The mechanism of osmotic adaptation in marine invertebraes. -Structural and functional characterization of taurine transporter

本研究旨在通过对海洋无脊椎动物牛磺酸转运体功能的研究，阐明海洋无脊椎动物具有较高的渗透适应能力。在第一年（1999年），我们研究了牡蛎暴露于环境渗透压变化下的游离氨基酸的变化，并指出牛磺酸是牡蛎中最重要的渗透压物。因此，我们尝试从海洋无脊椎动物中克隆编码牛磺酸转运蛋白的基因。因此，我们从海洋贻贝中克隆出了与哺乳动物牛磺酸转运体高度同源的部分cDNA片段，而在牡蛎中却无法克隆出该片段。第二年（2000年），我们获得了贻贝转运蛋白的全长cDNA，显示出与牛磺酸和甜菜碱/GABA转运蛋白的高度同源性。此外，northern blot分析表明，该基因的从头转录可由低渗透压和高渗透压诱导。在去年（2001年），我们尝试明确克隆转运蛋白的底物。为了分析克隆转运蛋白的底物特异性，我们检测了在爪蟾卵母细胞中表达的转运蛋白对[^3H]氨基酸、牛磺酸和[^<14>C]甜菜碱的摄取活性。结果表明，克隆的转运蛋白表达的卵母细胞对牛磺酸具有摄取活性，表明克隆的cDNA编码牛磺酸转运蛋白。本研究首次从海洋无脊椎动物中克隆出牛磺酸转运体，表明其参与渗透适应。我们的研究结果为阐明海洋无脊椎动物的高渗透适应能力迈出了第一步。

The aim of this study is to elucidate the high ability of osmo-adaptation of marine invertebrates by investigation into the function of taurine transporter of marine invertebrates.In the first year (1999), we investigated into the change in free amino acid in the oyster exposed to the change in the ambient osmolality, and indicated that taurine was most important as an osmdyte in the oyster. Therefore, we made an attempt to clone the gene coding taurine transporter from marine invertebrates. As a result, we cloned a partial cDNA fragment showing high homology with taurine transporter of mammals from the marine mussel, while we could not clone that from the oyster.In the second year (2000), we obtained the full length of cDNA of mussel transporter showing high homology with taurine and betaine/GABA transporter. In addition, as a result of northern blot analysis, it is indicated that de novo transcription of this gene is induced by both hypo- and hyper-osmolality.In the last year (2001), we made an attempt to make the substrate of cloned transporter clear. In order to analyze the substrate spedficity of cloned transporter, we examined uptake activity of [^3H]amino acids, taurine and [^<14>C]betaine in the transporter expressed in the Xenopus oocyte. As a result, cloned transporter expressed oocyte showed the uptake activity to taurine and it is indicated cloned cDNA codes taurine transporter. In this study, taurine transporter was cloned from marine invertebrate for the first time, and it's partidpate in osmo-adaptation wasindicated. Our result is the first step toward the elucidation of the high ability in osmo-adaptation of marine invertebrates.