Identification by Differential Display of a
Hypertonicity-inducible Inward Rectifier Potassium Channel Highly
Expressed in Chloride Cells*
Yoshiro
Suzuki
,
Makoto
Itakura,
Masahide
Kashiwagi,
Nobuhiro
Nakamura,
Taizo
Matsuki,
Hidenari
Sakuta,
Nobuko
Naito§,
Koji
Takano¶,
Toshiro
Fujita¶, and
Shigehisa
Hirose
From the Department of Biological Sciences, Tokyo Institute of
Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, the
§ Showa University College of Medical Sciences,
1865 Tokaichiba-cho, Midori-ku, Yokohama 226-8555, and the
¶ Fourth Department of Internal Medicine, University of Tokyo
School of Medicine, Tokyo 112-0015, Japan
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ABSTRACT |
By using differential mRNA display to monitor
the molecular alterations associated with adaptation of euryhaline eels
to different salinities, we identified a cDNA fragment strongly
induced in seawater eel gills. Cloning of a full-length cDNA and
its expression in COS-7 cells indicated that the clone codes for an
inward rectifier K+ channel (eKir) of 372 amino acid
residues, which has two transmembrane segments and a typical
pore-forming region (H5). Only low sequence similarities are present,
except the H5 region, compared with other members of the inward
rectifier K+ channel family (Kir). Consistent with this
divergence in the amino acid sequence, a phylogenetic analysis
indicated early divergence and independent evolution of eKir from other
members; it is only distantly related to the Kir5.0 subfamily members.
RNase protection analysis showed that eKir is highly expressed in the
seawater eel gill, kidney, and posterior intestine but very weakly in
freshwater eels. Immunohistochemistry of gill sections revealed dense
localization of eKir in the chloride cells. Immunoelectron microscopy
indicated that eKir is mainly present in the microtubular system in the chloride cell. This location and its salt-inducible nature suggest that
the eKir channel cloned here is a novel member of the Kir5.0 subfamily
of the Kir family and is implicated in osmoregulation.
 |
INTRODUCTION |
Euryhaline fishes such as eel, salmon, tilapia, and flounder can
live both in freshwater and in seawater. To tolerate a wide range of
salinities and move between freshwater and seawater, they developed
special structures and mechanisms for osmoregulation, one of which is
the chloride cells in the gill. The chloride cells or the ionocytes are
rich in mitochondria and actively absorb salt in freshwater and pump
out excess salt in seawater to help maintain body fluid homeostasis.
Understanding the molecular basis of these extraordinary abilities of
euryhaline species has long been a major goal in animal physiology.
Previous studies were mainly focused on the morphological,
electrophysiological, and hormonal aspects. Although such conventional
approaches have revealed a number of interesting facts concerning the
osmoregulatory processes, detailed mechanisms and the molecules
involved remain to be clarified. We employed, in the present study, the
recently developed RNA arbitrary primed polymerase chain reaction
(RAP-PCR)1 to identify the
mRNA species whose expressions are regulated by the environmental
salinities. RAP-PCR is a method closely related to the differential
display method that provides an attractive tool for the isolation of
differentially expressed genes (1, 2). Our attempt using mRNA
preparations from freshwater and seawater eels resulted in
identification of several mRNA species that exhibit markedly
altered expression. One of them was, as detailed below, a
K+ channel that belongs to the inward rectifier
K+ channel family and shows a unique tissue distribution as
being confined to osmoregulatory organs such as the gill, kidney, and intestine.
K+ channels are a diverse group of membrane proteins.
Recent molecular cloning has established the presence of two
structurally and functionally distinct families of the channel as
follows: 1) the voltage-gated K+ channels (Kv) that have
six transmembrane spans within each subunit and are activated by
depolarization in a steeply voltage-dependent manner, and
2) the inward rectifier K+ channels (Kir) that have only
two transmembrane segments and conduct inward current more readily than
outward current. Although the members of these K+ channel
families are different in their subunit structures,
electrophysiological properties, and mechanisms of activation, they are
considered to be assembled with multiple (probably four) homologous or
heterologous subunits to make up the K+-selective pore
(3-6). All of the channel subunits cloned so far including those of
animals (7-10) and even microorganisms (11) share a highly conserved
sequence of about 17 amino acid residues called the pore region or H5,
which has been shown to be part of the K+-selective pore
(12-14). The presence of this H5 sequence is therefore considered the
signature of a K+ channel protein.
