Localization of an organic anion transporter-GFP fusion construct (rROAT1-GFP) in intact proximal tubules

Am J Physiol. 1999 Jun;276(6):F864-73. doi: 10.1152/ajprenal.1999.276.6.F864.


The organic anion transporter, rROAT1, is a dicarboxylate/organic anion exchanger, a function associated with the basolateral membrane in rat proximal tubule. To directly establish the subcellular localization of rROAT1 in renal epithelia, we made a rROAT1-green fluorescent protein (GFP) fusion construct (rROAT1-GFP). Plasma membrane-associated fluorescence was observed in rROAT1-GFP-expressing Xenopus oocytes examined by confocal microscopy. Uptake of 3H-labeled p-aminohippurate (PAH) increased 2. 5-fold in rROAT1-GFP-expressing Xenopus oocytes, and this increase was abolished by 1 mM probenecid. Thus the construct was capable of specific organic anion transport. Cultured renal epithelial cell lines (MDCK and LLC-PK1) transfected with the vector pEGFP-C3 showed a diffuse, evenly distributed cytoplasmic signal. However, when transfected with pEGFP-C3/rROAT1 (vector coding for rROAT1-GFP), both cell lines showed predominantly plasma membrane fluorescence. The expression and distribution of rROAT1-GFP in intact renal proximal tubules was also investigated. Isolated killifish (Fundulus heteroclitus) renal tubules transfected with pEGFP-C3/rROAT1 showed marked basal and lateral membrane-associated fluorescence, but no detectable signal in the nucleus or the apical pole of tubule cells. Tubules transfected with pEGFP-C3 showed diffuse cytoplasmic fluorescence. Function of the rROAT1-GFP construct was demonstrated in transfected killifish tubules by fluorescein transport assay. These results demonstrate the basolateral subcellular localization of rROAT1 in polarized renal epithelia and validate a new technique for localizing cloned transporters within intact renal tubules.

MeSH terms

  • Animals
  • Anion Transport Proteins
  • Carrier Proteins / genetics*
  • Cell Line
  • Dogs
  • Green Fluorescent Proteins
  • Indicators and Reagents*
  • Kidney Tubules, Proximal / metabolism*
  • Killifishes
  • LLC-PK1 Cells
  • Luminescent Proteins / genetics*
  • Oocytes / metabolism
  • Rats
  • Recombinant Fusion Proteins / pharmacokinetics*
  • Swine
  • Tissue Distribution
  • Xenopus


  • Anion Transport Proteins
  • Carrier Proteins
  • Indicators and Reagents
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins