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Expression Cloning and Characterization of ROAT1

1997; Elsevier BV; Volume: 272; Issue: 48 Linguagem: Inglês

10.1074/jbc.272.48.30088

1083-351X

Douglas H. Sweet, Natascha A. Wolff, John B. Pritchard,

Metabolism and Genetic Disorders

Expression cloning in Xenopus laevisoocytes 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 μm [3H]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 α-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 nottrans-stimulate. Finally, ROAT1-mediated PAH transport was saturable, with an estimated K m of 70 μm. Each of these properties is identical to those previously described for the basolateral α-ketoglutarate/PAH exchanger in isolated membrane vesicles or intact renal tubules. Expression cloning in Xenopus laevisoocytes 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 μm [3H]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 α-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 nottrans-stimulate. Finally, ROAT1-mediated PAH transport was saturable, with an estimated K m of 70 μm. Each of these properties is identical to those previously described for the basolateral α-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. 1; for review, see Ref. 1Pritchard J.B. Miller D.S. Physiol. Rev. 1993; 73: 765-796Crossref PubMed Scopus (462) Google Scholar). Transport across the basolateral membrane is energetically uphill. It is accomplished by a tertiary active process in which (a) Na+,K+-ATPase establishes the out > in Na+ gradient, (b) Na+/α-ketoglutarate cotransport driven by the movement of Na+ down its concentration gradient, in concert with intracellular metabolic α-ketoglutarate generation, sustains an out < in dicarboxylate gradient, and (c) dicarboxylate/organic anion exchange moves the organic anion substrate into the cell (2Shimada H. Moewes B. Burckhardt G. Am. J. Physiol. 1987; 253: F795-F801PubMed Google Scholar, 3Pritchard J.B. Am. J. Physiol. 1988; 255: F597-F604PubMed Google Scholar). This cascade of events indirectly links organic anion transport to metabolic energy and the Na+ gradient, allowing entry of negatively charged substrate against both its chemical concentration gradient and the electrical potential of the cell. Once inside the cell, organic anions are subject to intracellular binding and to sequestration within vesicular structures (4Holohan P.D. Pessah N.I. Ross C.R. J. Pharmacol. Exp. Ther. 1975; 195: 22-33PubMed Google Scholar, 5Miller D.S. Stewart D.E. Pritchard J.B. Am. J. Physiol. 1993; 264: R882-R890PubMed Google Scholar). Finally, luminal exit is thought to occur by anion exchange and/or facilitated diffusion (6Aronson P.S. Annu. Rev. Physiol. 1989; 51: 419-441Crossref PubMed Scopus (90) Google Scholar, 7Werner D. Martinez F. Roch Ramel F. J. Pharmacol. Exp. Ther. 1990; 252: 792-799PubMed Google Scholar, 8Martinez F. Manganel M. Montrose Rafizadeh C. Werner D. Roch Ramel F. Am. J. Physiol. 1990; 258: F1145-F1153PubMed Google Scholar).In contrast to the physiology of the organic anion transport system, precise information about the structural properties of the transport proteins that make up this system are not yet available. However, considerable progress has recently been made for a variety of other transport proteins through the application of expression cloning techniques, leading to increased understanding of the regulation of their expression, identification of substrate binding sites, and a much more complete appreciation of their mechanisms of action (9Coady M.J. Pajor A.M. Toloza E.M. Wright E.M. Arch. Biochem. Biophys. 1990; 283: 130-134Crossref PubMed Scopus (16) Google Scholar, 10Hediger M.A. Coady M.J. Ikeda T.S. Wright E.M. Nature. 1987; 330: 379-381Crossref PubMed Scopus (800) Google Scholar, 11Grundemann D. Gorboulev V. Gambaryan S. Veyhl M. Koepsell H. Nature. 1994; 372: 549-552Crossref PubMed Scopus (604) Google Scholar). We report here the successful isolation and characterization of a cDNA encoding the organic anion transporter from rat kidney, ROAT1.RESULTSScreening of cDNA LibraryTotal poly(A)+ RNA was isolated from rat kidney and size-fractionated on a linear sucrose gradient (14Wolff N.A. Philpot R.M. Miller D.S. Pritchard J.B. Mol. Cell. Biochem. 