Department of Life Sciences, National Chung-Hsing University, Taichung 402, Taiwan.
J Exp Biol. 2010 Mar 1;213(5):683-93. doi: 10.1242/jeb.040212.
Previous studies have reported the mechanisms of ion absorption and secretion by diverse membrane transport proteins in gills of various teleostean species. To date, however, the chloride channel expressed in the basolateral membrane of mitochondrion-rich (MR) cells for Cl(-) uptake in freshwater (FW) fish is still unknown. In this study, the combination of bioinformatics tools [i.e. National Center for Biotechnology Information (NCBI) database, Tetraodon nigroviridis (spotted green pufferfish) genome database (Genoscope), BLAT and BLASTn] were used to identify the gene of ClC-3 (TnClC-3), a member of the CLC chloride channel family in the T. nigroviridis genome. RT-PCR analysis revealed that the gene encoding for the ClC-3 protein was widely expressed in diverse tissues (i.e. gill, kidney, intestine, liver and brain) of FW- and seawater (SW)-acclimated pufferfish. In whole-mount double immunofluorescent staining, branchial ClC-3-like immunoreactive protein was localized to the basolateral membrane of Na(+)/K(+)-ATPase (NKA) immunoreactive cells in both the FW- and SW-acclimated pufferfish. In response to salinity, the levels of transcript of branchial TnClC-3 were similar between FW and SW fish. Moreover, the membrane fraction of ClC-3-like protein in gills was 2.7-fold higher in FW compared with SW pufferfish. To identify whether the expression of branchial ClC-3-like protein specifically responded to lower environmental [Cl(-)], the pufferfish were acclimated to artificial waters either with a normal (control) or lower Cl(-) concentration (low-Cl). Immunoblotting of membrane fractions of gill ClC-3-like protein showed the expression was about 4.3-fold higher in pufferfish acclimated to the low-Cl environment than in the control group. Furthermore, branchial ClC-3-like protein was rapidly elevated in response to acute changes of environmental salinity or [Cl(-)]. Taken together, pufferfish ClC-3-like protein was expressed in the basolateral membrane of gill MR cells, and the protein amounts were stimulated by hyposmotic and low-Cl environments. The enhancement of ClC-3-like protein may trigger the step of basolateral Cl(-) absorption of the epithelium to carry out iono- and osmoregulatory functions of euryhaline pufferfish gills.
先前的研究报告了不同鱼类鳃中多种膜转运蛋白的离子吸收和分泌机制。然而，迄今为止，淡水鱼类中富含线粒体（MR）细胞基底外侧膜中用于摄取氯离子（Cl(-)）的氯离子通道仍然未知。在这项研究中，组合使用生物信息学工具（即国家生物技术信息中心（NCBI）数据库、Tetraodon nigroviridis（斑点绿河豚）基因组数据库（Genoscope）、BLAT 和 BLASTn）来鉴定 T. nigroviridis 基因组中氯离子通道家族成员 ClC-3（TnClC-3）的基因。RT-PCR 分析显示，编码 ClC-3 蛋白的基因在 FW 和 SW 适应的河豚的多种组织（即鳃、肾脏、肠、肝和脑）中广泛表达。在整体双免疫荧光染色中，鳃 ClC-3 样免疫反应蛋白定位于 FW 和 SW 适应的河豚中 Na(+)/K(+)-ATPase（NKA）免疫反应细胞的基底外侧膜。对盐度的响应，FW 和 SW 鱼类鳃中的 TnClC-3 转录水平相似。此外，FW 河豚的鳃 ClC-3 样蛋白的膜部分比 SW 河豚高 2.7 倍。为了确定鳃 ClC-3 样蛋白的表达是否专门响应较低的环境 [Cl(-)]，将河豚适应于正常（对照）或较低 Cl(-)浓度（低-Cl）的人工水中。对鳃 ClC-3 样蛋白的膜部分进行免疫印迹显示，在适应低-Cl 环境的河豚中，表达水平比对照组高约 4.3 倍。此外，鳃 ClC-3 样蛋白对环境盐度或 [Cl(-)]的急性变化迅速升高。总之，河豚 ClC-3 样蛋白在鳃 MR 细胞的基底外侧膜中表达，并且蛋白质含量受到低渗和低-Cl 环境的刺激。ClC-3 样蛋白的增强可能触发上皮细胞基底外侧 Cl(-)吸收的步骤，以执行广盐性河豚鳃的离子和渗透调节功能。
