The thiazide sensitive sodium chloride co-transporter NCC is modulated by site-specific ubiquitylation

Abstract

The renal sodium chloride cotransporter, NCC, in the distal convoluted tubule is important for maintaining body Na+ and K+ homeostasis. Endogenous NCC is highly ubiquitylated, but the role of individual ubiquitylation sites is not established. Here, we assessed the role of 10 ubiquitylation sites for NCC function. Transient transfections of HEK293 cells with human wildtype (WT) NCC or various K to R mutants identified greater membrane abundance for K706R, K828R and K909R mutants. Relative to WT-NCC, stable tetracycline inducible MDCKI cell lines expressing K706R, K828R and K909R mutants had significantly higher total and phosphorylated NCC levels at the apical plasma membrane under basal conditions. Low chloride stimulation increased membrane abundance of all mutants to similar or greater levels than WT-NCC. Under basal conditions K828R and K909R mutants had less ubiquitylated NCC in the plasma membrane, and all mutants displayed reduced NCC ubiquitylation following low chloride stimulation. Thiazide-sensitive sodium-22 uptakes were elevated in the mutants and internalization from the plasma membrane was significantly less than WT-NCC. K909R had increased half-life, whereas chloroquine or MG132 treatment indicated that K706 and K909 play roles in lysosomal and proteasomal NCC degradation, respectively. In conclusion, site-specific ubiquitylation of NCC plays alternative roles for NCC function.

Regulation of the Renal NaCl Cotransporter and Its Role in Potassium Homeostasis

“…In this model, Kir4.1 deletion in the kidney of adult mice caused depolarization of the basolateral membrane of DCT1 cells along with decreased SPAK phosphorylation and diminished NCC protein abundance and activity, confirming a central role of Kir4.1 in setting the membrane potential and controlling NCC activation in the DCT (363). Why total NCC levels are diminished in this model is unclear, but may be linked to increased ubiquitylation of NCC when in a dephosphorylated form (267,269). Interestingly, the Kir4.1 channel is also required to sense and control plasma K ϩ (59, 361), which will be reviewed in greater detail in section IIB.…”

“…Overall NCC function is regulated by phosphorylation of threonine (Thr) and serine (Ser) residues along its NH 2terminal cytoplasmic domain (228,258,266,396), coupled to altered apical membrane expression (276) (FIG-URE 3). Studies in cell lines and Xenopus laevis oocytes have indicated that phosphorylation of NCC most likely does not mediate trafficking to the cell surface (266,267,269), but it occurs directly in the plasma membrane resulting in increased NCC transport capacity and decreased NCC endocytosis from the plasma membrane (228,268). Thus increased phosphorylation of NCC is often used as a surrogate marker indicating an overall increase in NCC-dependent NaCl transport in the DCT.…”

Regulation of the renal NaCl cotransporter by the WNK/SPAK pathway: lessons learned from genetically altered animals

“…In vitro experiments suggest that NCC is a substrate of the HECT-type ubiquitin ligase Nedd4-2, which also regulates ENaC (5), by ubiquitinating specific sites on the cotransporter (56). A conditional knockout model to eliminate the Nedd4-2 expression in the nephron was associated with increased expression and phosphorylation of NCC (55).…”

Role of the alternative splice variant of NCC in blood pressure control

“…According to recent large-scale proteomics studies, NCC is ubiquitylated on several conserved lysine residues [30,31]. Generally, NCC ubiquitylation is associated with increased endocytosis from the plasma membrane, as well as protein degradation [32,33]. A recent study pointed out that NCC exhibits site-specific ubiquitylation exerting different roles on NCC function, via modulating NCC phosphorylation or its total abundance at the plasma membrane [33].…”

“…Generally, NCC ubiquitylation is associated with increased endocytosis from the plasma membrane, as well as protein degradation [32,33]. A recent study pointed out that NCC exhibits site-specific ubiquitylation exerting different roles on NCC function, via modulating NCC phosphorylation or its total abundance at the plasma membrane [33]. Interestingly, several studies indicate that phosphorylation of NCC can interfere with its ubiquitylation, thereby playing a dual role in modulating NCC function [34,35].…”