1992; 114: 35-41Crossref PubMed Scopus (12) Google Scholar). Fractions corresponding to 12.3% and 11.1% sucrose were shown to support probenecid-sensitive uptake of 50 μm[3H]PAH that was 2–3-fold higher than that observed for the original unfractionated starting material when injected intoX. laevis oocytes (Fig.2 A). The 11.1% sucrose fraction mRNA was used as template for the construction of a cDNA library, which was screened by expression cloning inXenopus oocytes, yielding a single purified clone that supported PAH uptake (Fig. 2 B). In the presence of 1 mm probenecid, PAH uptake by oocytes expressing the cloned transporter was always reduced to the level observed for water-injected oocytes. Electrophoresis of the in vitro cRNA synthesis product showed a band ∼2.3 kb in size, which corresponds with the previously reported size range for kidney mRNA that supported PAH uptake (14Wolff N.A. Philpot R.M. Miller D.S. Pritchard J.B. Mol. Cell. Biochem. 1992; 114: 35-41Crossref PubMed Scopus (12) Google Scholar). We refer to this cDNA clone as renalorganic anion transporter1, or ROAT1.Figure 2Expression cloning of a cDNA encoding an organic anion transporter. A rat kidney cDNA library was screened for the ability to support PAH uptake. cRNA transcribed from library plasmid DNA was injected into Xenopus oocytes (20 ng/oocyte) and allowed to express. Two days after injection, the oocytes were incubated for 1 h in 50 μm[3H]PAH in the absence (No Inhibitor) or presence (+ Probenecid) of 1 mm probenecid. Each column shows the mean value ± S.E. for 2 animals (10 oocytes/treatment/animal). A, evaluation of rat kidney poly(A)+ RNA sucrose gradient fractions; B, response from a single positive clone (ROAT1).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Molecular Characterization of ROAT1The complete DNA sequence of both strands of ROAT1 was determined, and the sense strand sequence is presented in Fig.3 A. The ROAT1 cDNA is 2,227 bp long, including 253 bp of 5′-untranslated region, a single, large open reading frame 1,656 bp long, and 318 bp of 3′-untranslated sequence. The ATG at position 254 has the strongest correlation with Kozak's consensus sequence for translation initiation (23Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4151) Google Scholar), giving a predicted protein length of 551 amino acids (Fig. 3 A). Hydropathy analysis with five different modeling programs yielded four different profiles for potential α-helical membrane-spanning domains (Fig. 3 B). Several regions were identified by all the modeling algorithms, and a few less well defined regions identified by some. However, all predictions agree that there is a large extracellular loop at approximately amino acid residues 40–136. Within this domain are several potential modification sites, including possible N-linked glycosylation sites at Asn-39, Asn-56, Asn-92, and Asn-113; four cysteine residues at positions 49, 78, 105, and 128 that could participate in disulfide bond formation; and a possible protein kinase C site at Ser-129. In addition, there are four more protein kinase C consensus sites located at Ser-271, Ser-278, Thr-284, and Thr-334 and potential casein kinase II sites at Ser-325, Thr-515, and Ser-544. These latter consensus sites may or may not be located intracellularly, depending on the model used.Figure 3DNA and predicted amino acid sequence of ROAT1. Both strands of the ROAT1 cDNA were sequenced completely. A, nucleotide sequence of the sense strand of ROAT1. The predicted amino acid sequence of the single large open reading frame is given. B, Kyte-Doolittle hydropathy analysis. Proposed transmembrane domains are indicated bynumbers above the hydropathy plot. The analysis package used to obtain each prediction is given.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A BLAST search (24Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69163) Google Scholar) of the GenBank data base (25Benson D.A. Boguski M.F. Lipman D.J. Ostell J. Nucleic Acids Res. 1997; 25: 1-6Crossref PubMed Scopus (198) Google Scholar) identified a single DNA sequence, NKT (accession number U52842; Ref. 26Lopez-Nieto C.E. You G. Bush K.T. Barros E.J. Beier D.R. Nigam S.K. J. Biol. Chem. 1997; 272: 6471-6478Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar), with significant homology to ROAT1. NKT was isolated from mouse kidney and described as a gene product related to the organic cation transporter family (26Lopez-Nieto C.E. You G. Bush K.T. Barros E.J. Beier D.R. Nigam S.K. J. Biol. Chem. 1997; 272: 6471-6478Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). A Smith and Waterman (27Smith T.F. Waterman M.S. Adv. Appl. Math. 