Expression cloning and characterization of ROAT1. The basolateral organic anion transporter in rat kidney.
Expression cloning in Xenopus laevis oocytes was used to isolate an organic anion transport protein from rat kidney. A cDNA library was constructed from size-fractionated poly(A)+ RNA and screened for probenecid-sensitive transport of p-aminohippurate (PAH). A 2, 227-base 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 mu M [3H]PAH was independent of imposed changes in membrane potential. Transport was significantly inhibited at 4 degrees 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 alpha-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 Km of 70 mu M. Each of these properties is identical to those previously described for the basolateral alpha-ketoglutarate/PAH exchanger in isolated membrane vesicles or intact renal tubules.
Sweet
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DH
Laboratory of Pharmacology and Chemistry, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA. pritchard@niehs.nih.gov
Wolff
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NA
Pritchard
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D027321:Anion Transport Proteins; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016764:Cell Polarity; D003001:Cloning, Molecular; D015870:Gene Expression; D007668:Kidney / Q000737:chemistry* / Q000378:metabolism; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009865:Oocytes; D027362:Organic Anion Transport Protein 1; D027361:Organic Anion Transporters; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment; D012995:Solubility; D013379:Substrate Specificity; D014018:Tissue Distribution; D014982:Xenopus laevis; D010130:p-Aminohippuric Acid / Q000378:metabolism
D027321:Anion Transport Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; D027362:Organic Anion Transport Protein 1; D027361:Organic Anion Transporters; D012333:RNA, Messenger; C105033:Slc22a6 protein, mouse; D010130:p-Aminohippuric Acid