ATP-sensitive K+ (KATP) channels play a crucial role in coupling cellular metabolism to membrane potential. In addition to the orthologs corresponding to Kir6.1 and Kir6.2 of mammals, we have identified a novel member, designated Kir6.3 (zKir6.3), of the inward rectifier K+ channel subfamily Kir6.x in zebrafish. zKir6.3 is a protein of 432 amino acids that shares 66% identity with mammalian Kir6.2 but differs considerably from mammalian Kir6.1 and Kir6.2 in the COOH terminus, which contain an Arg-Lys-Arg (RKR) motif, an endoplasmic reticulum (ER) retention signal. Single-channel recordings of reconstituted channels show that zKir6.3 requires the sulfonylurea receptor 1 (SUR1) subunit to produce KATP channel currents with single-channel conductance of 57.5 pS. Confocal microscopic analysis shows that zebrafish Kir6.3 requires the SUR1 subunit for its trafficking to the plasma membrane. Analyses of chimeric protein between human Kir6.2 and zKir6.3 and a COOH-terminal deletion of zKir6.3 indicate that interaction between the COOH terminus of zKir6.3 and SUR1 is critical for both channel activity and trafficking to the plasma membrane. We also identified zebrafish orthologs corresponding to mammalian SUR1 (zSUR1) and SUR2 (zSUR2) by the genomic database. Both Kir6.3 and SUR1 are expressed in embryonic brain of zebrafish, as assessed by whole mount in situ hybridization. These data indicate that Kir6.3 and SUR1 form functional KATP channels at the plasma membrane in zebrafish through a mechanism independent from ER retention by the RKR motif.
atp-sensitive k+ (KATP)channels play important roles in many physiological processes, including cytoprotection of neurons and cardiomyocytes and regulation of hormone release and vascular tonus (3, 14, 22, 29). The KATP channel is an octamer, formed by the physical association of Kir6.x subunits, members of the inwardly rectifying K+ (Kir) channel family, and regulatory sulfonylurea receptor subunits (SURx) (21). Different combinations of these subunits comprise the KATP channels in pancreatic β-cells (Kir6.2 plus SUR1), cardiac myocytes (Kir6.2 plus SUR2A), and vascular smooth muscles (Kir6.1 plus SUR2B) (21). Kir6.x subunits form the pore and confer channel inhibition by ATP, whereas SURx subunits confer activation by MgATP and sensitivity to sulfonylureas, widely used in treatment of diabetes mellitus (21). Kir6.x channel subunits contain two transmembrane segments (M1 and M2) (19) and a highly conserved pore-forming region (10). Within the pore-forming region, Kir6.x has a Gly-Phe-Gly (GFG) motif, whereas other Kir members have a Gly-Tyr-Gly (GYG) motif (15). In mammals, an Arg-Lys-Arg (RKR) motif, an endoplasmic reticulum (ER) retention sequence, is present in the COOH terminus of Kir6.x subunits (30). The Kir6.x subunit associates with the ER by its RKR motif. However, when coexpressed with SUR1 subunits, the RKR motif is masked, allowing trafficking of the Kir6.x-SUR1 complex to the cell surface (23, 30).
Comparative analysis of the KATP channels of different species can provide valuable insight into their structure-function relationships. In invertebrates, Kir channel genes (dKirI-III) and a SUR gene (Dsur) have been isolated and characterized in Drosophila melanogaster (6, 16). dKirIII has a GFG motif within its pore-forming region and is expressed in the embryonic hindgut; however, expression of dKirIII alone results in no functional channel current (6). Unlike dKirIII, Dsur is expressed specifically in the developing tracheal system and dorsal vessels. In addition, no KATP channel subunits have been identified in invertebrates except for Drosophila. These findings suggest that KATP channels may not exist in invertebrates. The zebrafish is used widely in genetic studies as a model of lower vertebrates. In the present study, we have found that there is a novel member, designated zKir6.3, of the inward rectifier K+ channel subfamily Kir6.x in zebrafish. To further clarify the structure-function relationships of KATP channels, we have characterized the Kir6.3-containing KATP channels in zebrafish.
MATERIALS AND METHODS
Screening of cDNA library.
An adult zebrafish brain cDNA library (gift of S. Okamoto, RIKEN, Japan) was used for cloning. By BLAST search of a fish expressed sequence tag database (fugu, medaka, zebrafish) using the human Kir6.2 cDNA sequence (GenBank/EBI Data Bank accession number D50582), several genes similar to Kir6.2 were identified. The gene-specific primers 5′-ttgcaatgtggcccacaaga-3′ (zebra-F) and 5′-tagtcgacagagtagcgtccatc-3′ (zebra-R) were designed within the highly conserved region. With this primer pair, an 800-bp fragment was amplified by PCR and a full-length cDNA encoding zebrafish Kir6.3 (zKir6.3; GenBank/EBI Data Bank accession number AB180939) was screened from the cDNA library. zKir6.2 and zKir6.1 were cloned from zebrafish heart cDNA by PCR.