Although the function of the voltage-gated K+ channels is
relatively simple and well established as being responsible for action potential repolarization and frequency encoding, the function of the
inward rectifier K+ channels is quite diverse. The inward
rectifier K+ channels are found in a wide variety of
tissues and cell types where they are involved not only in the
maintenance of the resting membrane potential and control of cell
excitability but also in processes not usually associated with
electrically excitable membranes such as hormone secretion,
K+ excretion, and buffering extracellular K+
concentrations or recycling K+. Several inward rectifier
K+ channels have recently been cloned from a number of
tissues and species by expression cloning (7, 8), low stringency
hybridization (15, 16), and PCR methods (10, 17, 18). Their sequencing and characterization have demonstrated structural and functional diversities of the channel proteins, and currently the molecular determinants of rectification are being identified. The next step will
be to clarify the physiological significance of the channel subtypes.
Here we report cloning of a unique member of the Kir family whose
expression is strongly induced in eels, especially in the chloride
cells of the gill, during adaptation to seawater and therefore
considered to play an essential role in osmoregulation.
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EXPERIMENTAL PROCEDURES |
Differential Display--
Differential display was carried out
as described (1, 19). Eels were adapted to seawater or freshwater for 2 weeks. Total RNA was prepared from the seawater- and freshwater-adapted
eel gills by the guanidinium thiocyanate/cesium chloride method (20), and mRNA was prepared using an oligo(dT)-cellulose column (Amersham Pharmacia Biotech) and then converted to cDNA as described (1) by a
single arbitrary primer. PCR was performed using 5 ng of cDNA, 1 µM same arbitrary primer, 0.5 mM dNTPs, and
2.5 units of Taq polymerase (Takara). The mixture was cycled
first at 94 °C for 1 min, 36 °C for 5 min, and 72 °C for 5 min
followed by 40 cycles at 94 °C for 1 min, 60 °C for 2 min, and
72 °C for 2 min. An aliquot of each amplification mixture was
subjected to electrophoresis in a 7.5% polyacrylamide gel, and DNA was
visualized by ethidium bromide staining. Differentially expressed bands
of interest were extracted from the gel and reamplified and then cloned
into pBluescript II vector (Stratagene). DNA sequence analysis from
both strands was performed using a SequiTherm cycle sequencing kit
(Epicentre Technologies). The DNA sequence was compared with the
GenBankTM/EMBL/DDBJ data bases using the BLAST network
service at the National Center for Biotechnological Information.
Northern Blot Analysis--
Ten micrograms of
poly(A)+ RNA from freshwater and seawater-adapted eel gills
were separated on 1.2% agarose-formaldehyde gel in 2.2 M
formamide, 20 mM Mops, 8 mM sodium acetate, and
1 mM EDTA, pH 7.0, and then transferred to a nylon membrane
(MagnaGraph, Micron Separations Inc.). The eKir cDNA was
32P-labeled by random priming and hybridized to the RNA
filters in 50% formamide, 5× SSPE, 2× Denhardt's solution, 0.5%
SDS, and 0.1 mg/ml herring sperm DNA at 42 °C overnight. After
hybridization, the filter was briefly washed in 2× SSC (SSC, 0.15 M NaCl, 50 mM sodium citrate), 0.1% SDS at
room temperature and twice with 0.1× SSC with 0.1% SDS at 65 °C
for 2 h. The filter was exposed to Kodak X-Omat film at
80 °C.
cDNA Library Screening--
The eel gill cDNA library in
ZAP II (Stratagene) was prepared as described (21). The library was
plated out at a density of 3 × 104 plaque-forming
units/150-mm plate. Phage plaques were lifted onto nitrocellulose
filters (Schleicher & Schuell), and the filters were prehybridized for
2 h at 42 °C in a solution containing 50% formamide, 5× SSPE
(SSPE, 0.15 M NaCl, 1 mM EDTA, and 10 mM NaH2PO4, pH 7.4), 0.1% SDS, 5×
Denhardt's solution, and 0.1 mg/ml herring sperm DNA. The probe was
labeled with [
-32P]dCTP (Amersham Pharmacia Biotech)
using random primers. Hybridization was performed for 16 h at
42 °C. To identify positive clones, filters were washed and then
exposed to Kodak X-Omat film at
80 °C overnight with intensifying
screens. Positive plaques were isolated and rescreened after dilution.
