Abstract
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 language | English |
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Pages (from-to) | 4482-4493 |
Number of pages | 12 |
Journal | The FASEB Journal |
Volume | 32 |
Issue number | 8 |
DOIs | |
Publication status | Published - Aug-2018 |
Keywords
- 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