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The American Journal of Physiology - Cell Physiology
Year: 2010  |  Volume: 298  |  Issue: 1  |  Page No.: 85 - 97

Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway

A Mykoniatis, L Shen, M Fedor Chaiken, J Tang, X Tang, R. T Worrell, E Delpire, J. R Turner, K. S Matlin, P Bouyer and J. B. Matthews    

Abstract:

In secretory epithelial cells, the basolateral Na+-K+-2Cl cotransporter (NKCC1) plays a major role in salt and fluid secretion. Our laboratory has identified NKCC1 surface expression as an important regulatory mechanism for Cl secretion in the colonic crypt cell line T84, a process also present in native human colonic crypts. We previously showed that activation of protein kinase C (PKC) by carbachol and phorbol 12-myristate 13-acetate (PMA) decreases NKCC1 surface expression in T84 cells. However, the specific endocytic entry pathway has not been defined. We used a Madin-Darby canine kidney (MDCK) cell line stably transfected with enhanced green fluorescent protein (EGFP)-NKCC1 to map NKCC1 entry during PMA exposure. At given times, we fixed and stained the cells with specific markers (e.g., dynamin II, clathrin heavy chain, and caveolin-1). We also used chlorpromazine, methyl-β-cyclodextrin, amiloride, and dynasore, blockers of the clathrin, caveolin, and macropinocytosis pathways and the vesicle "pinchase" dynamin, respectively. We found that PMA caused dose- and time-dependent NKCC1 endocytosis. After 2.5 min of PMA exposure, ~80% of EGFP-NKCC1 endocytic vesicles colocalized with clathrin and ~40% colocalized with dynamin II and with the transferrin receptor, the uptake of which is also mediated by clathrin-coated vesicles. We did not observe significant colocalization of EGFP-NKCC1 endocytic vesicles with caveolin-1, a marker of the caveolae-mediated endocytic pathway. We quantified the effect of each inhibitor on PMA-induced EGFP-NKCC1 endocytosis and found that only chlorpromazine and dynasore caused significant inhibition compared with the untreated control (61% and 25%, respectively, at 2.5 min). Together, these results strongly support the conclusion that PMA-stimulated NKCC1 endocytosis is associated with a clathrin pathway.

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