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Research Article

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

¹The University of Chicago ²University of Cincinnati ³Vanderbilt University Med. Ctr.

Submitted 16 March 2009 ; revised 13 October 2009 ; accepted in final form 27 October 2009

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 chloride 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. However, the specific endocytic entry pathway is still not defined. We used a Madin-Darby canine kidney (MDCK) cell line stably transfected with EGFP-NKCC1 to map NKCC1 entry during PMA exposure. At given times, we fixed and stained the cells with antibodies against specific markers (e.g. dynamin II, clathrin heavy chain, caveolin-1). Furthermore, we used chlorpromazine, methyl-β-cyclodextrin, amiloride, and dynasore, blockers respectively of the clathrin, caveolin, and macropinocytosis pathways, and the vesicle "pinchase" dynamin. We found that PMA caused dose and time dependent NKCC1 endocytosis. After 2.5 min exposure to PMA, ~80% of EGFP-NKCC1 endocytic vesicles colocalized with clathrin, and ~40% colocalized with dynamin II and with the transferrin receptor, whose uptake 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 only chlorpromazine and dynasore caused significant inhibition compared to the untreated control (61% and 25% respectively at 2.5 min). Together, these results strongly support the conclusion that NKCC1 endocytosis stimulated by PMA is associated with a clathrin pathway.

NKCC1; chlorpromazine; internalization; transferrin receptor