1981; 2: 482-489Crossref Scopus (565) Google Scholar) alignment of the two DNA sequences showed a 95% identity, whether the 5′- and 3′-untranslated regions were included or not. Needleman and Wunsch (28Needleman S.B. Wunsch C.D. J. Mol. Biol. 1970; 48: 443-453Crossref PubMed Scopus (7656) Google Scholar) analysis of the predicted amino acid sequences for the two proteins yielded a 96% similarity and 95% identity. The two peptides differ at 27 positions, and there is a six-amino acid gap in NKT corresponding to residues 85–90 in ROAT1 (Fig. 4).Figure 4Amino acid sequence alignment of ROAT1 and NKT. The peptide sequence alignment of these two proteins illustrates their high degree of homology. The two sequences differ at 27 positions, as well as a six-amino acid gap present in NKT corresponding to residues 85–90 in ROAT1. These differences are indicated by boxes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The only other known cloned organic anion transporter is oatp from rat liver (22Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (544) Google Scholar). Comparison of ROAT1 and oatp revealed no homology between the two at the DNA or peptide level (27Smith T.F. Waterman M.S. Adv. Appl. Math. 1981; 2: 482-489Crossref Scopus (565) Google Scholar, 28Needleman S.B. Wunsch C.D. J. Mol. Biol. 1970; 48: 443-453Crossref PubMed Scopus (7656) Google Scholar). Comparison between the peptide sequence of ROAT1 and those of the organic cation transporters OCT1 (11Grundemann D. Gorboulev V. Gambaryan S. Veyhl M. Koepsell H. Nature. 1994; 372: 549-552Crossref PubMed Scopus (604) Google Scholar) and OCT2 (29Okuda M. Saito H. Urakami Y. Takano M. Inui K. Biochem. Biophys. Res. Commun. 1996; 224: 500-507Crossref PubMed Scopus (324) Google Scholar), and another liver transporter of unknown substrate specificity, NLT (30Simonson G.D. Vincent A.C. Roberg K.J. Huang Y. Iwanij V. J. Cell. Sci. 1994; 107: 1065-1072PubMed Google Scholar), showed some homology: ROAT1/OCT1, 40% similarity and 33% identity; ROAT1/OCT2, 41% similarity and 31% identity; ROAT1/NLT, 48% similarity and 38% identity. No obvious regions of highly conserved sequence were identified, even in the putative membrane-spanning domains. Rather, the peptides seem to share a low level of identity throughout their sequence. There are, however, three motifs conserved among ROAT1, NKT, OCT1, OCT2, and NLT: the positioning of four cysteine residues and a protein kinase C consensus site within the extracellular loop between predicted membrane-spanning domains 1 and 2, and two casein kinase II consensus sites (TableI). Whether any of these features is involved in transporter function is unknown at this time.Table IConserved peptide motifs in cloned organic anion and cation transporterscDNACysteine residuesPKC consensus sitesCKII consensus sitesROAT149, 78, 105, 128Ser-271Ser-325, Thr-515NKT49, 78, 99, 122Ser-265Ser-319, Thr-509OCT150, 89, 122, 143Ser-286Ser-334, Thr-525OCT250, 89, 122, 143Ser-286Ser-334, Thr-525NLT49, 80, 113, 136Ser-279Ser-331, Thr-521Table lists amino acid residues conserved among these transport proteins with corresponding position numbers within their respective peptide chains. PKC, protein kinase C; CKII, casein kinase II. Open table in a new tab Southern and Northern AnalysisA Southern blot of cDNA library fractions performed during the course of the screen demonstrated that the positive library fractions containing ROAT1 did not contain any sequence homologous to a 643-bp probe from oatp (22Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (544) Google Scholar), the only other known organic anion transporter (Fig. 5, left panel). Subsequent reprobing of the same blot with a 1,368-bp ROAT1 probe confirmed that the ROAT1 probe bound only to the library fractions known to support PAH uptake, and did not bind to oatp (Fig. 5,right panel). Therefore, ROAT1 was determined to be unique from oatp. This was confirmed by DNA sequence analysis (see above).Figure 5Southern blot of cDNA library fractions. Left panel, the blot was probed with a 643-bp fragment from the liver organic anion transporter oatp (22Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (544) Google Scholar). As shown, the probe recognizes itself, but nothing in any of the kidney cDNA library fractions. Right panel, the same blot was probed with a 1,368-bp ROAT1 fragment, demonstrating that it does not recognize the liver transporter, nor any library fractions that did not