Evidence for an apical Na-Cl cotransporter involved in ion uptake in a teleost fish
Evidence for an apical Na-Cl cotransporter involved in ion uptake in a teleost fish
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DOI：
10.1242/jeb.018663
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发表时间：
2008-08-15
影响因子：
2.8
通讯作者：
Kaneko, Toyoji
中科院分区：
文献类型：
--
作者：
Hiroi, Junya;Yasumasu, Shigeki;Kaneko, Toyoji
阳离子-氯共转运蛋白，如Na（+）/K（+）/2Cl（-）共转运蛋白（NKCC）和Na（+）/Cl（-）共转运蛋白（NCC），定位于上皮细胞的顶侧或基底侧质膜，并参与活性离子的吸收或分泌。本研究的目的是克隆和鉴定广盐性莫桑比克罗非鱼（Oreochromis mossambicus）的“淡水型”和“海水型”阳离子-氯离子协同转运蛋白，并确定其在富钙细胞（MRCs）内的细胞内定位模式。从罗非鱼鳃中克隆了4个与人阳离子-氯离子共转运蛋白同源的全长cDNA，分别命名为罗非鱼NKCC 1a、NKCC 1b、NKCC 2和NCC。在4个候选人中，编码NKCC 1a的mRNA在海水适应鱼的卵黄囊膜和鳃（MRC定位的位点）中高度表达，而编码NCC的mRNA仅在淡水适应鱼的卵黄囊膜和鳃中表达。然后，我们制备了罗非鱼NKCC 1a和NCC的特异性抗体，并对NKCC 1a和NCC以及Na（+）/K（+）-ATP酶、囊性纤维化跨膜传导调节因子（CFTR）和Na（+）/H（+）交换因子3（NHE 3）在淡水或海水驯化的罗非鱼胚胎卵黄囊膜上进行了整装免疫荧光染色。同时五色免疫荧光染色使我们能够将MRCs清楚地分为四种类型：I型，II型，III型和IV型。NKCC 1a免疫反应定位于海水特异性IV型MRCs的基底外侧膜，而NCC免疫反应仅限于淡水特异性II型MRCs的顶膜。考虑到这些数据在mRNA和蛋白质水平，我们推断，NKCC 1a是海水型共转运蛋白参与离子分泌的IV型MRCs和NCC是淡水型共转运蛋白参与离子吸收的II型MRCs。我们提出了一个新的离子吸收模型MRCs在淡水中，包括位于顶部的NCC。我们还重新评估了传统的离子吸收模型，将NHE 3的mRNA在淡水中高度表达，并在其他淡水特异性MRCs的顶端膜的免疫反应性。
Cation-chloride cotransporters, such as the Na(+)/K(+)/2Cl(-) cotransporter (NKCC) and Na(+)/Cl(-) cotransporter (NCC), are localized to the apical or basolateral plasma membranes of epithelial cells and are involved in active ion absorption or secretion. The objectives of this study were to clone and identify 'freshwater-type' and 'seawater-type' cation-chloride cotransporters of euryhaline Mozambique tilapia (Oreochromis mossambicus) and to determine their intracellular localization patterns within mitochondria-rich cells (MRCs). From tilapia gills, we cloned four full-length cDNAs homologous to human cation-chloride cotransporters and designated them as tilapia NKCC1a, NKCC1b, NKCC2 and NCC. Out of the four candidates, the mRNA encoding NKCC1a was highly expressed in the yolk-sac membrane and gills (sites of the MRC localization) of seawater-acclimatized fish, whereas the mRNA encoding NCC was exclusively expressed in the yolk-sac membrane and gills of freshwater-acclimatized fish. We then generated antibodies specific for tilapia NKCC1a and NCC and conducted whole-mount immunofluorescence staining for NKCC1a and NCC, together with Na(+)/K(+)-ATPase, cystic fibrosis transmembrane conductance regulator (CFTR) and Na(+)/H(+) xchanger 3 (NHE3), on the yolk- sac membrane of tilapia embryos acclimatized to freshwater or seawater. The simultaneous quintuple-color immunofluorescence staining allowed us to classify MRCs clearly into four types: types I, II, III and IV. The NKCC1a immunoreactivity was localized to the basolateral membrane of seawater-specific type-IV MRCs, whereas the NCC immunoreactivity was restricted to the apical membrane of freshwater-specific type-II MRCs. Taking account of these data at the level of both mRNA and protein, we deduce that NKCC1a is the seawater-type cotransporter involved in ion secretion by type-IV MRCs and that NCC is the freshwater-type cotransporter involved in ion absorption by type-II MRCs. We propose a novel ion-uptake model by MRCs in freshwater that incorporates apically located NCC. We also reevaluate a traditional ion-uptake model incorporating NHE3; the mRNA was highly expressed in freshwater, and the immunoreactivity was found at the apical membrane of other freshwater-specific MRCs.