title: Phosphorylation regulates NCC stability and transporter activity in vivo.
abstract: 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.
authors.affiliation: Division of Nephrology, Department of Medicine, Tri-Service General Hospital, Taipei, Taiwan;
authors.forename: Sung-Sen
authors.initials: SS
authors.lastname: Yang
authors.forename: Yu-Wei
authors.initials: YW
authors.lastname: Fang
authors.forename: Min-Hua
authors.initials: MH
authors.lastname: Tseng
authors.forename: Pei-Yi
authors.initials: PY
authors.lastname: Chu
authors.forename: I-Shing
authors.initials: IS
authors.lastname: Yu
authors.forename: Han-Chung
authors.initials: HC
authors.lastname: Wu
authors.forename: Shu-Wha
authors.initials: SW
authors.lastname: Lin
authors.forename: Tom
authors.initials: T
authors.lastname: Chau
authors.forename: Shinichi
authors.initials: S
authors.lastname: Uchida
authors.forename: Sei
authors.initials: S
authors.lastname: Sasaki
authors.forename: Yuh-Feng
authors.initials: YF
authors.lastname: Lin
authors.forename: Huey-Kang
authors.initials: HK
authors.lastname: Sytwu
authors.forename: Shih-Hua
authors.initials: SH
authors.lastname: Lin
mesh_terms: D000818:Animals; D016022:Case-Control Studies; D004285:Dogs; D005260:Female; D055879:Gene Knock-In Techniques; D053579:Gitelman Syndrome / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D010641:Phenotype; D010766:Phosphorylation / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011546:Pseudohypoaldosteronism / Q000378:metabolism; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism*; D049992:Sodium Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1 / Q000235:genetics / Q000378:metabolism
chemical_list: D011955:Receptors, Drug; D049992:Sodium Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; C064415:thiazide receptor; C486407:Stk39 protein, mouse; D017346:Protein Serine-Threonine Kinases