Phosphorylation regulates NCC stability and transporter activity in vivo

Sung Sen Yang, Yu Wei Fang, Min Hua Tseng, Pei Yi Chu, I. Shing Yu, Han Chung Wu, Shu Wha Lin, Tom Chau, Shinichi Uchida, Sei Sasaki, Yuh Feng Lin, Huey Kang Sytwu, Shih Hua Lin

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27 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1587-1597
Number of pages11
JournalJournal of the American Society of Nephrology
Volume24
Issue number10
DOIs
Publication statusPublished - Oct 2013

ASJC Scopus subject areas

  • Nephrology

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    Yang, S. S., Fang, Y. W., Tseng, M. H., Chu, P. Y., Yu, I. S., Wu, H. C., Lin, S. W., Chau, T., Uchida, S., Sasaki, S., Lin, Y. F., Sytwu, H. K., & Lin, S. H. (2013). Phosphorylation regulates NCC stability and transporter activity in vivo. Journal of the American Society of Nephrology, 24(10), 1587-1597. https://doi.org/10.1681/ASN.2012070742