H+ exchanger NHE-1 possesses N- and O-linked glycosylation restricted to the first N-terminal extracellular domain.
Biochemistry
33:
10463-10469,
1994[Medline].
18.
Counillon, L.,
W. Scholz,
H. J. Lang,
and
J. Pouyssegur.
Pharmacological characterization of stably transfected Na+/H+ antiporter isoforms using amiloride analogs and a new inhibitor exhibiting anti-ischemic properties.
Mol. Pharmacol.
44:
1041-1045,
1993[Abstract].
19.
Cox, G. A.,
C. M. Lutz,
C. Yang,
D. Biemesderfer,
R. T. Bronson,
A. Fu,
P. S. Aronson,
J. L. Noebels,
and
W. N. Frankel.
Sodium/hydrogen exchanger gene defect in slow-wave epilepsy mutant mice.
Cell
91:
139-148,
1997[Medline].
20.
Fafournoux, P.,
J. Noel,
and
J. Pouyssegur.
Evidence that Na+/H+ exchanger isoforms NHE1 and NHE3 exist as stable dimers in membranes with a high degree of specificity for homodimers.
J. Biol. Chem.
269:
2589-2596,
1994[Abstract/Free Full Text].
21.
Feinberg, A. P.,
and
B. Vogelstein.
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.
Anal. Biochem.
132:
6-13,
1983[Medline].
22.
Felsenstein, J.
Inferring phylogenies from protein sequences by parsimony, distance, and likelihood methods.
Methods Enzymol.
266:
418-427,
1996[Medline].
23.
Harris, S. P.,
T. V. Strong,
N. Wys,
N. W. Richards,
J. Pouyssegur,
S. A. Ernst,
and
D. C. Dawson.
Epithelial localization of a reptilian Na+/H+ exchanger homologous to NHE-1.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G1594-G1606,
1997[Abstract/Free Full Text].
24.
Horvat, B.,
S. Taheri,
and
A. Salihagic.
Tumour cell proliferation is abolished by inhibitors of Na+/H+ and HCO
3/Cl
exchange.
Eur. J. Cancer
29A:
132-137,
1993.
25.
Huang, X. Y.,
A. D. Morielli,
and
E. G. Peralta.
Tyrosine kinase-dependent suppression of a potassium channel by the G protein-coupled m1 muscarinic acetylcholine receptor.
Cell
75:
1145-1156,
1993[Medline].
26.
Johnson, J. D.,
and
D. Epel.
Intracellular pH and activation of sea urchin eggs after fertilisation.
Nature
262:
661-664,
1976[Medline].
27.
Klanke, C. A.,
Y. R. Su,
D. F. Callen,
Z. Wang,
P. Meneton,
N. Baird,
R. A. Kandasamy,
J. Orlowski,
B. E. Otterud,
M. Leppert,
G. E. Shull,
and
A. G. Menon.
Molecular cloning and physical and genetic mapping of a novel human Na+/H+ exchanger (NHE5/SLC9A5) to chromosome 16q22.1.
Genomics
25:
615-622,
1995[Medline].
28.
Kyte, J.,
and
R. F. Doolittle.
A simple method for displaying the hydropathic character of a protein.
J. Mol. Biol.
157:
105-132,
1982[Medline].
29.
Matthews, C.,
B. Froman,
R. Carlsen,
and
F. A. Gorin.
Nerve-dependent factors regulating transcript levels of glycogen phosphorylase in skeletal muscle.
Cell. Mol. Neurobiol.
18:
319-338,
1998[Medline].
30.
Murer, H.,
U. Hopfer,
and
R. Kinne.
Sodium/proton antiport in brush border-membrane vesicles isolated from rat small intestine and kidney.
Biochem. J.
154:
597-604,
1976[Medline].
31.
Noel, J.,
and
J. Pouyssegur.
Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms.
Am. J. Physiol.
268 (Cell Physiol. 37):
C283-C296,
1995[Abstract/Free Full Text].
32.
Orlowski, J.
Heterologous expression and functional properties of amiloride high affinity (NHE-1) and low affinity (NHE-3) isoforms of the rat Na/H exchanger.
J. Biol. Chem.
268:
16369-16377,
1993[Abstract/Free Full Text].
33.
Orlowski, J.,
and
S. Grinstein.
Na+/H+ exchangers of mammalian cells.
J. Biol. Chem.
272:
22373-22376,
1997[Free Full Text].
34.
Orlowski, J.,
and
R. A. Kandasamy.
Delineation of transmembrane domains of the Na+/H+ exchanger that confer sensitivity to pharmacological antagonists.
J. Biol. Chem.
271:
19922-19927,
1996[Abstract/Free Full Text].
35.
Orlowski, J.,
R. A. Kandasamy,
and
G. E. Shull.
Molecular cloning of putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, and mRNA tissue expression of the rat Na/H exchanger NHE-1 and two structurally related proteins.
