Abstract
The thiazide-sensitive sodium chloride cotransporter, NCC, is the major NaCl transport protein in the distal convoluted tubule (DCT). The transport activity of NCC can be regulated by phosphorylation, but knowledge of modulation of NCC trafficking by phosphorylation is limited. In this study, we generated novel tetracycline-inducible Madin-Darby canine kidney type I (MDCKI) cell lines expressing NCC to examine the role of NCC phosphorylation and ubiquitylation on NCC endocytosis. In MDCKI-NCC cells, NCC was highly glycosylated at molecular weights consistent with NCC monomers and dimers. NCC constitutively cycles to the apical plasma membrane of MDCKI-NCC cells, with 20-30% of the membrane pool of NCC internalized within 30 min. The use of dynasore, PitStop2, methyl-β-cyclodextrin, nystatin, and filipin (specific inhibitors of either clathrin-dependent or -independent endocytosis) demonstrated that NCC is internalized via a clathrin-mediated pathway. Reduction of endocytosis resulted in greater levels of NCC in the plasma membrane. Immunogold electron microscopy confirmed the association of NCC with the clathrin-mediated internalization pathway in rat DCT cells. Compared with controls, inducing phosphorylation of NCC via low chloride treatment or mimicking phosphorylation by replacing Thr-53, Thr-58, and Ser-71 residues with Asp resulted in increased membrane abundance and reduced rates of NCC internalization. NCC ubiquitylation was lowest in the conditions with greatest NCC phosphorylation, thus providing a mechanism for the reduced endocytosis. In conclusion, our data support a model where NCC is constitutively cycled to the plasma membrane, and upon stimulation, it can be phosphorylated to both increase NCC activity and decrease NCC endocytosis, together increasing NaCl transport in the DCT.
Highlights
The sodium chloride cotransporter NCC mediates NaCl reabsorption in the kidney distal convoluted tubule
We generated various Madin-Darby canine kidney type I (MDCKI) cell lines with tetracycline-inducible NCC expression and characterized them based on cell morphology, the degree of NCC expression and glycosylation, and polarized trafficking of NCC to the apical plasma membrane
In the MDCKI-NCC cells, NCC in total cell extracts was detected as various protein moieties as follows: a protein smear centered at 140 kDa, a single protein entity at ϳ100 kDa, and an equivalent banding pattern at higher molecular weights
Summary
The sodium chloride cotransporter NCC mediates NaCl reabsorption in the kidney distal convoluted tubule. Results: NCC internalization from the plasma membrane is clathrin-mediated and regulated by NCC phosphorylation and ubiquitylation. The thiazide-sensitive sodium chloride cotransporter, NCC, is the major NaCl transport protein in the distal convoluted tubule (DCT). Our data support a model where NCC is constitutively cycled to the plasma membrane, and upon stimulation, it can be phosphorylated to both increase NCC activity and decrease NCC endocytosis, together increasing NaCl transport in the DCT. Phosphorylation of NCC at Thr, Thr-58, and Ser-71 occurs directly in the DCT plasma membrane and is unlikely to play a major role in NCC forward trafficking (exocytosis) [18, 19]. Our studies indicate that NCC undergoes constitutive trafficking to/from the apical plasma membrane via a clathrin-dependent mechanism that can modulate overall NCC cell surface expression. We demonstrate that phosphorylation of NCC can regulate the apical membrane abundance of NCC via reducing NCC ubiquitylation and subsequent endocytosis
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