Deletion mutants and chimeric constructs of zKir6.3 and human Kir6.2 were generated with a PCR-based method. The cDNAs were subcloned into the expression vector pFLAG-CMV1 (Sigma, St Louis, MO).
Cell transfection and culture.
Human embryonic kidney (HEK) 293T cells were cultured in high-glucose DMEM supplemented with 10% fetal calf serum. The cells were transfected with construct plasmids alone or cotransfected with human SUR1 (hSUR1/pcMV6) (25), using LipofectAMINE plus reagent (Invitrogen, San Diego, CA). For electrophysiological recording, an enhanced green fluorescent protein vector (pEGFP; Clontech, Oxford, UK) was used for detecting transfected cells.
Whole cell recordings of ATP-sensitive K+ current were performed as described previously (15). The extracellular solution contained (in mM) 135 NaCl, 5 KCl, 5 CaCl2, 2 MgSO4, 5 HEPES, and 3 glucose (pH 7.4). The pipette solution contained (in mM) 107 KCl, 11 EGTA, 2 MgSO4, 1 CaCl2, and 11 HEPES (pH 7.2). Single-channel recordings were done in the excised inside-out membrane patch configuration, as described previously (9). The intracellular solution contained (in mM) 140 KCl, 2 MgCl2, 1 EGTA, and 10 HEPES (pH 7.3), with 1 μM ATP. The pipette solution contained (in mM) 140 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES (pH 7.3).
HEK293T cells were fixed with 4% formaldehyde in PBS (1 h at 4°C), treated with 0.2% Triton X-100 for 30 min at room temperature (RT), blocked with 10% goat serum in PBS (30 min at RT), and labeled with monoclonal mouse anti-FLAG M2 primary antibody (0.66 μg/ml). After the primary antibody was washed out, the cells were labeled with Cy3-conjugated goat anti-mouse secondary antibody (Chemicon International, Temecula, CA; 1:500).
In situ hybridization.
A full protein coding sequence of zKir6.3 and a partial cDNA fragment of the zebrafish SUR1 gene (zSUR1) (GenBank/EBI Data Bank accession number AB180940) were used as probes for whole mount in situ hybridization. The probe for zSUR1 (396 bp) was obtained by PCR using the primer pair zSUR1/F (5′-aactacctgaactggatggtgc-3′) and zSUR1/R (5′-aatggagaagcgtgacctcag-3′). Digoxigenin-labeled riboprobes were synthesized with linearized DNA templates. The transcription reactions were carried out according to the manufacturer's instructions, using Sp6 and T7 RNA polymerase (Promega, Heidelberg, Germany). Subsequently, whole mount in situ hybridization was performed as described previously (2).
zKir6.3 is a novel member of the Kir6.x family in zebrafish.
We isolated a cDNA clone encoding a mammalian Kir6.2-like protein from an adult zebrafish brain cDNA library.1Database search revealed three members of the Kir6.x family in zebrafish. Comparison of the amino acid sequences of these three zebrafish members with those of mammalian Kir6.1 and Kir6.2 indicates that the Kir6.2-like protein identified is a novel member of the Kir6.x family. Accordingly, we designated it zKir6.3 (GenBank/EBI Data Bank accession number AB180939) (Fig. 1A). zKir6.1 is a protein of 416 amino acids having 79% amino acid identity to human Kir6.1. zKir6.2 is a protein of 381 amino acids having 73% amino acid identity to human Kir6.2. zKir6.3 is a protein of 432 amino acids having 62% and 66% amino acid identity to human Kir6.1 and Kir6.2, respectively (Fig. 1B). The NH2-terminal region [amino acids (aa) 1–338] of zKir6.3 is homologous to mammalian Kir6.2, whereas the COOH terminus (aa 339∼432) is considerably divergent. zKir6.3 has two transmembrane segments (M1 and M2) and a pore-forming region (H5). Within the H5 region, there is a GFG motif that is conserved in mammalian Kir6.1 and Kir6.2, supporting inclusion of zKir6.3 in the Kir6.x family. In humans, the COOH-terminal 36 amino acids of Kir6.2 contain an ER retention signal (RKR motif) that is critical in membrane trafficking of the channels (28, 30). Serine at residue 372 (S372) following the RKR motif is responsible for PKA phosphorylation (4). zKir6.3 lacks the RKR motif in the COOH terminus (Fig. 1A).
Previous studies of Kir6.2 mutants revealed several amino acids that are involved in determining ATP sensitivity, phosphatidylinositol 4,5-bisphosphate (PIP2) binding, and channel activity (7, 8, 15, 18, 20, 24, 27, 30). Comparison of amino acid sequences between zKir6.3 and mammalian Kir6.2 indicates that most of these 24 residues are conserved in zKir6.3 (Fig. 1A).
zKir6.3 requires the SUR1 subunit to form functional KATP channels.
To investigate the electrophysiological properties of zKir6.3, we performed single-channel recordings of HEK293T cells transfected with pFLAG-zKir6.3 alone or together with human SUR1 subunit (hSUR1). When zKir6.3 was expressed alone, no significant current was detected (data not shown). However, when coexpressed with hSUR1, zKir6.3 exhibited KATP channel currents (Fig. 2B) with single-channel conductance of 57.5 pS, similar to that of mammalian Kir6.2-containing KATP channels (1). The activity of zKir6.3/hSUR1 channels was inhibited by ATP in a dose-dependent manner (IC50 3 μM; Fig. 2D).
Subcellular localization of zKir6.3 in HEK293T cells.
Cell surface expression of zKir6.3 was examined by confocal microscopy (Fig. 3). When HEK293T cells were transfected with zKir6.3 alone, zKir6.3 was present intracellularly (Fig. 3C). When coexpressed with hSUR1, zKir6.3 was clearly localized at the membrane (Fig. 3D). These distribution patterns are similar to those of cells expressing hKir6.2 alone (Fig. 3A) and cells coexpressing hKir6.2 and hSUR1 (Fig. 3B).
zKir6.3 is targeted to the plasma membrane through interaction of its COOH terminus with SUR1.
The COOH terminus of mouse Kir6.2 has been shown to contain an ER retention signal, an RKR motif, that prevents its trafficking to membrane (30). Because zKir6.3 lacks an RKR motif, we investigated the possibility that the COOH terminus of zKir6.3 contains another retention signal. We constructed a chimeric protein between human Kir6.2 and zKir6.3 (hKir6.2/zKir6.3) and examined its electrophysiological properties and subcellular localization (Fig. 4A). hKir6.2/zKir6.3 is a fusion protein of the NH2-terminal half of hKir6.2 (aa 1–336) and the COOH-terminal half of zKir6.3 (aa 339∼432). When HEK293T cells were transfected with hKir6.2/zKir6.3 alone, hKir6.2/zKir6.3 protein was expressed intracellularly and no KATP channel current could be detected (data not shown). However, when cotransfected with hSUR1, hKir6.2/zKir6.3 was strongly expressed at the plasma membrane (Fig. 4B). Importantly, functional KATP channel currents were detected in the transfected cells (20 of 25 patches; Fig. 4C). In addition, when the COOH terminus (aa 356–432) of zKir6.3 was deleted (zKir6.3ΔC) (Fig. 5A), zKir6.3ΔC failed to traffic to the plasma membrane (Fig. 5,B and C). Moreover, neither zKir6.3ΔC alone nor coexpression with hSUR1 elicited KATP channel activity (data not shown).
Kir6.3 and SUR1 mRNA are coexpressed in zebrafish embryo.
Because zKir6.3 generates KATP channel currents in the presence of the hSUR1 subunit, there may well be functional KATP channels in zebrafish. We examined the expression patterns of zKir6.3 and zSUR1 in zebrafish embryo by whole mount in situ hybridization (Fig. 6). Both zKir6.3 and zSUR1 mRNA expressions were detected in forebrain, midbrain, and hindbrain, suggesting coexpression of zKir6.3 and zSUR1 mRNAs in the brain.
Kir6.3 and SUR1 are localized on different chromosomes in zebrafish.
By BLAST search of the zebrafish sequence database, we found two paired genes clustered on zebrafish chromosomes 4 and 25 (Fig. 7). We identified the Kir6.1 gene (zKir6.1) and the SUR2 gene (zSUR2) on chromosome 4 and the SUR1 gene (zSUR1) and the Kir6.2 gene (zKir6.2) on chromosome 25 (Fig. 7). zSUR2 shares 71% identity with human SUR2A (69% with SUR2B and 58% with SUR1 of human). zKir6.1 has two transmembrane regions linked by a highly conserved pore-forming region containing the GFG motif. Notably, zKir6.1 has an RKR motif in the COOH terminus. zSUR1 shares 57% identity with hSUR1 and 52% with hSUR2. zKir6.2 gene has a single exon. zKir6.3 gene is localized on chromosome 15 (Fig. 7).