J. Biol. Chem.
267:
9331-9339,
1992[Abstract/Free Full Text].
36.
Piper, H. M.,
C. Balser,
Y. V. Ladilov,
M. Schafer,
B. Siegmund,
M. M. Ruiz,
and
D. D. Garcia.
The role of Na+/H+ exchange in ischemia-reperfusion.
Basic Res. Cardiol.
91:
191-202,
1996[Medline].
37.
Rotin, D.,
and
S. Grinstein.
Impaired cell volume regulation in Na+-H+ exchange-deficient mutants.
Am. J. Physiol.
257 (Cell Physiol. 26):
C1158-C1165,
1989[Abstract/Free Full Text].
38.
Saitou, N.,
and
M. Nei.
The neighbor-joining method: a new method for reconstructing phylogenetic trees.
Mol. Biol. Evol.
4:
406-425,
1987[Abstract].
39.
Sambrook, J.,
E. F. Fritsch,
and
T. Maniatis.
Selection of poly(A)+ RNA.
In: Molecular Cloning: A Laboratory Manual (2nd ed.). Plainview, NY: Cold Spring Harbor Laboratory Press, 1989, chapt. 7, p. 7.26-7.30.
40.
Sardet, C. S.,
A. Franchi,
and
J. Pouyssegur.
Molecular cloning, primary structure and expression of the human growth factor-activatable Na+/H+ antiporter.
Cell
56:
271-280,
1989[Medline].
41.
Schaefer, S.,
and
R. Ramasamy.
Short-term inhibition of the Na-H exchanger limits acidosis and reduces ischemic injury in the rat heart.
Cardiovasc. Res.
34:
329-336,
1997[Medline].
42.
Siffert, W.,
and
R. Dusing.
Na+/H+ exchange in hypertension and in diabetes mellitus: facts and hypotheses.
Basic Res. Cardiol.
91:
179-190,
1996[Medline].
43.
Soleimani, M.,
and
G. Singh.
Physiologic and molecular aspects of the Na+/H+ exchangers in health and disease processes.
J. Investig. Med.
43:
419-430,
1995[Medline].
44.
Thomas, R. C.
The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurons.
J. Physiol. (Lond.)
273:
317-338,
1977[Medline].
45.
Tse, C. M.,
S. A. Levine,
C. H. Yun,
S. R. Brant,
J. Pouyssegur,
M. H. Montrose,
and
M. Donowitz.
Functional characteristics of a cloned epithelial Na+/H+ exchanger (NHE3): resistance to amiloride and inhibition by protein kinase C.
Proc. Natl. Acad. Sci. USA
90:
9110-9114,
1993[Abstract].
46.
Tse, C. M.,
S. A. Levine,
C. H. C. Yun,
M. H. Montrose,
P. Little,
J. Pouyssegur,
and
M. Donowitz.
Cloning and expression of a rabbit cDNA encoding a serum-activated ethylisopropyl-amiloride-resistant epithelial Na+/H+ exchanger isoform (NHE-2).
J. Biol. Chem.
268:
11917-11924,
1993[Abstract/Free Full Text].
47.
Von Heijne, G.
A new method for predicting signal sequence cleavage sites.
Nucleic Acids Res.
14:
4683-4690,
1986[Abstract].
48.
Wakabayashi, S.,
B. Bertrand,
M. Shigekawa,
P. Fafournoux,
and
J. Pouyssegur.
Growth factor activation and "H(+)-sensing" of the Na+/H+ exchanger isoform 1 (NHE1). Evidence for an additional mechanism not requiring direct phosphorylation.
J. Biol. Chem.
269:
5583-5588,
1994[Abstract/Free Full Text].
49.
Wakabayashi, S.,
P. Fafournoux,
C. Sardet,
and
J. Pouyssegur.
The Na+/H+ antiporter cytoplasmic domain mediates growth factor signals and controls H+-sensing.
Proc. Natl. Acad. Sci. USA
89:
2424-2428,
1992[Abstract].
50.
Wakabayashi, S.,
M. Shigekawa,
and
J. Pouyssegur.
Molecular physiology of vertebrate Na+/H+ exchangers.
Physiol. Rev.
77:
51-74,
1997[Abstract/Free Full Text].
51.
Wang, D.,
D. F. Balkovetz,
and
D. G. Warnock.
Mutational analysis of transmembrane histidines in the amiloride-sensitive Na+/H+ exchanger.
Am. J. Physiol.
269 (Cell Physiol. 38):
C392-C402,
1995[Abstract/Free Full Text].
52.
Yun, C. H.,
C. M. Tse,
S. K. Nath,
S. A. Levine,
S. R. Brant,
and
M. Donowitz.
Mammalian Na+/H+ exchanger gene family: structure and function studies.
Am. J. Physiol.
269 (Gastrointest. Liver Physiol. 32):
G1-G11,
1995[Abstract/Free Full Text].
Am J Physiol Cell Physiol 276(5):C1025-C1037
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society