NKCC2 Surface Expression in Mammalian Cells

Abstract

Apical bumetanide-sensitive Na؉ -K ؉ -2Cl ؊ co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at understanding the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.

A Highly Conserved Motif at the COOH Terminus Dictates Endoplasmic Reticulum Exit and Cell Surface Expression of NKCC2

“…20 mM NH 4 Cl was then added to the medium, inducing a very rapid initial cellular alkalinization followed by a pH i recovery. The initial rate of intracellular pH recovery (dpH i /dt) was measured over the first 20 s of records, as reported earlier (23,25). The ⌬pH i caused by NH 4 Cl addition was used to calculate the cell buffer capacity, which was not different between the studied groups (data not shown).…”

“…Calibration of the 2Ј,7Ј-bis(carboxyethyl)-5,6-carboxyfluorescein excitation ratio was accomplished using the nigericin technique. Na-K-2Cl cotransport activity was measured as bumetanide-sensitive NH 4 influx, as previously described (23,24). Cells were first bathed at 37°C in a CO 2 -free Hepes/Tris-buffered medium containing 135 mM NaCl, 10 mM Hepes (pH 7.4), 5 mM KCl, 1 mM MgCl 2 , 0.8 mM KH 2 PO 4 , 0.2 mM K 2 HOP 4 , 1 mM CaCl 2 , and 10 mM BaCl 2 to measure base-line pH i .…”

“…To characterize the expression and function of WT NKCC2 and Y998X proteins in renal epithelial cell lines, we transiently transfected NH 2 -terminally tagged WT and truncated NKCC2 into cultured renal cells. We showed previously that NH 2 -terminally tagging NKCC2 does not affect co-transporter trafficking and function (23).…”

“…Ϫ transport activity was assessed by estimating the rate of intracellular acidification caused by entry into the cells of NH 4 ϩ via this transport mechanism after abrupt application of NH 4 Cl to the cells (23,24). As illustrated in Fig.…”

“…We were recently able to express NKCC2 protein in mammalian cells (23), providing therefore a powerful tool to study and understand the molecular mechanisms underlying the cotransporter expression and regulation in renal cells. This allowed us, in this study, to take the advantage of the existence of natural mutants altering the COOH-terminal tail of the cotransporter to investigate the role of the COOH terminus in the biogenesis of NKCC2 and to explore possible mechanisms implicated in BS1.…”