Conditions for secondary and tertiary screening were identical to
primary screening.
Cloning of Eel Kir Family cDNA--
Polymerase chain
reaction (PCR) was performed with degenerated primers deduced from
those highly conserved among Kir channels. Primer
5'-ACCAT(C/T)GGCTA(C/T)GG(A/C/T)TTC(A/C)G-3' corresponds to the amino
acid sequence TIGYG(F/Y)R, and the following were used as antisense
primers: 5'-TGA(A/G)TAGTCCAC(C/T)TTGTAGT-3', 5'-TAGGA(A/C/G)GTGCG(A/C/G)ACCTGGCA-3',
5'-GTAGCCCCA(A/C/G)AGGATCTCCT-3', and
5'-TCCACCAT(C/G)CCCTC(C/T)AG(A/G)AT-3'. The PCR conditions were as
follows: 1 min at 94 °C, 1 min at 55 °C, and 2 min at 72 °C,
35 cycles. The amplified fragments were subcloned into pBluescript II
vector, and their sequences were determined.
Cell Culture and Plasmid Transfection--
COS-7 cells were
grown in Dulbecco's modified Eagle's medium supplemented with 10%
(v/v) fetal bovine serum, 10 mM Hepes, penicillin (50 units/ml), and streptomycin (50 µg/ml) at 37 °C in 5%
CO2. The cloned cDNA was introduced into the
pcDL-SR
296 vector and transfected to the COS-7 cells using DOSPER
liposomal transfection reagent (Boehringer Mannheim). Two days after
the transfection, the cells were used for electrophysiological experiments.
Electrophysiology--
For electrophysiological experiments, the
whole-cell variation of the patch clamp technique (22) was employed.
The EPC-9 amplifier (HEKA) was used to record the membrane current. The patch electrode solution contained (in mM) 95 potassium
aspartate, 40 KCl, 1 MgCl2, 5 EGTA (K salt) and 20 Hepes
(sodium salt, pH 7.4). The electrode resistance ranged between 3 and 5 megohms. The composition of the extracellular solution was (in
mM) 140 KCl, 1 MgCl2, 10 Hepes (sodium salt, pH
7.4). Extracellular solutions containing 40 mM
K+ and 20 mM K+ were made by
substituting KCl with isoosmotic NaCl.
RNase Protection Assay--
A 590-bp PCR fragment was subcloned
into the pBluescript II vector and linearized by digestion with
BstPI and was used as a template to generate a
300-nucleotide antisense RNA probe. In vitro transcription
was carried out using an RNA transcription kit (Stratagene) with
[32P]rUTP. Template DNA was then digested with RNase-free
DNase I. The radiolabeled probe (105 cpm/sample) was mixed
with 10 µg of total RNA from various eel tissues and hybridized
overnight in 80% formamide, 40 mM Pipes, pH 6.4, 1 mM EDTA, and 0.4 M NaCl. RNase digestion was
performed for 2 h at 37 °C with a mixture of RNase A (40 µg/ml) and RNase T1 (2 µg/ml). Protected fragments were
electrophoresed on 5% polyacrylamide, 8 M urea denaturing
gels and visualized by autoradiography.
Expression and Purification of MBP-eKir Fusion Protein--
To
express the eKir-MBP (maltose-binding protein) fusion protein, a 590-bp
fragment of eKir (nucleotides 544-1135) was prepared by PCR and
subcloned into the pMAL-p vector (New England Biolabs). Escherichia coli strain XL1-Blue harboring the recombinant
plasmid was grown in TB (Terrific Broth), induced with 0.1 mM isopropyl-1-thio-b-D-galactopyranoside for 2 h, and harvested by centrifugation. After cell lysis, the lysate was centrifuged, and the supernatant was subjected to affinity chromatography on amylose resin as described by the manufacturer.
