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Abstract: SA-OR061

Differential Effects of STCH and HSP70 on the Stability and Maturation of NKCC2

Session Information

Category: Fluid, Electrolytes, and Acid-Base

  • 704 Fluid, Electrolyte, Acid-Base Disorders

Authors

  • Seaayfan, Elie, INSERM-U1138, UPMC, CNRS-ERL8228, Paris, France
  • Demaretz, Sylvie, INSERM-U1138, UPMC, CNRS-ERL8228, Paris, France
  • Kömhoff, Martin, Philipps Univ Marburg, Marburg, Germany
  • Laghmani, Kamel, INSERM-U1138, UPMC, CNRS-ERL8228, Paris, France
Background

Mutations in the apically located Na-K-2Cl cotransporter, NKCC2, lead to type I Bartter syndrome, a life-threatening kidney disorder associated with salt wasting, hypokalemia, and metabolic alkalosis. Conversely, increased expression of NKCC2 promotes hypertension. We previously showed that export from the ER constitutes the limiting step in the maturation and cell surface expression of NKCC2 and its disease causing mutants. Yet the molecular mechanisms involved in this process remain obscure.

Methods

To identify the protein partners involved in ER associated degradation of NKCC2, we screened a kidney cDNA library through yeast two-hybrid using NKCC2 C-terminus as bait. NKCC2 protein expression was monitored in transiently transfected HEK cells, using immunoblot and confocal imaging. NKCC2 stability was assessed by cycloheximide chase assay.

Results

We identified STCH, a constitutively expressed member of the heat shock protein 70 family, as a specific binding partner of NKCC2. STCH is known to be a microsome-associated chaperone. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-STCH interaction in HEK cells. Interestingly, they also identified the cytoplasmic and stress-inducible heat shock protein 70 (HSP70) as an interactor with NKCC2. STCH and HSP70 binding to NKCC2 involve mainly the immature form of the co-transporter and takes place at the ER. STCH co-expression heavily decreased total cellular WT NKCC2 protein and its disease associated folding mutants, in a dose dependent fashion, whereas HSP70 co-expression had the opposite effect. Cycloheximide chase assay showed that in cells over-expressing STCH, NKCC2 stability and maturation are heavily impaired. In contrast to STCH, HSP70 co-expression increased strikingly NKCC2 expression and maturation.

Conclusion

Our results are consistent with STCH and HSP70 having differential and antagonistic effects with regard to NKCC2 biogenesis, in particular under ER stress conditions. Most importantly, they may have an impact on our understanding and potential treatment of diseases related to aberrant NKCC2 trafficking and expression.

Funding

  • Government Support - Non-U.S.