Identification by Differential Display of a Hypertonicity-inducible Inward Rectifier Potassium Channel Highly Expressed in Chloride Cells*

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.

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 ϩ channel that belongs to the inward rectifier K ϩ channel family and shows a unique tissue distribution as being confined to osmoregulatory organs such as the gill, kidney, and intestine.
K ϩ 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 ϩ 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 ϩ channels (Kir) that have only two transmembrane segments and conduct inward current more readily than outward current. Although the members of these K ϩ 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 ϩ -selective pore (3)(4)(5)(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 ϩ -selective pore (12)(13)(14). The presence of this H5 sequence is therefore considered the signature of a K ϩ channel protein.
Although the function of the voltage-gated K ϩ channels is relatively simple and well established as being responsible for action potential repolarization and frequency encoding, the function of the inward rectifier K ϩ channels is quite diverse. The inward rectifier K ϩ 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 ϩ excretion, and buffering extracellular K ϩ concentrations or recycling K ϩ . Several inward rectifier K ϩ 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.

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 M 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 Se-quiTherm cycle sequencing kit (Epicentre Technologies). The DNA sequence was compared with the GenBank TM /EMBL/DDBJ data bases using the BLAST network service at the National Center for Biotechnological Information.
Northern Blot Analysis-Ten micrograms of poly(A) ϩ 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 32 P-labeled by random priming and hybridized to the RNA filters in 50% formamide, 5ϫ SSPE, 2ϫ 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 2ϫ SSC (SSC, 0.15 M NaCl, 50 mM sodium citrate), 0.1% SDS at room temperature and twice with 0.1ϫ SSC with 0.1% SDS at 65°C for 2 h. The filter was exposed to Kodak X-Omat film at Ϫ80°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 ϫ 10 4 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, 5ϫ SSPE (SSPE, 0.15 M NaCl, 1 mM EDTA, and 10 mM NaH 2 PO 4 , pH 7.4), 0.1% SDS, 5ϫ Denhardt's solution, and 0.1 mg/ml herring sperm DNA. The probe was labeled with [␣-32 P]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 Ϫ80°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. 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 g/ml) at 37°C in 5% CO 2 . 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 wholecell 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 MgCl 2 , 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 MgCl 2 , 10 Hepes (sodium salt, pH 7.4). Extracellular solutions containing 40 mM K ϩ and 20 mM K ϩ 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 [ 32 P]rUTP. Template DNA was then digested with RNase-free DNase I. The radiolabeled probe (10 5 cpm/sample) was mixed with 10 g 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 g/ml) and RNase T 1 (2 g/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 g/ml pepstatin, and 10 g/ml leupeptin. The homogenates were centrifuged at 5000 ϫ 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.
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 m. The eKir on the sections was detected by use of the pre-embedding 1-nm gold particlesilver 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.

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 (ϳ70% similar); SW250 represents Rho-type GTPaseactivating protein (p190RhoGAP, ϳ70% similar to mammalian homologs), a potent GTPase activator for various Rho-type GTPases, and SW300, an inward rectifier K ϩ channel (eKir). Fish P450s have been demonstrated to undergo marked upregulation in response to water pollution regardless of osmotic environments (25); therefore, we did not analyze the FW220derived 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 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/GenBank TM accession number is AB009669.
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.
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 ϫ 10 5 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 membranespanning hydrophobic segments, M1 and M2, and shares significant sequence conservation in the pore-forming H5 region with previously cloned inward rectifier K ϩ channels (7-9, 15, 18) (Fig. 3). Overall similarities to other inward rectifier K ϩ 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). 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 (ϳ51% 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.
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).
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-trans-  (33), and rat ROMK1 (Kir1.1a) (7). Gaps are inserted to achieve maximum similarity. Amino acid identities are indicated by background shading.

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 ϩ channel (residues 144 -296 of eKir) using the neighbor-joining method. The sequence of rat voltage-gated K ϩ channel Kv1.3 was used as an outgroup. The GenBank TM accession numbers of the compared proteins are as follows: 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 proteincoupled 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. fected 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 ϩ . The eKir-transfected cells produced large inward currents (n ϭ 10) that were not seen in mock-transfected cells (n ϭ 10). Almost identical currents were seen in eKir-transfected GH 3 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 Ba 2ϩ (1 mM, closed squares) and Cs ϩ (1 mM, closed triangles), as described previously for inward rectifier K ϩ channel (Kir) family members (7,8). Fig. 6C shows the extracellular K ϩ dependence of the reversal potentials. These reversal potentials were close to the equilibrium potentials of K ϩ predicted by the Nernst's equation which suggests a high selectivity for K ϩ . These data indicate that the eKir constitutes a K ϩ channel that belongs to the Kir family.
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. 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. The membrane currents were evoked by pulse steps to ϩ20, 0, Ϫ20, Ϫ40, Ϫ60, Ϫ80, Ϫ100, Ϫ120 mV from the holding potential of 0 mV. B, I-V relationships of the membrane current from the eKirtransfected cell in 140 mM K ϩ (closed circles) and in the presence of 1 mM Ba 2ϩ (closed squares) and 1 mM Cs ϩ (closed triangles). C, the extracellular K ϩ dependence of the reversal potentials. Potassium was replaced by equimolar sodium, such 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.

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 ϩ ,K ϩ -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. 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 ϩ 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 ϩ ,K ϩ -ATPase reflecting their extraordinary power of ion transport, eKir cloned here may be an osmoregulatory component working cooperatively with Na ϩ ,K ϩ -ATPase, for example, by recycling K ϩ . 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. 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.