Preparation of Antiserum and Western Blotting--
Antiserum to
eKir was raised in a Japanese White rabbit. The MBP-eKir fusion protein
was emulsified with Freund's adjuvant and injected several times.
Western blotting was used to establish the specificity of the
antiserum. Freshwater and seawater eel gills were homogenized in 100 mM Tris buffer containing 0.9% NaCl, 0.2 mM
phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, and 10 µg/ml
leupeptin. The homogenates were centrifuged at 5000 × g for 20 min, and the pellets were resuspended in the same
buffer. These procedures were repeated three times at 4 °C. The
membrane proteins were separated by SDS-polyacrylamide gel
electrophoresis and transferred to a membrane. After blocking in the
buffer containing 5% nonfat milk, the membrane was incubated with
anti-eKir antiserum (1:3000). The channel-antibody complexes were
visualized using alkaline phosphatase-conjugated secondary antibody
with substrates for alkaline phosphatase.
Immunohistochemistry--
Frozen sections were cut in a
cryostat, washed in phosphate-buffered saline (100 mM NaCl,
10 mM NaH2PO4, pH 7.4), and fixed in acetone containing 5% H2O2 to inactivate
endogenous peroxidase. These sections were stained using the
avidin-biotin complex (ABC) method. After an overnight incubation with
anti-eKir antiserum (1:1000), they were incubated with biotinylated
goat anti-rabbit IgG (1:500). The bound antibodies were visualized
using 3,3'-diaminobenzidine tetrahydrochloride and 0.02%
H2O2 in 50 mM Tris-HCl, pH 7.4.
Immunoelectron Microscopy--
Eels, adapted in seawater for 14 days, were perfused under anesthesia through the ventral aorta with a
fixative containing 4% paraformaldehyde in phosphate-buffered saline,
pH 7.4. After the perfusion, gill arches were dissected out and
immersed for 3 h in the same fixative at ice-cold temperature and
rinsed in phosphate-buffered saline containing 8% sucrose. The fixed
tissues were cryoprotected through a range of increasing sucrose
concentrations up to 30%, quick frozen, and cut on a cryostat at 10 µm. The eKir on the sections was detected by use of the pre-embedding
1-nm gold particle-silver enhancement method (23) slightly modified as
described (24). Briefly, sections were incubated for 48 h with the
anti-eKir (1:500) at 4 °C and then incubated overnight with a goat
anti-rabbit IgG labeled with 1-nm gold particles (1:100) (Nanogold,
Nanoprobes Inc.) at 4 °C. Tissue-bound gold particles were enhanced
by incubation with a silver developer (HQ silver, Nanoprobes Inc.) at
18 °C for 10 min in the dark. The sections were postfixed with
osmium tetroxide, dehydrated through ethanol, and embedded in epoxy
resin. Ultrathin sections were stained with uranyl acetate and lead citrate.
 |
RESULTS |
Identification of Differentially Expressed Genes by
RAP-PCR--
In an attempt to isolate genes that may be responsible
for freshwater or seawater adaptation of euryhaline fishes, we carried out RAP-PCR, a recently developed technique for detecting altered gene
expression, using poly(A)-rich RNA preparations obtained from
freshwater- and seawater-adapted eel gills. This method is based on the
use of arbitrary primers to generate fingerprints by polymerase chain
reaction from closely matched RNA populations. The RAP-PCR products
were separated by agarose gel electrophoresis and compared for
differences in their band intensities. Although both mRNA samples
showed very similar display patterns, a number of bands were affected
by the changes in salinity. We therefore isolated, from among the
differentially displayed bands, the following three major ones and
characterized them as follows: a band of about 220 bp that was
amplified to a greater degree in freshwater samples (FW220) and two
seawater-specific bands of about 250 and 300 bp (SW250 and SW300). DNA
sequencing followed by a data base search using BLAST indicated that
the major component of FW220 is an eel homolog of P450 (~70%
similar); SW250 represents Rho-type GTPase-activating protein
(p190RhoGAP, ~70% similar to mammalian homologs), a potent GTPase
activator for various Rho-type GTPases, and SW300, an inward rectifier
K+ channel (eKir). Fish P450s have been demonstrated to
undergo marked up-regulation in response to water pollution regardless of osmotic environments (25); therefore, we did not analyze the
FW220-derived clone any further. Gross morphological changes of the
chloride cells in the gill have been shown to occur when euryhaline
fishes face osmotic challenges (26), and the control of the actin
cytoskeleton and cell morphology appear to be mediated by members of
the Rho family (27, 28). In this context, the p190RhoGAP clone
contained in the SW250 band seemed to be very interesting, but its
characterization will be the subject of another report. The third
clone, termed eKir, was selected for further characterization.
