Phosphorylation regulates NCC stability and transporter activity in vivo.
Phosphorylation regulates NCC stability and transporter activity in vivo.
复制标题
DOI：
10.1681/asn.2012070742
复制
发表时间：
2013-10
期刊：
影响因子：
--
通讯作者：
S. Yang;Yu-Wei Fang;Min-Hua Tseng;P. Chu;I. Yu;Han‐Chung Wu;Shu-Wha Lin;T. Chau;S. Uchida;S. Sasaki;Yuh-Feng Lin;H. Sytwu;Shih-Hua Lin
中科院分区：
文献类型：
--
作者：
S. Yang;Yu-Wei Fang;Min-Hua Tseng;P. Chu;I. Yu;Han‐Chung Wu;Shu-Wha Lin;T. Chau;S. Uchida;S. Sasaki;Yuh-Feng Lin;H. Sytwu;Shih-Hua Lin
噻嗪敏感性氯化钠协同转运蛋白（NCC）的T60 M突变在Gitelman综合征（GS）患者中很常见。该突变阻止了Ste 20相关的脯氨酸和富含丙氨酸的激酶（SPAK）/氧化应激反应激酶-1（OSR 1）介导的NCC磷酸化，并改变了体外NCC转运蛋白活性。在这里，我们研究了体内NCC磷酸化的生理作用，使用一种新的Ncc T58 M（人T60 M）敲入小鼠模型。Ncc（T58 M/T58 M）小鼠表现出典型的GS特征，对噻嗪类利尿剂的反应迟钝。尽管表达了正常水平的Ncc mRNA，但这些小鼠的总Ncc和p-Ncc蛋白水平较低，而低盐饮食增加了p-Spak。与野生型Ncc（其定位于远曲小管细胞的顶膜）相反，T58 M Ncc主要定位于胞质区域，并引起晚期远曲小管体积的增加。在MDCK细胞中，磷酸化缺陷型NCC突变体的外源表达降低了总蛋白表达水平和膜稳定性。此外，我们的分析发现，在具有纯合NCC T60 M突变的GS患者队列中，总尿NCC排泄减少。当Wnk 4（D561 A/+）小鼠（表达活化Spak/Osr 1-Ncc的假性醛固酮减少症II型模型）与Ncc（T58 M/T58 M）小鼠杂交时，总Ncc和p-Ncc蛋白水平降低，GS表型持续超过高血压表型。总的来说，这些数据表明，SPAK介导的磷酸化NCC在T60调节NCC的稳定性和功能，并在此残基的磷酸化缺陷纠正假性醛固酮减少症II型的表型。
A T60M mutation in the thiazide-sensitive sodium chloride cotransporter (NCC) is common in patients with Gitelman's syndrome (GS). This mutation prevents Ste20-related proline and alanine-rich kinase (SPAK)/oxidative stress responsive kinase-1 (OSR1)-mediated phosphorylation of NCC and alters NCC transporter activity in vitro. Here, we examined the physiologic effects of NCC phosphorylation in vivo using a novel Ncc T58M (human T60M) knock-in mouse model. Ncc(T58M/T58M) mice exhibited typical features of GS with a blunted response to thiazide diuretics. Despite expressing normal levels of Ncc mRNA, these mice had lower levels of total Ncc and p-Ncc protein that did not change with a low-salt diet that increased p-Spak. In contrast to wild-type Ncc, which localized to the apical membrane of distal convoluted tubule cells, T58M Ncc localized primarily to the cytosolic region and caused an increase in late distal convoluted tubule volume. In MDCK cells, exogenous expression of phosphorylation-defective NCC mutants reduced total protein expression levels and membrane stability. Furthermore, our analysis found diminished total urine NCC excretion in a cohort of GS patients with homozygous NCC T60M mutations. When Wnk4(D561A/+) mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/Osr1-Ncc, were crossed with Ncc(T58M/T58M) mice, total Ncc and p-Ncc protein levels decreased and the GS phenotype persisted over the hypertensive phenotype. Overall, these data suggest that SPAK-mediated phosphorylation of NCC at T60 regulates NCC stability and function, and defective phosphorylation at this residue corrects the phenotype of pseudohypoaldosteronism type II.