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

Omar A Z Tutakhel, Frans Bianchi, Daniël A Smits, René J M Bindels, Joost G J Hoenderop, Jenny van der Wijst

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The NaCl cotransporter (NCC) is essential for electrolyte homeostasis and control of blood pressure. The human SLC12A3 gene, which encodes NCC, gives rise to 3 isoforms, of which only the shortest isoform [NaCl cotransporter isoform 3 (NCC3)] has been studied extensively. All NCC isoforms share key phosphorylation sites at T55 and T60 that are essential mediators of NCC function. Recently, a novel phosphorylation site at S811 was identified in isoforms 1 and 2 [NaCl cotransporter splice variant (NCCSV)], which are only present in humans and higher primates. The aim of the current study, therefore, is to investigate the role of S811 phosphorylation in the regulation of NCC by a combination of biochemical and fluorescent microscopy analyses. We demonstrate that hypotonic low-chloride buffer increases S811 phosphorylation, whereas phosphorylation-deficient S811A mutant hinders phosphorylation at T55 and T60 in NCCSV and NCC3. NCCSV S811A impairs NCC3 activity in a dominant-negative fashion, although it does not affect plasma membrane abundance. This effect may be explained by the heterodimerization of NCCSV with NCC3. Taken together, our study highlights the dominant-negative effect of NCCSV on T55 and T60 phosphorylation and NCC activity. Here, we reveal a new function of NCCSV in humans that broadens the understanding on NCC regulation in blood pressure control.-Tutakhel, O. A. Z., Bianchi, F., Smits, D. A., Bindels, R. J. M., Hoenderop, J. G. J., van der Wijst, J. Dominant functional role of the novel phosphorylation site S811 in the human renal NaCl cotransporter.

Original languageEnglish
Pages (from-to)4482-4493
Number of pages12
JournalThe FASEB Journal
Issue number8
Publication statusPublished - Aug-2018


  • Blood Pressure/physiology
  • Cell Line
  • HEK293 Cells
  • Humans
  • Kidney/metabolism
  • Phosphorylation/physiology
  • Protein Isoforms/metabolism
  • Protein-Serine-Threonine Kinases/metabolism
  • Solute Carrier Family 12, Member 3/metabolism

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