The expression pattern of eKir observed in the differential mRNA
display was confirmed by Northern blot analysis (Fig.
1). A single eKir mRNA species of 2 kilobase pairs was detected, which was strongly induced in the gill of
seawater eels.

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Fig. 1.
Differential expression of eKir mRNA in
gills of seawater- and freshwater-adapted eels. Northern blot
analysis was performed using seawater- and freshwater-adapted eel
mRNA. Poly(A)+ RNA from seawater and freshwater eel
gills were electrophoresed on a 1.2% agarose gel, transferred to a
nylon membrane, and hybridized with an eKir 32P-labeled
cDNA probe. Positions of 28 S and 18 S ribosomal RNAs are as
noted in figure. Hybridization to an eel -actin probe (corresponding
to nucleotides 206-343 in rat sequence) demonstrated equal loading of
the lanes. FW, freshwater eels; SW, seawater
eels.
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Cloning and Sequence Analysis of Full-length eKir--
On Northern
blot analysis, the SW300-derived eKir probe hybridized to a 2-kilobase
pair transcript. To isolate eKir cDNA clones of this size, we
constructed a seawater eel gill cDNA library in the
ZAP II
vector and obtained 3 positive clones by screening 2 × 105 recombinants. The nucleotide sequence of the longest
clone, consisting of 1692 bp, is shown in Fig.
2 together with the deduced amino acid
sequence. The open reading frame encodes a 372-amino acid protein that
has two putative membrane-spanning hydrophobic segments, M1 and M2, and
shares significant sequence conservation in the pore-forming H5 region
with previously cloned inward rectifier K+ channels (7-9,
15, 18) (Fig. 3). Overall similarities to other inward rectifier K+ channels are, however, very low.
No N-terminal hydrophobic signal sequence is present as in the cases of
the other members of the Kir family, favoring a topology with a
cytoplasmic N terminus. There is no potential externally faced
N-glycosylation site such as detected in some members
(29).

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Fig. 2.
Nucleotide and deduced amino acid sequences
of eKir cDNA. The nucleotide sequence of the longest clone and
its amino acid sequence are shown. Numbers to the
left refer to the first amino acids on the lines, and the
numbers to the right refer to the last
nucleotides on the lines. Two putative membrane-spanning regions
(M1 and M2) and pore-forming region (H5) are
underlined. Potential phosphorylation sites for protein
kinase C are indicated by #, and putative polyadenylation
signals are boxed. The DDBJ/EMBL/GenBankTM
accession number is AB009669.
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Fig. 3.
Sequence identity between the eKir amino acid
sequence and four members of the inward rectifier K+
channel family. The aligned sequences are eKir, rat channel
subunit BIR9 (Kir5.1) (17), mouse IRK1 (Kir2.1) (8), mouse GIRK1
(Kir3.1) (33), and rat ROMK1 (Kir1.1a) (7). Gaps are
inserted to achieve maximum similarity. Amino acid identities are
indicated by background shading.
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Fig. 4 shows a phylogenetic tree of the
known members of the Kir family. The position of the eKir in the tree
is far from those of the other members (~51% similarity). It seems
reasonable, therefore, to assume that the eKir cloned here is a
previously unreported member of the Kir family that diverged early
during evolution from the Kir5.0 subfamily. However the possibility
remains that the sequence divergence is simply due to the species
difference. We therefore cloned, by polymerase chain reaction (PCR),
eel homologs of the Kir family members and compared their sequences
with those of the mammalian counterparts. The eel homologs of Kir1.3,
Kir2.1, Kir3.4, and Kir4.1 (eKir1.3, eKir2.1, eKir3.4, and
eWIRK in Fig. 4) share a high degree of similarity with
their mammalian counterparts (70-90%, Fig. 4) despite the
evolutionary distance between the eel and mammals, supporting our
assumption that eKir represents a new member of the Kir5.0
subfamily.

