Expression Cloning and Characterization of ROAT1

Douglas H. Sweet, Natascha A. Wolff, John B. Pritchard
1997 Journal of Biological Chemistry  
Expression cloning in Xenopus laevis oocytes was used to isolate an organic anion transport protein from rat kidney. A cDNA library was constructed from sizefractionated poly(A) ؉ RNA and screened for probenecidsensitive transport of p-aminohippurate (PAH). A 2,227base pair cDNA clone containing a 1,656-base pair open reading frame coding for a peptide 551 amino acids long was isolated and named ROAT1. ROAT1-mediated transport of 50 M [ 3 H]PAH was independent of imposed changes in membrane
more » ... ntial. Transport was significantly inhibited at 4°C, or upon incubation with other organic anions, but not by the organic cation tetraethylammonium, by the multidrug resistance ATPase inhibitor cyclosporin A, or by urate. External glutarate and ␣-ketoglutarate (1 mM), both counterions for basolateral PAH exchange, also inhibited transport, suggesting that ROAT1 is functionally similar to the basolateral PAH carrier. Consistent with this conclusion, PAH uptake was trans-stimulated in oocytes preloaded with glutarate, whereas the dicarboxylate methylsuccinate, which is not accepted by the basolateral exchanger, did not trans-stimulate. Finally, ROAT1-mediated PAH transport was saturable, with an estimated K m of 70 M. Each of these properties is identical to those previously described for the basolateral ␣-ketoglutarate/PAH exchanger in isolated membrane vesicles or intact renal tubules.
doi:10.1074/jbc.272.48.30088 pmid:9374486 fatcat:c6yt6gidq5djlht4qss255nu2m