Aim: 

The renal NKCC2 cotransporter is selectively expressed in the apical membranes of cells of thick ascending limb (TAL) of the mammalian kidney where it is the target of the clinically important loop diuretics. In contrast, the "secretory" NKCC1 cotransporter is localized in the basolateral membranes of many epithelia where is involved in Cl^- secretion and cell volume regulation. The aim of this work is to identify the sorting signal(s) that direct apical or basolateral trafficking of NKCCs.

Methods: 

We generated chimeras between the two isoforms of the cotransporter. Then we expressed these constructs in polarized renal epithelial cell lines and analyzed their membrane localization by Immunofluorescence Confocal Microscopy and biotinylation experiments. To assess whether the chimeras retained their physiological activity, we performed functional experiments by ⁸⁶Rb⁺ influx assay.

Results: 

Chimeras made by exchanging the three large portions of the cotransporter (N-terminal, C-terminal, and Transmembrane domains) revealed that NKCC2 C-terminus was able to induce the apical localization of NKCC1 backbone. The analysis of subsequent C-terminal chimeras resulted in the identification of an amino acid stretch in NKCC2 containing confomation dependent apical sorting determinants.and a dileucine motif in NKCC1 as the essential component of its basolateral sorting signal. All chimeras resulted functional active demonstrating that the constructs were correctly folded.

Conclusion: 

The cytoplasmic C-terminus of both NKCC1 and NKCC2 contains information necessary for sorting.