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Fig. 4.
Phylogenetic tree of the members of the
inward rectifier K+ channel family. A phylogenetic
tree was constructed by comparing the amino acid sequences of inward
rectifier K+ channel (residues 144-296 of eKir) using the
neighbor-joining method. The sequence of rat voltage-gated
K+ channel Kv1.3 was used as an outgroup. The
GenBankTM accession numbers of the compared proteins are as
follows: hKir1.1 (U03884), hKir1.2/4.1 (U73193),
hKir1.3 (U73191), sWIRK (D83537),
hKir2.1 (U12507), hKir3.1 (U50964),
hKir3.4 (L47208), XIR (U42207),
rKir6.1 (D42145), rKir6.2 (D86039),
rKir5.1 (X83581), rKir7.1 (AB013890), and
rKv1.3 (X16001). The tree also reveals seven clusters of the
channel proteins Kir1.0- Kir7.0. The members of a cluster are also
more similar to each other than to non-cluster members in the function
and mechanisms of activation and regulation; for example, the members
of the Kir1.0, Kir4.0, and Kir6.0 subfamilies are likely to be
ATP-sensitive; the Kir2.0 subfamily members are mainly localized in the
excitatory cells; the Kir3.0 subfamily members are G protein-coupled
channels; the Kir5.0 is inactive by itself but becomes active when
coexpressed with Kir4.0 probably because of formation of a
heteromultimer (6); and our eKir, proposed to be a new member of Kir5.0
subfamily appears to be a channel implicated in osmoregulation.
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Immunochemical Characterization of eKir--
An antiserum was
raised against eKir produced in E. coli as a fusion protein
(MBP-eKir). On Western blot analysis of seawater eel gill membrane
proteins, the antiserum stained a band of 42 kDa (Fig.
5, lane 2). No bands were
detected in freshwater eel gill (lane 1). The 42-kDa band
may therefore represent eKir whose calculated molecular mass is 41 kDa.
The identity of the band was further confirmed using eKir expressed in
mammalian cells. When extracts of COS-7 cells transfected with the
expression construct SR
296-eKir were subjected to Western blot
analysis, a single band of 42 kDa was detected (Fig. 5, lane
3), which was not present in mock-transfected cells (lane
4).

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Fig. 5.
Western blot analysis of the eKir
protein. Eel gill membrane proteins were electrophoresed and
blotted to polyvinylidene difluoride membrane and stained with
antiserum against recombinant eKir. A 42-kDa band was observed in
seawater eel (lane 2) but not in freshwater (lane
1). This antiserum recognized a 42-kDa protein in COS-7 cells
transfected with the eKir expression construct (lane 3) but
not in vector-transfected cells (lane 4). Positions of
molecular mass markers are shown on the left.
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Characterization of eKir by Functional Expression in COS-7
Cells--
The electrophysiological properties of eKir were determined
by expressing the cloned channel protein in COS-7 cells using pcDL-SR
296 as a vector and cationic liposome as a transfection reagent. pEGFP-N2 (CLONTECH) was co-transfected to
select cells for recordings. The expression levels of eKir were
assessed by Western blot analysis (Fig. 5). Fig.
6A shows the membrane currents from an eKir-transfected cell (left) and a cell transfected
with the mock vector (right) under voltage clamp in the
extracellular solution containing 140 mM K+.
The eKir-transfected cells produced large inward currents
(n = 10) that were not seen in mock-transfected cells
(n = 10). Almost identical currents were seen in
eKir-transfected GH3 cells (data not shown). The
current-voltage (I-V) relationship of an eKir current is plotted in
Fig. 6B (closed circles). This current had sensitivity to Ba2+ (1 mM, closed
squares) and Cs+ (1 mM, closed
triangles), as described previously for inward rectifier
K+ channel (Kir) family members (7, 8). Fig. 6C
shows the extracellular K+ dependence of the reversal
potentials. These reversal potentials were close to the equilibrium
potentials of K+ predicted by the Nernst's equation which
suggests a high selectivity for K+. These data indicate
that the eKir constitutes a K+ channel that belongs to the
Kir family.

