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Abstract: FR-PO617

N-Terminal Phosphorylation of Kidney Na-K-2Cl-Cotransporter Attenuates Its Endocytosis

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Mutig, Kerim, Charité Universitätsmedizin Berlin, Berlin, Germany
  • Munk, Anna-Lena, Charité Universitätsmedizin Berlin, Berlin, Germany
  • Bachmann, Sebastian, Charité Universitätsmedizin Berlin, Berlin, Germany
Background

The renal Na-K-2Cl-cotransporter (NKCC2) of the thick ascending limb (TAL) is critical for renal salt handling. Its activity is stimulated by phosphorylation of conserved N-terminal threonine and serine residues (T96, T101, T114, and S126), although the underlying mechanisms are not entirely clear. We hypothesized that NKCC2 phosphorylation interferes with its clathrin-mediated endocytosis.

Methods

Cellular distribution of NKCC2 and its phosphorylated form (pNKCC2) was evaluated in rat kidney and cultured rat TAL cells by high-resolution immunofluorescence, electron microscopy and biochemical tissue fractionation by sucrose gradient. Association of NKCC2 and pNKCC2 with clathrin was studied by binding assays.

Results

Labeling of rat kidney sections for NKCC2 revealed its even distribution between the luminal membrane and apical vesicular compartment, whereas pNKCC2 resided predominantly in the luminal membrane. Analysis of NKCC2, pNKCC2 and clathrin distribution in apical membrane fragments obtained from cultured TAL cells using the rip/flip technique showed regular co-localization of NKCC2- and clathrin immunogold signals, whereas pNKCC2 signal localized to clathrin-negative electron-dense membrane domains containing the lipid raft marker flotilin-1. In line with this, isolation of detergent-resistant membrane rafts from rat kidney tissue using extraction with Triton X-100 and subsequent sucrose gradient centrifugation revealed co-distribution of pNKCC2 and flotilin-1 signals in rafts-containing low-density gradient fractions, whereas clathrin signal was present in non-raft high-density fractions. GST pull down assays showed interactions of clathrin with recombinant N-terminal NKCC2 mutants mimicking its dephosphorylation (S/T->A), whereas mutants mimicking the phosphorylated N-terminus (S/T->D) did not bind clathrin. Acute in vivo stimulation of NKCC2 phosphorylation by treating vasopressin-deficient Brattleboro rats with desmopressin (1ng/kg body weight for 30 min) attenuated its co-immunoprecipitation with clathrin (-72%, p<0.05) and increased NKCC2 surface expression (+24%, p<0.05).

Conclusion

In sum, these results suggest that NKCC2 phosphorylation inhibits its clathrin-mediated NKCC2 endocytosis resulting in increased NKCC2 surface expression and activity.

Funding

  • Government Support - Non-U.S.