Ion and diuretic specificity of chimeric proteins between apical Na(+)-K(+)-2Cl(-) and Na(+)-Cl(-) cotransporters.
Ion and diuretic specificity of chimeric proteins between apical Na(+)-K(+)-2Cl(-) and Na(+)-Cl(-) cotransporters.
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DOI：
10.1152/ajprenal.00124.2004
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发表时间：
2004-05
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影响因子：
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通讯作者：
C. Tovar-Palacio;N. Bobadilla;P. Cortés;C. Plata;Paola de los Heros;N. Vázquez;G. Gamba
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文献类型：
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作者：
C. Tovar-Palacio;N. Bobadilla;P. Cortés;C. Plata;Paola de los Heros;N. Vázquez;G. Gamba
哺乳动物肾脏布美他尼敏感性Na（+）-K（+）-2Cl（-）和噻嗪敏感性Na（+）-Cl（-）共转运蛋白分别是Henle袢和远曲小管粗升支重吸收盐的主要途径。这些协同转运蛋白作为袢型和噻嗪型利尿剂的受体，相应基因的失活突变分别与I型Bartter综合征和Gitleman病的发生相关。离子移位和利尿剂结合特异性的结构要求尚不清楚。作为分析这些共转运蛋白中赋予离子或利尿剂偏好的结构决定因素的初步方法，我们利用Na（+）-K（+）-2Cl（-）和Na（+）-Cl（-）共转运蛋白之间的功能差异和结构相似性来设计和研究嵌合蛋白，其中NH（2）-末端和/或COOH-末端结构域彼此转换。因此，产生了六种嵌合蛋白。利用非洲爪蟾卵母细胞的异源表达系统，我们观察到四个嵌合体表现出功能活性。结果表明，Na（+）-K（+）-2Cl（-）协同转运蛋白的离子转运和利尿剂结合特异性由其中心疏水区决定。因此，NH（2）-末端和COOH-末端结构域在定义这些性质中不起作用。对于Na（+）-Cl（-）协同转运蛋白也可以得出类似的结论。
The mammalian kidney bumetanide-sensitive Na(+)-K(+)-2Cl(-) and thiazide-sensitive Na(+)-Cl(-) cotransporters are the major pathways for salt reabsorption in the thick ascending limb of Henle's loop and distal convoluted tubule, respectively. These cotransporters serve as receptors for the loop- and thiazide-type diuretics, and inactivating mutations of corresponding genes are associated with development of Bartter's syndrome type I and Gitleman's disease, respectively. Structural requirements for ion translocation and diuretic binding specificity are unknown. As an initial approach for analyzing structural determinants conferring ion or diuretic preferences in these cotransporters, we exploited functional differences and structural similarities between Na(+)-K(+)-2Cl(-) and Na(+)-Cl(-) cotransporters to design and study chimeric proteins in which the NH(2)-terminal and/or COOH-terminal domains were switched between each other. Thus six chimeric proteins were produced. Using the heterologous expression system of Xenopus laevis oocytes, we observed that four chimeras exhibited functional activity. Our results revealed that, in the Na(+)-K(+)-2Cl(-) cotransporter, ion translocation and diuretic binding specificity are determined by the central hydrophobic domain. Thus NH(2)-terminal and COOH-terminal domains do not play a role in defining these properties. A similar conclusion can be suggested for the Na(+)-Cl(-) cotransporter.