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.
journal_name
Sci Repjournal_title
Scientific reportsauthors
Rosenbaek LL,Rizzo F,Wu Q,Rojas-Vega L,Gamba G,MacAulay N,Staub O,Fenton RAdoi
10.1038/s41598-017-12819-0subject
Has Abstractpub_date
2017-10-11 00:00:00pages
12981issue
1issn
2045-2322pii
10.1038/s41598-017-12819-0journal_volume
7pub_type
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