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Fig. 6.
Characterization of eKir. A,
membrane currents from an eKir-transfected cell (left) and
mock-transfected cell (right) under voltage clamp in the
extracellular solution containing 140 mM K+.
The membrane currents were evoked by pulse steps to +20, 0, 20, 40,
60, 80, 100, 120 mV from the holding potential of 0 mV.
B, I-V relationships of the membrane current from the
eKir-transfected cell in 140 mM K+
(closed circles) and in the presence of 1 mM
Ba2+ (closed squares) and 1 mM
Cs+ (closed triangles). C, the
extracellular K+ dependence of the reversal potentials.
Potassium was replaced by equimolar sodium, such that
[K+]o + [Na+]o = 140 mM.
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Tissue Distribution and Time Course of Induction--
To determine
the tissue distribution of eKir mRNA and to compare its expression
levels in freshwater and seawater eels, we performed RNase protection
analysis using total RNA preparations from various tissues of
freshwater and seawater eels including the gill, brain, heart, liver,
stomach, posterior intestine, anterior intestine, kidney, and head
kidney. A strong signal was detected in the gill, posterior intestine,
and kidney but not in the other tissues examined (Fig.
7). Comparison between the freshwater and seawater eel samples indicated that there was a 3-5-fold increase in
the eKir mRNA expression during adaptation to seawater.

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Fig. 7.
eKir mRNA levels in various eel tissues
under different salinity conditions. Eels were adapted to seawater
or freshwater for 2 weeks, and total RNA were isolated from the eels,
and then RNase protection analysis was performed as described under
"Experimental Procedures". FW, freshwater-adapted eels;
SW, seawater-adapted eels.
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Fig. 8 shows the time course of the
induction of eKir mRNA expression in the gill following transfer of
eels from freshwater to seawater. The changes occurred over a time
course of hours to days.

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Fig. 8.
Time course of eKir expression in eel gills
under the conditions of hypertonic stress. Freshwater eels were
adapted to seawater and their RNA were isolated at the time indicated
in figure. RNase protection assay was carried out as described under
"Experimental Procedures." Two eels were processed separately at
each time point; each lane, therefore, represents mRNA from one
eel.
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Cellular and Subcellular Localization of eKir in the Gill--
On
immunohistochemistry, the anti-eKir antiserum specifically stained
chloride cells that are located near the basal regions of the secondary
lamella (Fig. 9). Chloride cells have a
complex microtubular system formed by extensive invaginations of the
basolateral plasma membranes that are heavily laden with channels and
transporters such as Na+,K+-ATPase (30). To
examine further the subcellular localization of eKir in the chloride
cells, we performed immunoelectron microscopy on seawater eel gill
sections using colloidal gold. Immunogold labeling was found in the
microtubular system (Fig. 10),
indicating that eKir is mainly present in the microtubular system of
the chloride cells.

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Fig. 9.
Immunohistochemical localization of eKir in
seawater adapted eel gill. Sections of gill were stained with
anti-eKir antiserum (A) or preimmune serum (B) at a 1:1000
dilution. Chloride cells were stained with antiserum but not with
preimmune serum. CC, chloride cells; SL,
secondary lamella; Ca, cartilage. Scale bars
represent 50 µm.
|
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Fig. 10.
Immunocytochemistry of eKir in an eel
chloride cell by the immunogold method. The gold particles
representing eKir are seen as black dots around the tubular
system, specifically located intracellular side (× 35,000).
M, mitochondria; T, tubular system.
