Heterologous expression and functional characterization of a mouse renal organic anion transporter in mammalian cells.
Organic anion transporters play an essential role in eliminating a wide range of organic anions including endogenous compounds, xenobiotics, and their metabolites from kidney, thereby preventing their potentially toxic effects within the body. The goal of this study was to extend our previous study on the functional characterization and post-translational modification of a mouse kidney organic anion transporter (mOAT), in a mammalian cell system, COS-7 cells. The transporter-mediated p-aminohippurate (PAH) uptake was saturable, probenecid-sensitive, and inhibited by a wide range of organic anions including vitamins, anti-hypertensive drugs, anti-tumor drugs, and anti-inflammatory drugs. Tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited the transport activity. Immunofluorescence provided evidence that most of the protein remained in the intracellular compartment in tunicamycin-treated cells. Diethyl pyrocarbonate (DEPC), a histidine residue-specific reagent, completely blocked PAH transport. The inhibitory effect by DEPC was significantly protected (90%) by pretreating the cells with excess unlabeled PAH, suggesting that the histidine residues may be close to the PAH binding sites. Finally, in situ mRNA localization was studied in postnatal mouse kidney. The expression was observed in proximal tubules throughout development. We conclude that COS-7 cells may be useful in pharmacological and molecular biological studies of this carrier. The carbohydrate moieties are necessary for the proper trafficking of mOAT to the plasma membrane, and histidine residues appear to be important for the transport function.
Kuze
K
K
Department of Medicine, The Mount Sinai School of Medicine, New York, New York 10029, USA.
Graves
P
P
Leahy
A
A
Wilson
P
P
Stuhlmann
H
H
You
G
G
D000818:Animals; D027321:Anion Transport Proteins; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000502:physiology*; D006031:Glycosylation; D006639:Histidine / Q000378:metabolism; D017403:In Situ Hybridization; D007668:Kidney / Q000187:drug effects / Q000254:growth & development*; D007700:Kinetics; D051379:Mice; D011339:Probenecid / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D012076:Renal Agents / Q000494:pharmacology; D013379:Substrate Specificity; D014415:Tunicamycin / Q000494:pharmacology; D010130:p-Aminohippuric Acid / Q000378:metabolism
D027321:Anion Transport Proteins; D002352:Carrier Proteins; D012076:Renal Agents; D014415:Tunicamycin; D006639:Histidine; D011339:Probenecid; D010130:p-Aminohippuric Acid