Abstract

The water channel aquaporin 2 (AQP2) is responsible for water reabsorption by kidney collecting duct cells. A substitution of amino acid leucine 137 to proline in AQP2 (AQP2-L137P) causes Nephrogenic Diabetes Insipidus (NDI). This study aimed to determine the cell biological consequences of this mutation on AQP2 function. Studies were performed in HEK293 and MDCK type I cells, transfected with wildtype (WT) AQP2 or an AQP2-L137P mutant. AQP2-L137P was predominantly detected as a high-mannose form of AQP2, whereas AQP2-WT was observed in both non-glycosylated and complex glycosylated forms. In contrast to AQP2-WT, the AQP2-L137P mutant did not accumulate on the apical plasma membrane following stimulation with forskolin. Ubiquitylation of AQP2-L137P was different from AQP2-WT, with predominance of non-distinct protein bands at various molecular weights. The AQP2-L137P mutant displayed reduced half-life compared to AQP2-WT. Treatment of cells with chloroquine increased abundance of AQP2-WT, but not AQP2-L137P. In contrast, treatment with MG132 increased abundance of AQP2-L137P but not AQP2-WT. Xenopus oocytes injected with AQP2-WT had increased osmotic water permeability when compared to AQP2-L137P, which correlated with lack of the mutant form in the plasma membrane. From the localization of the mutation and nature of the substitution it is likely that AQP2-L137P causes protein misfolding, which may be responsible for the observed functional defects. The data suggest that the L137P mutation results in altered AQP2 protein maturation, increased AQP2 degradation via the proteasomal pathway and limited plasma membrane expression. These combined mechanisms are likely responsible for the phenotype observed in this class of NDI patients.

中文翻译：

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导致严重形式的肾性尿崩症的aquaporin-2突变的分子表征

抽象的

水通道水通道蛋白2（AQP2）负责肾脏收集导管细胞对水的重吸收。氨基酸亮氨酸137取代AQP2（AQP2-L137P）中的脯氨酸会引起肾原性尿崩症（NDI）。这项研究旨在确定此突变对AQP2功能的细胞生物学影响。在用野生型（WT）AQP2或AQP2-L137P突变体转染的HEK293和MDCK I型细胞中进行了研究。AQP2-L137P主要被检测为高甘露糖形式的AQP2，而AQP2-WT以非糖基化形式和复杂糖基化形式均被观察到。与AQP2-WT相反，在用福司可林刺激后，AQP2-L137P突变体并未积聚在顶质膜上。AQP2-L137P的泛素化不同于AQP2-WT，在不同分子量下主要具有不明显的蛋白质条带。与AQP2-WT相比，AQP2-L137P突变体显示出降低的半衰期。用氯喹处理细胞可增加AQP2-WT的丰度，但不会增加AQP2-L137P的丰度。相反，用MG132处理可增加AQP2-L137P的丰度，但不会增加AQP2-WT的丰度。与AQP2-L137P相比，注射AQP2-WT的非洲爪蟾卵母细胞具有增加的渗透水渗透性，这与质膜中缺乏突变形式有关。从突变的位置和取代的性质来看，AQP2-L137P可能导致蛋白质错误折叠，这可能是观察到的功能缺陷的原因。数据表明，L137P突变导致改变的AQP2蛋白成熟，增加的AQP2通过蛋白酶体途径的降解和有限的质膜表达。这些组合的机制可能是导致此类NDI患者中观察到的表型的原因。