A di‐arginine‐based ER retention motif regulates the stability and maturation of NKCC2 and its disease causing mutants
Authors: Shaukat Irfan , Demartez Sylvie , Laghmani Kamel 
Published: June 2021

Mutations in the apically located Na‐K‐2Cl cotransporter, NKCC2, lead to type I Bartter syndrome, a life‐threatening kidney disease. Yet very little is known about NKCC2 biogenesis. We previously identified multiple evolutionarily conserved di‐leucine like motifs in the carboxyl terminus that control the anterograde trafficking of NKCC2. Here, we sought to identify an ER retention and/or retrieval signal that could participate in the quality control of NKCC2 and its disease causing mutants. Interestingly, sequence analysis of NKCC2 protein revealed the presence of a potential di‐arginine ER retention motif (57RLR59) in the cytoplasmic N‐terminal region of the cotransporter. We therefore studied the role of this potential RXR‐type ER retention signal in the stability and the biosynthetic processing of wild‐type (WT) NKCC2 and its ER retained mutants E368G, Y477N and A628D, using a cycloheximide decay assay in HEK293 cells transiently expressing the cotransporter. Mutating the 57RLR59 motif in WT NKCC2 increased the stability of the core‐glycosylated form of the cotransporter and strikingly facilitated its maturation, resulting in higher levels of the mature and hence membrane‐expressed and functional form of NKCC2. Importantly, mutation of 57RLR59 in the disease‐associated NKCC2 folding mutant Y477N increased its stability and restored the expression of its mature form. Intriguingly, mutating this RXR motif in the ER‐retained mutants E368G and A628D strikingly increased also their expression and the stability of their core glycosylated forms but failed to significantly improve their maturation, suggesting that a mutant specific method would be required to rescue the functional expression of each NKCC2 mutant. To better explore the molecular mechanisms involved in 57RLR59 action on NKCC2 stability and maturation, we checked its effect on NKCC2 interaction with OS9, an ER resident protein that mediates the ER associated degradation (ERAD) of the cotransporter. Co‐immunoprecipitation assays revealed that mutation of 57RLR59 motif inhibits the interaction of OS9 with NKCC2. Furthermore, OS9 knockdown reproduced the effect of 57RLR59 mutation on NKCC2 expression, suggesting a functional interaction between OS9 and this RXR motif in the ERAD of the cotransporter.

In summary, we showed evidence for the presence of an RXR‐type ER retention signal in the cytoplasmic N‐terminal of NKCC2 that regulates the ER associated degradation of WT NKCC2 and its disease causing mutants, thereby revealing a new mode of regulating the co‐transporter biogenesis and intracellular trafficking. Elucidating the molecular mechanisms of the motiffacilitated ER retention and/or export may help to develop therapeutic strategies targeting NKCC2 transport from the ER to the cell surface.

Support or Funding Information

This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale the Centre National de la Recherche Scientifique, and l'Agence Nationale de la Recherche grant ANR‐17‐CE14‐0040/Pheno2Geno; Paris, France.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.