Suzuki Y, Itakura M, Kashiwagi M, Nakamura N, Matsuki T, Sakuta H, Naito N, Takano K, Fujita T, Hirose S
Department of Biological Sciences, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
J Biol Chem. 1999 Apr 16;274(16):11376-82. doi: 10.1074/jbc.274.16.11376.
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.
通过使用差异mRNA显示技术来监测广盐性鳗鱼适应不同盐度时相关的分子变化，我们鉴定出一个在海水鳗鱼鳃中强烈诱导表达的cDNA片段。全长cDNA的克隆及其在COS-7细胞中的表达表明，该克隆编码一个由372个氨基酸残基组成的内向整流钾通道（eKir），它有两个跨膜片段和一个典型的孔形成区域（H5）。与内向整流钾通道家族（Kir）的其他成员相比，除了H5区域外，只有很低的序列相似性。与氨基酸序列的这种差异一致，系统发育分析表明eKir与其他成员早期分化并独立进化；它与Kir5.0亚家族成员的关系非常疏远。核糖核酸酶保护分析表明，eKir在海水鳗鱼的鳃、肾脏和后肠中高度表达，但在淡水鳗鱼中表达非常微弱。鳃切片的免疫组织化学显示eKir在氯细胞中密集定位。免疫电子显微镜表明，eKir主要存在于氯细胞的微管系统中。这种定位及其盐诱导特性表明，这里克隆的eKir通道是Kir家族Kir5.0亚家族的一个新成员，并且与渗透调节有关。
