Sabath Ernesto, Meade Patricia, Berkman Jennifer, de los Heros Paola, Moreno Erika, Bobadilla Norma A, Vázquez Norma, Ellison David H, Gamba Gerardo
Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Tlalpan 14000, Mexico City, Mexico.
Am J Physiol Renal Physiol. 2004 Aug;287(2):F195-203. doi: 10.1152/ajprenal.00044.2004. Epub 2004 Apr 6.
Most of the missense mutations that have been described in the human SLC12A3 gene encoding the thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC, NCC, or NCCT), as the cause of Gitelman disease, block TSC function by interfering with normal protein processing and glycosylation. However, some mutations exhibit considerable activity. To investigate the pathogenesis of Gitelman disease mediated by such mutations and to gain insights into structure-function relationships on the cotransporter, five functional disease mutations were introduced into mouse TSC cDNA, and their expression was determined in Xenopus laevis oocytes. Western blot analysis revealed immunoreactive bands in all mutant TSCs that were undistinguishable from wild-type TSC. The activity profile was: wild-type TSC (100%) > G627V (66%) > R935Q (36%) = V995M (32%) > G610S (12%) > A585V (6%). Ion transport kinetics in all mutant clones were similar to wild-type TSC, except in G627V, in which a small but significant increase in affinity for extracellular Cl(-) was observed. In addition, G627V and G610S exhibited a small increase in metolazone affinity. The surface expression of wild-type and mutant TSCs was performed by laser-scanning confocal microscopy. All mutants exhibited a significant reduction in surface expression compared with wild-type TSC, with a profile similar to that observed in functional expression analysis. Our data show that biochemical and functional properties of the mutant TSCs are similar to wild-type TSC but that the surface expression is reduced, suggesting that these mutations impair the insertion of a functional protein into the plasma membrane. The small increase in Cl(-) and thiazide affinity in G610S and G627V suggests that the beginning of the COOH-terminal domain could be implicated in defining kinetic properties.
在编码噻嗪类敏感型钠氯协同转运蛋白（TSC、NCC或NCCT）的人类SLC12A3基因中，已被描述的大多数错义突变作为吉特曼病的病因，通过干扰正常蛋白质加工和糖基化来阻断TSC功能。然而，一些突变表现出相当的活性。为了研究由这类突变介导的吉特曼病的发病机制，并深入了解协同转运蛋白的结构-功能关系，将五个功能性疾病突变引入小鼠TSC cDNA，并在非洲爪蟾卵母细胞中测定它们的表达。蛋白质印迹分析显示，所有突变型TSC中均出现了与野生型TSC无法区分的免疫反应条带。活性概况为：野生型TSC（100%）＞G627V（66%）＞R935Q（36%） = V995M（32%）＞G610S（12%）＞A585V（6%）。除G627V外，所有突变克隆中的离子转运动力学均与野生型TSC相似，在G627V中观察到对细胞外氯离子的亲和力有小幅但显著的增加。此外，G627V和G610S对美托拉宗的亲和力有小幅增加。通过激光扫描共聚焦显微镜对野生型和突变型TSC的表面表达进行检测。与野生型TSC相比，所有突变体的表面表达均显著降低，其概况与功能表达分析中观察到的相似。我们的数据表明，突变型TSC的生化和功能特性与野生型TSC相似，但表面表达降低，这表明这些突变损害了功能性蛋白插入质膜的过程。G610S和G627V中氯离子和噻嗪类亲和力的小幅增加表明，COOH末端结构域的起始部分可能与确定动力学特性有关。
