title: Functionomics of NCC mutations in Gitelman syndrome using a novel mammalian cell-based activity assay.
abstract: 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.
authors: Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.|Programa Regional en Doctorado en Biotecnología, Universidad Autónoma de Sinaloa, Sinaloa, Mexico.
authors: Marco A
authors: MA
authors: Valdez-Flores
authors: Department of Genetics, Hôpital Européen Georges Pompidou, Paris, France; and.
authors: Rosa
authors: R
authors: Vargas-Poussou
authors: Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
authors: Sjoerd
authors: S
authors: Verkaart
authors: Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
authors: Omar A Z
authors: OA
authors: Tutakhel
authors: Programa Regional en Doctorado en Biotecnología, Universidad Autónoma de Sinaloa, Sinaloa, Mexico.
authors: Angel
authors: A
authors: Valdez-Ortiz
authors: Clinical Research Center, Hôpital Européen Georges Pompidou, Paris, France.
authors: Anne
authors: A
authors: Blanchard
authors: Department of Genetics, Hôpital Européen Georges Pompidou, Paris, France; and.
authors: Cyrielle
authors: C
authors: Treard
authors: Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
authors: Joost G J
authors: JG
authors: Hoenderop
authors: Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands; rene.bindels@radboudumc.nl.
authors: René J M
authors: RJ
authors: Bindels
authors: Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
authors: Sabina
authors: S
authors: Jeleń
mesh_terms: D001681:Biological Assay / Q000379:methods; D053579:Gitelman Syndrome / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D064486:Solute Carrier Family 12, Member 3 / Q000235:genetics* / Q000378:metabolism
chemical_list: D064486:Solute Carrier Family 12, Member 3
keywords: Gitelman syndrome; NCC; SLC12A3; halide; sodium transport