摘要
A T60Mmutation in the thiazide-sensitive sodiumchloride cotransporter (NCC) is common in patientswith 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. NccT58M/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, T58MNcc 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 Wnk4D561A/+ mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/Osr1-Ncc, were crossed with NccT58M/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.
| 原文 | 英語 |
|---|---|
| 頁（從 - 到） | 1587-1597 |
| 頁數 | 11 |
| 期刊 | Journal of the American Society of Nephrology |
| 卷 | 24 |
| 發行號 | 10 |
| DOIs | |
| 出版狀態 | 已發佈 - 十月 2013 |
指紋
ASJC Scopus subject areas
- Nephrology
引用此文
Phosphorylation regulates NCC stability and transporter activity in vivo. / Yang, Sung Sen; Fang, Yu Wei; Tseng, Min Hua; Chu, Pei Yi; Yu, I. Shing; Wu, Han Chung; Lin, Shu Wha; Chau, Tom; Uchida, Shinichi; Sasaki, Sei; Lin, Yuh Feng; Sytwu, Huey Kang; Lin, Shih Hua.
於: Journal of the American Society of Nephrology, 卷 24, 編號 10, 10.2013, p. 1587-1597.研究成果: 雜誌貢獻 › 文章
}
TY - JOUR
T1 - Phosphorylation regulates NCC stability and transporter activity in vivo
AU - Yang, Sung Sen
AU - Fang, Yu Wei
AU - Tseng, Min Hua
AU - Chu, Pei Yi
AU - Yu, I. Shing
AU - Wu, Han Chung
AU - Lin, Shu Wha
AU - Chau, Tom
AU - Uchida, Shinichi
AU - Sasaki, Sei
AU - Lin, Yuh Feng
AU - Sytwu, Huey Kang
AU - Lin, Shih Hua
PY - 2013/10
Y1 - 2013/10
N2 - A T60Mmutation in the thiazide-sensitive sodiumchloride cotransporter (NCC) is common in patientswith 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. NccT58M/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, T58MNcc 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 Wnk4D561A/+ mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/Osr1-Ncc, were crossed with NccT58M/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.
AB - A T60Mmutation in the thiazide-sensitive sodiumchloride cotransporter (NCC) is common in patientswith 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. NccT58M/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, T58MNcc 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 Wnk4D561A/+ mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/Osr1-Ncc, were crossed with NccT58M/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.
UR - http://www.scopus.com/inward/record.url?scp=84885027369&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84885027369&partnerID=8YFLogxK
U2 - 10.1681/ASN.2012070742
DO - 10.1681/ASN.2012070742
M3 - Article
C2 - 23833262
AN - SCOPUS:84885027369
VL - 24
SP - 1587
EP - 1597
JO - Journal of the American Society of Nephrology : JASN
JF - Journal of the American Society of Nephrology : JASN
SN - 1046-6673
IS - 10
ER -