In mammals, 15 Kir channel subunits have been identified and classified into 7 subfamilies (19). All Kir channel subunits have a conserved pore-forming region (H5) flanked by two transmembrane segments (M1 and M2). In addition, Kir6.x members are unique in having a GFG motif rather than a GYG motif in the H5 region. In lower organisms, two Kir channel genes are present in Caenorhabditis elegans [nIRK1 (U40947) and nIRK2 (U58730)] and three Kir channel genes are present in D. melanogaster [dKirI (AJ344344), dKirII (AJ344345 and AJ344346), and dKirIII (AJ344347)]. Among these, only dKirIII has the GFG motif. The SUR subunit has also been isolated (Dsur, AF167431) from D. melanogaster (16). However, the dKirIII and Dsur subunits are expressed in different tissues (6, 16), suggesting that there are no heteromultimeric KATP channels present in D. melanogaster. Several Kir family members have been cloned in lower vertebrates, including eKir from seawater eel gills (26) and sWIRK from masu salmon (12). ATP-sensitive K+ currents have been detected in several species of vertebrates, including lizard, frog, and fish (17). In the present study, we isolated zKir6.3 from an adult zebrafish brain cDNA library. zKir6.3 possesses the GFG motif that is found in the pore-forming region of mammalian Kir6.x.
Amino acid residues critical in the function of mammalian Kir6.2-containing KATP channels (7, 8, 15, 18, 20, 24, 27, 30) are highly conserved in zKir6.3 (Fig. 1). This indicates that zKir6.3 may function as the pore-forming subunit of these KATP channels. Electrophysiological examination shows that the zKir6.3 subunit, by coupling with the SUR1 subunit, produces KATP channels with properties similar to those of mammalian KATP channels reconstituted by hKir6.2 and hSUR1. Together with the findings of whole mount in situ hybridization, these data demonstrate that there are KATP channels in zebrafish brain possessing both Kir6.3 and SUR1 subunits. RT-PCR analysis also shows coexpression of Kir6.3 and SUR1 in adult zebrafish heart (data not shown).
Zerangue et al. (30) reported that the RKR motif in the COOH terminus of mammalian Kir6.2 acts as an ER retention signal that prevents its trafficking to the plasma membrane. When mammalian Kir6.2 is coexpressed with a SUR1 subunit, the RKR motif in Kir6.2 is masked and the Kir6.2/SUR1 complex is sorted to the plasma membrane (30). In zebrafish, although zKir6.3 lacks an RKR motif, it requires SUR1 for its expression on the plasma membrane to elicit KATP channel activity. However, Kir6.3 has a long COOH terminus that is divergent from mammalian Kir6.2 and might contain a retention signal other than the RKR motif present in mammals.
In mammals, the Kir6.x and SUR genes are paired on chromosomes: Kir6.2 and SUR1 are adjacent on human chromosome 11 and mouse chromosome 7; Kir6.1 and SUR2 are near each other on human chromosome 12 and mouse chromosome 6. In zebrafish, Kir6.1 and SUR2-like genes are adjacent on chromosome 4 and Kir6.2 and SUR1 are adjacent on chromosome 25 (Fig. 7). However, although zKir6.3 is located on chromosome 15, no SUR-related gene is present on the same chromosome. In addition, the protein coding region of zKir6.3 is coded by more than 3 exons, as assessed by the genomic structures in the database. The findings that Kir6.3 and SUR1 constitute functional KATP channels in a reconstituted system and that they are coexpressed in brain and heart suggest that Kir6.3-containing KATP channels may play a physiological role in native tissues. As the KATP channels in the brain and heart of mammals act as metabolic sensors in the regulation of cellular excitability (22), the Kir6.3-containing channels in zebrafish may also be involved in such regulation.
This study was supported by a Grant-in-Aid for Specially Promoted Research and a Research Grant from the Ministry of Education, Culture, Sports, Science and Technology and by a Grant-in Aid for Core Research for Evolutional Science and Technology (CREST).
We thank Dr. T. Gonoi (Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University) for suggestions. We are grateful to Dr. H. Okamoto (Brain Science Institute, RIKEN, Japan) for his gift of the adult zebrafish brain cDNA library.
↵1 The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB180939, AB180940, AB231936, AB231937, AB231938, and AB231939.
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: S. Seino, Division of Cellular and Molecular Medicine, Kobe Univ. Graduate School of Medicine, Kobe 650-0017, Japan (e-mail:).
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