Expression Cloning and Characterization of ROAT1

Abstract

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 2,227-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 M [ 3 H]PAH was independent of imposed changes in membrane potential. 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.Renal organic anion transport has been widely studied for more than a century, both as a prototypic transport process and as a primary means for removal of xenobiotics from the body. Because many foreign chemicals, including plant and animal toxins, drugs, and pesticides, are organic anions or are metabolized to organic anions, the renal organic anion secretory system plays a critical role in limiting or preventing their toxicity. Over the last decade, a great deal of progress has been made toward understanding the physiology of this system, particularly its coupling to metabolic energy. Thus, it is now well established that organic anion secretion is a complex process involving distinctly different proteins at the apical and basolateral membranes of the proximal tubule (Fig.

Identification of Glutathione as a Driving Force and Leukotriene C4 as a Substrate for oatp1, the Hepatic Sinusoidal Organic Solute Transporter

“…One family comprises the organic anion/dicarboxylate exchangers, and includes kidney oat1 (or roat1) (35)(36)(37). Cellular uptake of organic anions on kidney oat1 was recently demonstrated to be directly coupled to efflux of ␣-ketoglutarate (35,36).…”

Functional characterization and immunolocalization of the transporter encoded by the life-extending gene Indy

“…In the mammalian kidney, dicarboxylate transporters are involved in the excretion of anionic xenobiotics, drugs, and environmental toxicants (18). The prototypic organic anion transport mechanism mediating xenobiotic secretion is the paminohippurate (PAH)͞dicarboxylate exchanger (19)(20)(21)(22). As shown in Fig.…”

Identification of a New Urate and High Affinity Nicotinate Transporter, hOAT10 (SLC22A13)

“…The family of OATs (SLC22A) was discovered in 1997 with the cloning of the renal rat and flounder OAT1 (rOAT1 (5,6) and fOAT (7)). Meanwhile, nine OATs were functionally identified (OAT1, OAT2 (8), OAT3 (9), OAT4 (10), Oat5 (11), Oat6 (12), OAT7 (13), Oat v 1 (14), and the urate transporter URAT1 (15)).…”