Basolateral localization of organic cation transporter 2 in intact renal proximal tubules

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 fluoresce nt protein (GFP) fusion construct (rOCT2-GFP) and determined its localizati on within Xenopus laevis oocytes and renal cells using confocal microscopy. Oocytes expressing rOCT2-GFP exhibited plasma membrane fluorescence as wel l as greatly increased specific, potential-driven uptake of [C-14] tetraeth ylammonium (TEA). Polarized monolayers of renal epithelial cell lines [LLC- PK1 and Madin-Darby canine kidney (MDCK)] transiently transfected with pEGF P-C3, which codes for a cytoplasmic GFP, showed a diffuse, evenly distribut ed cytoplasmic signal with no plasma membrane fluorescence. In contrast, ce lls transiently transfected with pEGFP-C3/rOCT2 (the vector coding for rOCT 2-GFP) showed predominantly plasma membrane fluorescence, which was most pr ominent in the lateral membrane. MDCK cells stably expressing rOCT2-GFP (MD CK/rOCT2-GFP) maintained in long-term culture showed a greatly increased ba sal 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 investig ated in isolated killifish (Fundulus heteroclitus) renal proximal tubules. On expression of rOCT2-GFP, transfected tubules showed marked basal and lat eral membrane fluorescence, with no detectable signal at the apical membran e. In contrast, tubules expressing a luminal sodium-dicarboxylate cotranspo rter (rbNaDC-1)-GFP construct showed apical membrane fluorescence, and tubu les expressing cytoplasmic GFP had a diffuse cytoplasmic fluorescence. Thes e results indicate that rOCT2 is basolateral in renal proximal tubule cells .