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|
 |
DISCUSSION |
Identification of the genes that are specifically expressed in
seawater eels but not in freshwater eels, or vice versa, is important
for understanding the molecular basis of the osmoregulation of
euryhaline fishes. In the present study, we applied the RAP-PCR technique to compare mRNA species from freshwater and seawater eel
gills, and we identified several differentially expressed cDNA
bands, one of which was fully characterized and shown to be an inward
rectifier K+ channel (eKir) highly expressed in the
osmoregulatory chloride cells in the gill. The amino acid sequence of
the channel protein is quite different from those of the currently
known Kir family members; the sequence identity is less than 51% when
compared with any of the members. Phylogenetic analysis suggested early divergence of the eKir channel from the other members (Fig. 4). The
sequence divergence appears not to be due to the species difference since other members of the eel, salmon, and chick Kir families (eel
Kir1.3, eel Kir2.1, eel Kir3.4, eel and salmon WIRK (16), and chick
cIRK1 (10)) share more than 70% sequence identity with the
corresponding mammalian homologs (Fig. 4). Based on these facts,
therefore, we proposed that eKir is a new member of the subfamily
Kir5.0. This conclusion is supported by its unique tissue distribution
and inducibility; the eKir channel is confined to the gill, kidney, and
posterior intestine, and its message levels are increased dramatically
in response to osmotic challenge. Such distributions and inductions are
not displayed by its predecessors.
The three locations of eKir (the gill, kidney, and intestine) are the
major organs involved in osmoregulation. These locations, particularly
the microtubular localization of eKir in the chloride cell (Fig. 10),
may provide an important clue regarding its physiological roles. Since
chloride cells are rich in Na+,K+-ATPase
reflecting their extraordinary power of ion transport, eKir cloned here
may be an osmoregulatory component working cooperatively with
Na+,K+-ATPase, for example, by recycling
K+. The localization in the posterior intestine but not in
the anterior intestine is also particularly interesting since, to date,
no significant difference in osmoregulatory roles has been found between the anterior and posterior parts of the intestine (31) that are
divided by the presence of a constriction due to a sphincter muscle.
Our finding is therefore expected to provide new avenues for further
research on the intestinal fluid and electrolyte transport.
Osmoregulatory mechanisms of euryhaline fishes are known to be under
endocrine control; for example, prolactin is the predominant osmoregulatory hormone in freshwater adaptation, and cortisol serves
this function in seawater adaptation. In euryhaline fishes, these
hormones seem to play a preparatory role and trigger the operation of
the osmoregulatory system to the appropriate direction since their
surge in serum levels precedes freshwater or seawater entry (32). In
contrast, the inward rectifier potassium channel eKir appears to be
downstream of these hormonal factors and plays an essential role for
the subsequent survival in seawater as suggested by its time course of
induction (Fig. 8). Our identification of an inducible channel protein,
eKir, on which euryhaline fishes are supposed to depend in the seawater
milieu may serve as a molecular clue for clarifying the molecular basis
for seawater adaptation. Future studies should reveal how hypertonicity
is recognized and how the recognition is converted to the activation of
the eKir gene as well as the physiological function of the channel in
association with other channels and transporters.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Yoshio Takei (Ocean Research
Institute, University of Tokyo, Japan) and Hiromi Hagiwara for
discussion and Setsuko Satoh and Kazuko Tanaka for their secretarial
and technical assistance. We also thank Dr. Yutaka Takebe (National
Institute of Health, Japan) for providing the pcDL-SR
296 expression vector.
 |
FOOTNOTES |
*
This work was supported in part by grants-in-aid for
scientific research from the Ministry of Education, Science, Sport and Culture of Japan, a research grant for cardiovascular diseases from the
Ministry of Health and Welfare of Japan, and an SRF grant for
Biomedical Research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB009669.
Supported by Research Fellowship for Young Scientists from the
Japan Society for the Promotion of Science.
To whom correspondence should be addressed. Tel.:
81-45-924-5726; Fax: 81-45-924-5824; E-mail:
shirose{at}bio.titech.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
RAP-PCR, RNA
arbitrary primed polymerase chain reaction;
eKir, eel inward rectifier
K+ channel;
Kv, voltage-gated K+ channel;
Kir, inward rectifier K+ channel;
MBP, maltose-binding protein;
bp, base pair(s);
Pipes, 1,4-piperazinediethanesulfonic acid;
Mops, 4-morpholinepropanesulfonic acid.
 |
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