Functionomics of NCC mutations in Gitelman syndrome using a novel mammalian cell-based activity assay

Am J Physiol Renal Physiol. 2016 Dec 1;311(6):F1159-F1167. doi: 10.1152/ajprenal.00124.2016. Epub 2016 Aug 31.


Gitelman syndrome (GS) is an autosomal recessive salt-wasting tubular disorder resulting from loss-of-function mutations in the thiazide-sensitive NaCl cotransporter (NCC). Functional analysis of these mutations has been limited to the use of Xenopus laevis oocytes. The aim of the present study was, therefore, to analyze the functional consequences of NCC mutations in a mammalian cell-based assay, followed by analysis of mutated NCC protein expression as well as glycosylation and phosphorylation profiles using human embryonic kidney (HEK) 293 cells. NCC activity was assessed with a novel assay based on thiazide-sensitive iodide uptake in HEK293 cells expressing wild-type or mutant NCC (N59I, R83W, I360T, C421Y, G463R, G731R, L859P, or R861C). All mutations caused a significantly lower NCC activity. Immunoblot analysis of the HEK293 cells revealed that 1) all NCC mutants have decreased NCC protein expression; 2) mutant N59I, R83W, I360T, C421Y, G463R, and L859P have decreased NCC abundance at the plasma membrane; 3) mutants C421Y and L859P display impaired NCC glycosylation; and 4) mutants N59I, R83W, C421Y, C731R, and L859P show affected NCC phosphorylation. In conclusion, we developed a mammalian cell-based assay in which NCC activity assessment together with a profiling of mutated protein processing aid our understanding of the pathogenic mechanism of the NCC mutations.

Keywords: Gitelman syndrome; NCC; SLC12A3; halide; sodium transport.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay / methods
  • Gitelman Syndrome / genetics*
  • Gitelman Syndrome / metabolism
  • Glycosylation
  • HEK293 Cells
  • Humans
  • Mutation
  • Phosphorylation
  • Solute Carrier Family 12, Member 3 / genetics*
  • Solute Carrier Family 12, Member 3 / metabolism


  • Solute Carrier Family 12, Member 3