Distribution and diversity of Na-K-Cl cotransport proteins: A study with monoclonal antibodies
Distribution and diversity of Na-K-Cl cotransport proteins: A study with monoclonal antibodies
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DOI：
10.1152/ajpcell.1995.269.6.c1496
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发表时间：
1995-12-01
影响因子：
5.5
通讯作者：
Forbush, B
中科院分区：
文献类型：
--
作者：
Lytle, C;Xu, JC;Forbush, B
Na-K-Cl协同转运蛋白（NKCC）存在于大多数动物细胞中，其中其在细胞体积稳态和上皮盐转运中起作用。我们开发了六种单克隆抗体（命名为T4，T8，T9，TIG，T12和T14），针对包含人结肠NKCC羧基末端310个氨基酸的融合蛋白片段。这些T抗体选择性地识别各种动物组织中的推定NKCC蛋白。从23种类型的细胞分离的膜的Western印迹分析确定的免疫反应性蛋白质的质量范围从146至205 kDa的单带。在这些细胞中检测到的免疫反应蛋白的量与袢利尿剂结合位点密度相关。先前通过袢利尿剂光亲和标记鉴定为Na-K-Cl共转运蛋白的蛋白质被多种T抗体相互识别。大多数T抗体有效地免疫沉淀NKCC蛋白的变性形式。免疫细胞化学研究表明，NKCC局限于腺泡细胞的基底外侧边缘，导管中没有NKCC，这与Na-K-Cl共转运在氯化物分泌中的中心作用雅阁。在兔肾脏中，NKCC定位于厚的升肢细胞的顶膜，与其在氯离子重吸收中的作用一致。
The Na-K-CI cotransporter (NKCC) is present in most animal cells where it functions in cell volume homeostasis and epithelial salt transport. We developed six monoclonal antibodies (designated T4, T8, T9, TIG, T12, and T14) against a fusion protein fragment encompassing the carboxy-terminal 310 amino acids of the human colonic NKCC. These T antibodies selectively recognized putative NKCC proteins in a diverse variety of animal tissues. Western blot analysis of membranes isolated from 23 types of cells identified single bands of immunoreactive protein ranging in mass from 146 to 205 kDa. The amount of immunoreactive protein detected in these cells correlated with loop diuretic binding site density. Proteins identified previously as Na-K-CI cotransporters by loop diuretic photoaffinity labeling were mutually recognized by multiple T antibodies. Mast of the T antibodies effectively immunoprecipitated the denatured form of the NKCC protein. Immunocytochemical studies on the rabbit parotid gland demonstrated that NKCC is restricted to the basolateral margin of the acinar cells and absent from the ducts, in accord with the central role of Na-K-Cl cotransport in chloride secretion. In the rabbit kidney, NKCC was localized to the apical membrane of thick ascending limb cells, consistent with its role in chloride reabsorption.