Real time monitoring of NKCC2 endocytosis by total internal reflection fluorescence microscopy (892.19)

The apical Na‐K‐2Cl cotransporter NKCC2 mediates NaCl reabsorption by the thick ascending limb (TAL). The amount of NKCC2 at the apical membrane of TALs is determined by dynamic endocytosis from the apical membrane mediated by clathrin and lipid rafts. However, the time resolution of previous methods used to measure endocytosis was low (5 min), and internalization of NKCC2 containing vesicles not measurable. We hypothesize that TIRF microscopy imaging of fluorescently labeled apical NKCC2 would allow us to monitor its endocytosis in real‐time. We transduced polarized MDCK cells (in Transwell filters) with an N‐terminus GFP‐tagged NKCC2 using adenoviruses. Similar to native TALs, GFP‐NKCC2 was located in the apical membrane, at and above tight junctions. Surface biotinylation confirmed targeting of mature GFP‐NKCC2 (≍190 kDa) to the apical membrane. For TIRF imaging, cells were placed in a chamber with the apical membrane facing the TIRF illumination field. To selectively label NKCC2 at the apical surface we generated NKCC2 containing a biotin acceptor domain of 16 aa between one of the extracellular loops. Once expressed in MDCK cells, we specifically biotinylated surface NKCC2 with recombinant biotin ligase. Labeling apical NKCC2 with streptavidin‐Qdot, or streptavidin‐AlexaFluor allowed us to monitor single endocytic events and apical surface NKCC2 distribution in real‐time. In the absence of biotin ligase we did not observe labeling, indicating specificity for NKCC2 labeling. The overall rate of constitutive NKCC2 endocytosis was 1.2±0.1 % per minute (n=6). In MDCK and TAL cells most apical NKCC2 was located within small discrete domains that exhibited little lateral movement. We conclude that TIRF microscopy of labeled NKCC2 allows the imaging of individual endocytic events at the apical membrane. Grant Funding Source : NIH‐AHA