Diacidic Motifs in the Carboxyl Terminus Are Required for ER Exit and Translocation to the Plasma Membrane of NKCC2

Mutations in the apical Na-K-2Cl co-transporter, NKCC2, cause type I Bartter syndrome (BS1), a life-threatening kidney disease. We have previously demonstrated that the BS1 variant Y998X, which deprives NKCC2 from its highly conserved dileucine-like motifs, compromises co-transporter surface delivery through ER retention mechanisms. However, whether these hydrophobic motifs are sufficient for anterograde trafficking of NKCC2 remains to be determined. Interestingly, sequence analysis of NKCC2 C-terminus revealed the presence of consensus di-acidic (D/E-X-D/E) motifs, ⁹⁴⁹EEE⁹⁵¹ and ¹⁰¹⁹DAELE¹⁰²³, located upstream and downstream of BS1 mutation Y998X, respectively. Di-acidic codes are involved in ER export of proteins through interaction with COPII budding machinery. Importantly, whereas mutating ⁹⁴⁹EEE⁹⁵¹ motif to ⁹⁴⁹AEA⁹⁵¹ had no effect on NKCC2 processing, mutating ¹⁰¹⁹DAE¹⁰²¹ to ¹⁰¹⁹AAA¹⁰²¹ heavily impaired complex-glycosylation and cell surface expression of the cotransporter in HEK293 and OKP cells. Most importantly, triple mutation of D, E and E residues of ¹⁰¹⁹DAELE¹⁰²³ to ¹⁰¹⁹AAALA¹⁰²³ almost completely abolished NKCC2 complex-glycosylation, suggesting that this mutant failed to exit the ER. Cycloheximide chase analysis demonstrated that the absence of the terminally glycosylated form of ¹⁰¹⁹AAALA¹⁰²³ was caused by defects in NKCC2 maturation. Accordingly, co-immunolocalization experiments revealed that ¹⁰¹⁹AAALA¹⁰²³ was trapped in the ER. Finally, overexpression of a dominant negative mutant of Sar1-GTPase abolished NKCC2 maturation and cell surface expression, clearly indicating that NKCC2 export from the ER is COPII-dependent. Hence, our data indicate that in addition to the di-leucine like motifs, NKCC2 uses di-acidic exit codes for export from the ER through the COPII-dependent pathway. We propose that any naturally occurring mutation of NKCC2 interfering with this pathway could form the molecular basis of BS1.