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.
Highlights
The renal sodium chloride cotransporter, NCC, in the distal convoluted tubule is important for maintaining body Na+ and K+ homeostasis
Ten conserved lysines were selected for individual mutational analysis (K to R) within human NCC to evaluate their selective role in NCC function
As we have previously demonstrated that increased apical membrane levels of NCC are associated with decreased NCC ubiquitylation[23], we focused our initial studies on identifying NCC mutants that had greater plasma membrane abundance
Summary
The renal sodium chloride cotransporter, NCC, in the distal convoluted tubule is important for maintaining body Na+ and K+ homeostasis. The sodium chloride cotransporter NCC (Slc12a3) of the distal convoluted tubule (DCT) plays a major role in determining body Na+ and K+ homeostasis[1] This essential role is emphasized by various diseases in which the activity of NCC is altered, leading to changes in blood pressure and electrolyte balance, e.g. Gitelman or Gordon syndrome[2,3,4,5,6]. NCC activity can be regulated by a variety of hormones, including angiotensin-2 (ANGII)[2,7], aldosterone[8] and arginine-vasopressin (AVP)[9] It is intensely debated whether the effects of these hormones are direct or indirect via secondary alterations in serum electrolyte levels[10,11], they all influence NCC activity to some degree by activating the WNK-SPAK signaling network in DCT cells[6]. Greater levels of NCC at the apical plasma membrane are associated with increased phosphorylation of NCC, alongside decreased NCC ubiquitylation and decreased NCC endocytosis[22,23]
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