Abstract: FR-OR067
Calcineurin Homologous Protein 1 Regulates the Renal Na-K-2Cl-Cotransporter
Session Information
- Fluids and Electrolytes: New Insights on Balance
October 26, 2018 | Location: 8, San Diego Convention Center
Abstract Time: 06:18 PM - 06:30 PM
Category: Fluid and Electrolytes
- 901 Fluid and Electrolytes: Basic
Authors
- Mutig, Kerim, Charité Universitätsmedizin Berlin, Berlin, Germany
- Paliege, Alexander, Universitätsklinikum Dresden, Dresden, Germany
- Bachmann, Sebastian, Charité Universitätsmedizin Berlin, Berlin, Germany
Background
The Na-K-2Cl-cotransporter (NKCC2) of the thick ascending limb (TAL) is essential for renal salt handling. Its activity depends on phosphorylation and dephosphorylation steps. Our previous work suggested that calcineurin is involved in dephosphorylation and deactivation of NKCC2. Calcineurin activity can be modulated by members of the calcineurin homologous protein family (CHP). Among these, CHP1 has been implicated in the regulation of several membrane proteins including ion transporters. We hypothesized that CHP1 participates in calcineurin-dependent regulation of NKCC2 activity.
Methods
Immunofluorescence, immunoblotting, co-immunoprecipitation and GST pull down assays were applied in rodent kidneys and cultured cells to characterize physical and functional interactions between NKCC2, CHP1 and calcineurin.
Results
Double labeling of CHP1 and NKCC2 revealed their close co-localization in rat TAL. Both products co-immunoprecipitated, suggesting their interaction. GST pull down assays with recombinant N-terminal NKCC2 mutants, mimicking its (de)phosphorylation at functionally relevant residues (T96, T101, T114, and S126), suggested that CHP1 and calcineurin may compete for the binding with phosphorylated NKCC2. Overexpression of CHP1 in cultured macula densa cells significantly increased NKCC2 phosphorylation at baseline and upon low-chloride stimulation. Acute stimulation of NKCC2 by administration of desmopressin in rats promoted the interaction of NKCC2 with CHP1, while attenuating its binding with calcineurin.
Conclusion
Our data suggests that CHP1 protects NKCC2 from calcineurin-dependent dephosphorylation and deactivation.