NKCC2 surface expression in mammalian cells: down-regulation by novel interaction with aldolase B.

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