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Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
The localization of organic cation transporter 2 (OCT2) within renal cells is the subject of considerable controversy, resulting in marked uncertainty as to its function. To resolve this issue, we made an OCT2/green fluorescent protein (GFP) fusion construct (rOCT2-GFP) and determined its localization within Xenopus laevis oocytes and renal cells using confocal microscopy. Oocytes expressing rOCT2-GFP exhibited plasma membrane fluorescence as well as greatly increased specific, potential-driven uptake of [14C]tetraethylammonium (TEA). Polarized monolayers of renal epithelial cell lines [LLC-PK1 and Madin-Darby canine kidney (MDCK)] transiently transfected with pEGFP-C3, which codes for a cytoplasmic GFP, showed a diffuse, evenly distributed cytoplasmic signal with no plasma membrane fluorescence. In contrast, cells transiently transfected with pEGFP-C3/rOCT2 (the vector coding for rOCT2-GFP) showed predominantly plasma membrane fluorescence, which was most prominent in the lateral membrane. MDCK cells stably expressing rOCT2-GFP (MDCK/rOCT2-GFP) maintained in long-term culture showed a greatly increased basal and lateral membrane fluorescence. When grown on porous supports, MDCK/rOCT2-GFP monolayers showed specific, potential-driven TEA uptake from the basal side. Finally, expression and distribution of rOCT2-GFP were investigated in isolated killifish (Fundulus heteroclitus) renal proximal tubules. On expression of rOCT2-GFP, transfected tubules showed marked basal and lateral membrane fluorescence, with no detectable signal at the apical membrane. In contrast, tubules expressing a luminal sodium-dicarboxylate cotransporter (rbNaDC-1)-GFP construct showed apical membrane fluorescence, and tubules expressing cytoplasmic GFP had a diffuse cytoplasmic fluorescence. These results indicate that rOCT2 is basolateral in renal proximal tubule cells.
green fluorescent protein; kidney; killifish; transfection; Madin-Darby canine kidney cells; LLC-PK1 cells
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