Abstract: SA-PO1035
SCAMP4, a New Player in the Regulation of NKCC2 Surface Expression
Session Information
- Fluid and Electrolytes: Basic - II
October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid and Electrolytes
- 901 Fluid and Electrolytes: Basic
Authors
- Demaretz, Sylvie, INSERM-U1138, CNRS-ERL8228, Paris, France
- Laghmani, Kamel, INSERM-U1138, CNRS-ERL8228, Paris, France
Background
The apically located Na-K-2l cotransporter is the pacemaker of NaCl reabsorption in the thick ascending limb (TAL) of the loop of Henle. We have previously shown that secretory carrier membrane protein 2 (SCAMP2) regulates exocytic insertion of NKCC2 into the cell membrane. The aim of the present study was to investigate the effect of SCAMP4, a member of the SCAMPs family that lacks the highly conserved N-terminal NPF repeats, on NKCC2 surface expression.
Methods
To examine the expression of SCAMP4 in TAL, we checked for the presence of its transcript in native TAL cells using real-time RT-PCR. Protein-protein interaction was assessed by co-immunopreciptation assay. NKCC2 surface expression was monitored in transiently transfected OKP and HEK cells, using protein biotinylation, immunoblot and confocal imaging.
Results
Real time PCR on renal microdissected tubules demonstrated the expression of SCAMP4 in TAL. Co-immunoprecipitation experiments showed robust interaction between SCAMP4 and the complex-glycosalyed form of NKCC2 in cultured renal cells, suggesting that the interaction takes a place at the post-Golgi level. Consistent with this notion, co-immunolocalization experiments revealed that similar to SCAMP2, SCAMP4 co-localizes with NKCC2 in recycling endosomes. However, in contrast to SCAMP2, SCAMP4 co-expression promoted NKCC2 surface expression, whereas SCAMP4 knock-down had the opposite effect. Interestingly, deleting the deleting N-terminal NPF repeats from SCAMP2, abolished its negative effect on NKCC2 surface expression, suggesting that this highly conserved domain plays a crucial in the differential regulation of NKCC2 surface expression by SCAMP2 and SCAMP4.
Conclusion
We identified SCAMP4 as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression. Most importantly, our results are consistent with SCAMP2 and SCAMP4 having differential and antagonistic effects with regard to NKCC2 transit through recycling endosomes, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking.
Funding
- Government Support - Non-U.S.