Kidney Week

Abstract: PO1078

Diacidic Motif Is Required for Efficient Transport of NKCC2 to the Plasma Membrane

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Basic
  November 04, 2021 | Location: On-Demand, Virtual Only
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid, Electrolyte, and Acid-Base Disorders

• 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

• Bakhos Al Douaihy, Dalal, Sorbonne Universite, Paris, Île-de-France, France

Dalal Bakhos Al Douaihy, PhD

• Seaayfan, Elie, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany

Elie Seaayfan,

• Demaretz, Sylvie, Sorbonne Universite, Paris, Île-de-France, France

Sylvie Demaretz,

• Laghmani, Kamel, Sorbonne Universite, Paris, Île-de-France, France

Kamel Laghmani,

• Kömhoff, Martin, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany

Martin Kömhoff,

Group or Team Name

• Inserm, CNRS-ERL8228

Background

Mutations in the apical Na-K-2Cl cotransporter, NKCC2, cause type I Bartter syndrome (BS1), a life-threatening kidney disease. We have previously demonstrated that BS1 nonsense mutation Y998X, which interferes with the highly conserved dileucine like motifs of NKCC2 C-terminus, compromises NKCC2 surface delivery through ER retention mechanisms. However, whether these dileucine like motifs are sufficient for anterograde trafficking of NKCC2 remained to be determined. Consequently, the aim of the present study was to investigate whether additional motifs are required for NKCC2 efficient transport to the plasma membrane.

Methods

NKCC2 protein expression was monitored in transiently transfected OKP and HEK cells, using immunoblot and confocal imaging. NKCC2 surface expression was assessed by cell surface biotinylation assay. NKCC2 stability and maturation was monitored by cycloheximide chase assay.

Results

Among the motifs identified as ER export signals in ion channels are the diacidic D/E-X-D/E motifs, which have been shown to promote interaction of cargo with the coat complex II (COPII) budding machinery. Interestingly, sequence analysis of NKCC2 C-terminus revealed the presence of two di-acidic motifs, ⁹⁴⁹EEE⁹⁵¹ and ¹⁰¹⁹DAE¹⁰²¹, located upstream and downstream of BS1 mutation Y998X, respectively. Importantly, mutation of ¹⁰¹⁹DAE¹⁰²¹ to ¹⁰¹⁹AAA¹⁰²¹ disrupted glycosylation and cell surface expression of NKCC2, whereas mutation of ⁹⁴⁹EEE⁹⁵¹ had no effect. Cycloheximide chase analysis demonstrated that the absence of the terminally glycosylated form of ¹⁰¹⁹AAA¹⁰²¹ was not due to increased rates of degradation of mutant co-transporters, but was instead caused by defect in maturation. Accordinglty, co-immunolocalization experiments showed that ¹⁰¹⁹AAA¹⁰²¹ was trapped in the ER. Finally, overexpression of dominant negative mutant of Sar1 GTPase completely abolished NKCC2 maturation, clearly indicating that NKCC2 exit from the ER is COPII dependent.

Conclusion

Our data indicate that in addition to highly conserved dileucine like motifs of NKCC2 C-terminus, the cotransporter uses also a di-acidic exit code for export from the ER and targeting to the cell surface. Elucidating the molecular mechanisms of the motif-facilitated ER export may help to develop therapeutic strategies targeting NKCC2 transport from the ER to the cell surface.

Funding

• Government Support – Non-U.S.