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.

Dominant functional role of the novel phosphorylation site S811 in the human renal NaCl cotransporter

“…NCC activity was performed as described in Valdez-Flores et al (22). NCC is able to transport NaI-like NaCl in a hydrochlorothiazide (HCTZ)-sensitive manner (22).…”

“…NCC activity was performed as described in Valdez-Flores et al (22). NCC is able to transport NaI-like NaCl in a hydrochlorothiazide (HCTZ)-sensitive manner (22). In brief, HEK293 cells were cotransfected with the halide-sensitive YFP-mKate construct (37) and the appropriate human NCC pCINeo-IRES-eCFP construct or pCINeo-IRES-eCFP mock.…”

“…The respective plasmids were cotransfected in HEK293 cells with halide-sensitive YFP that was coupled to the halide-insensitive fluorescent red protein, mKate. Before measurements, transfected HEK293 cells were incubated in hypotonic low Cl 2 buffer to maximally increase NCC phosphorylation and activity (22). After 30 s, NaI was added to the medium, which induced a significant reduction in the YFP-to-mKate ratio in NCC 3 -expressing cells, which was indicative of NCC transport activity (Fig.…”

“…The importance of NCC in the regulation of blood pressure is illustrated by such diseases as Gitelman syndrome (OMIM: 263800) (20)(21)(22) and familial hyperkalemic ABBREVIATIONS: DCT, distal convoluted tubule; eGFP, enhanced green fluorescent protein; ENaC, epithelial Na + channel; FHHt, familial hyperkalemic hypertension; FLIM, fluorescence lifetime imaging microscopy; FRET, Förster resonance energy transfer; HA, hemagglutinin; HCTZ, hydrochlorothiazide; HEK293, human embryonic kidney 293; ICS, image cytometry standard; NCC 3 , NaCl cotransporter isoform 3; NCC, NaCl cotransporter; NCC SV , NaCl cotransporter isoform splice variant; SPAK, ste20-related proline-alanine-rich kinase; TBS-T, Tris-buffered saline and Tween-20; tNCC, total NaCl cotransporter; WNK, with no lysine; YFP, yellow fluorescent protein hypertension (FHHt; OMIM: 145260) (23)(24)(25)(26). Gitelman syndrome is caused by loss-of-function mutations in the SLC12A3 gene that encodes NCC (20)(21)(22), which results in renal Na + wasting and hypotension (18,27). In contrast, FHHt is a form of hypertension that results from the gain of function of NCC as a consequence of mutations in genes that code for key signaling molecules (23,28).…”

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

“…NCC is an important pharmacological target in the treatment of hypertension as thiazide-type diuretics, which specifically block NCC, are considered first-line therapy [12,13]. Furthermore, over 180 loss-of-function mutations have been identified in the SLC12A3 gene, encoding NCC, in relation to Gitelman syndrome (OMIM 263,800) [14–16]. This is an autosomal recessive disease that is portrayed by hypocalciuria, hypomagnesemia, hypokalemic metabolic alkalosis, sodium wasting and lower blood pressure levels in comparison to their age-matched unaffected relatives [17,18].…”