Secretory Carrier Membrane Protein 2 Regulates Exocytic Insertion of NKCC2 into the Cell Membrane

Nancy Zaarour,Nadia Defontaine,Sylvie Demaretz,Anie Azroyan,Lydie Cheval,Kamel Laghmani

doi:10.1074/jbc.m110.166546

Abstract

The renal-specific Na-K-2Cl co-transporter, NKCC2, plays a pivotal role in regulating body salt levels and blood pressure. NKCC2 mutations lead to type I Bartter syndrome, a life-threatening kidney disease. Regulation of NKCC2 trafficking behavior serves as a major mechanism in controlling NKCC2 activity across the plasma membrane. However, the identities of the protein partners involved in cell surface targeting of NKCC2 are largely unknown. To gain insight into these processes, we used a yeast two-hybrid system to screen a kidney cDNA library for proteins that interact with the NKCC2 C terminus. One binding partner we identified was SCAMP2 (secretory carrier membrane protein 2). Microscopic confocal imaging and co-immunoprecipitation assays confirmed NKCC2-SCAMP2 interaction in renal cells. SCAMP2 associated also with the structurally related co-transporter NCC, suggesting that the interaction with SCAMP2 is a common feature of sodium-dependent chloride co-transporters. Heterologous expression of SCAMP2 specifically decreased cell surface abundance as well as transport activity of NKCC2 across the plasma membrane. Co-immunolocalization experiments revealed that intracellularly retained NKCC2 co-localizes with SCAMP2 in recycling endosomes. The rate of NKCC2 endocytic retrieval, assessed by the sodium 2-mercaptoethane sulfonate cleavage assay, was not affected by SCAMP2. The surface-biotinylatable fraction of newly inserted NKCC2 in the plasma membrane was reduced by SCAMP2, demonstrating that SCAMP2-induced decrease in surface NKCC2 is due to decreased exocytotic trafficking. Finally, a single amino acid mutation, cysteine 201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, which is believed to regulate exocytosis, abolished SCAMP2-mediated down-regulation of the co-transporter. Taken together, these data are consistent with a model whereby SCAMP2 regulates NKCC2 transit through recycling endosomes and limits the cell surface targeting of the co-transporter by interfering with its exocytotic trafficking.

Highlights

NKCC2 belongs to the cation-chloride co-transporter (CCC)3 family, which comprises two principal branches of membrane proteins [1]
In contrast to results obtained with the proximal region of NKCC2 C terminus, no growth was observed when AH109 yeast cells were transformed with SCAMP2 and NKCC2 C2-term (Fig. 1A, right), indicating that the interaction is specific for the first 108 aa of the NKCC2 C terminus
Our results indicate that SCAMP2 interaction with NKCC2 is specific and depends on the first 108 aa of the co-transporter C terminus

Summary

Introduction

NKCC2 belongs to the cation-chloride co-transporter (CCC)3 family, which comprises two principal branches of membrane proteins [1]. These control experiments were performed to examine the possibility that SCAMP2 may decrease the surface expression of NKCC2 by generally interfering with the migration of proteins through the trafficking pathway leading to the plasma membrane.

Results

Conclusion