EXPRESSION CLOSING AND CHARACTERIZATION OF ROAT1 - THE BASOLATERAL ORGANIC ANION TRANSPORTER IN RAT-KIDNEY

Expression cloning in Xenopus laevis oocytes was used to isolate an or ganic anion transport protein from rat kidney, A cDNA library was cons tructed from size-fractionated poly(A)(+) RNA and screened for probene cid-sensitive transport of p-aminohippurate (PAH), A 2,227-base pair c DNA clone containing a 1,656-base pair open reading frame coding for a peptide 551 amino acids long was isolated and named ROAT1, ROAT1-medi ated transport of 50 mu M [H-3]PAH was independent of imposed changes in membrane potential, Transport was significantly inhibited at 4 degr ees C, or upon incubation with other organic anions, but nea, by the o rganic cation tetraethyl-ammonium, by the multidrug resistance ATPase inhibitor cyclosporin A, or by urate, External glutarate and alpha-ket oglutarate (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 upt ake was trans-stimulated in oocytes preloaded with glutarate, whereas the dicarboxylate methylsuccinate, which is not accepted by the basola teral exchanger, did not trans-stimulate. Finally, ROAT1-mediated PAH transport was saturable, with an estimated K-m of 70 mu M. Each of the se properties is identical to those previously described for the basol ateral alpha-ketoglutarate/PAH exchanger in isolated membrane vesicles or intact renal tubules.