support PAH transport (i.e. ALf1, ALf9.2.4.3.2, and ALf9.2.4.3.3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The same ROAT1 probe was used for a Northern blot and detected a strong signal in rat kidney (Fig. 6). ROAT1 transcript was not observed in rat heart, brain, spleen, lung, liver, skeletal muscle, or testis. The major transcript detected in kidney was ∼2.4 kb in size; however, a second, far less abundant transcript ∼4.2 kb in size was also seen in longer exposures. The blot was stripped and reprobed with a human β-actin probe, confirming that there was viable mRNA present in each lane of the blot (data not shown).Figure 6Northern blot analysis of ROAT1 tissue distribution. A rat multiple tissue mRNA blot was probed with the 1,368-bp ROAT1 probe. A highly expressed 2.4-kb transcript was detected in kidney, as well as a much less abundant 4.2-kb transcript. There was no signal detected in rat heart, brain, spleen, lung, liver, skeletal muscle, or testis. The blot was stripped and reprobed with a human β-actin control probe, confirming the presence of viable mRNA in all lanes of the blot (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Functional Characterization of ROAT1 TransportPotential DependenceAs a first step in determining the identity of ROAT1 (see Fig. 1), the effect of altered membrane potential on PAH transport in ROAT1-injected oocytes was assessed. The luminal facilitated diffusion system is markedly dependent upon membrane potential (7Werner D. Martinez F. Roch Ramel F. J. Pharmacol. Exp. Ther. 1990; 252: 792-799PubMed Google Scholar, 8Martinez F. Manganel M. Montrose Rafizadeh C. Werner D. Roch Ramel F. Am. J. Physiol. 1990; 258: F1145-F1153PubMed Google Scholar), whereas both the basolateral and luminal exchangers are not (3Pritchard J.B. Am. J. Physiol. 1988; 255: F597-F604PubMed Google Scholar, 31Blomstedt J.W. Aronson P.S. J. Clin. Invest. 1980; 65: 931-934Crossref PubMed Scopus (82) Google Scholar). Potential was altered by raising external K+, a condition previously shown to depolarize the plasma membrane of the oocyte (11Grundemann D. Gorboulev V. Gambaryan S. Veyhl M. Koepsell H. Nature. 1994; 372: 549-552Crossref PubMed Scopus (604) Google Scholar). When ROAT1-expressing oocytes were incubated in OR-2 with a high potassium ion concentration (102.5 mm), there was no reduction in PAH transport (Fig.7). As a positive control, oocytes injected with OCT2 cRNA, an organic cation transporter known to have a large potential-sensitive transport component (32Sweet D.H. Walsh R.C. Pritchard J.B. FASEB Abstr. 1996; 10: A127Google Scholar), showed a 67% drop in transport of [14C]TEA when exposed under the same conditions (Fig. 7). Water-injected oocytes showed no uptake under either condition. Therefore, ROAT1-mediated PAH transport is independent of membrane potential.Figure 7Effect of membrane potential on substrate uptake. The 60-min uptake of 50 μm[3H]PAH by water- or ROAT1 cRNA-injected oocytes was compared under normal (2.5 mm K+) and membrane depolarizing conditions (102.5 mm K+; (11Grundemann D. Gorboulev V. Gambaryan S. Veyhl M. Koepsell H. Nature. 1994; 372: 549-552Crossref PubMed Scopus (604) Google Scholar)), in the absence (No Inhibitor) or presence (+ Probenecid) of 1 mm probenecid. We have documented the influence of potential on both ROAT1 and OCT2 in 6 animals; however, the data presented here are from a representative animal because the experiment has only been done with both transporters in the same animal on two occasions. Columns represent mean values ± S.E. from 10 oocytes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Inhibition ProfileThe effects of various compounds and reduced temperature on PAH uptake were also examined (Fig.8). Incubation at 4 °C reduced uptake to just 3% of that seen at room temperature. The organic anions probenecid (1 mm), α-ketoglutarate (α-KG; 1 mm), bromcresol green (1 mm), and excess unlabeled PAH (1 mm) each reduced [3H]PAH uptake by 80–90%. In addition, like basolateral PAH/α-KG exchange (3Pritchard J.B. Am. J. Physiol. 1988; 255: F597-F604PubMed Google Scholar, 33Werner D. Roch-Ramel F. Am. J. Physiol. 1991; 261: F265-F272PubMed Google Scholar), but in contrast with luminal exchange (31Blomstedt J.W. Aronson P.S. J. Clin. Invest. 1980; 65: 931-934Crossref PubMed Scopus (82) Google Scholar), ROAT1-mediated PAH uptake was not inhibited by urate (1 mm).Figure 8ROAT1 substrate specificity. cRNA from ROAT1 was injected into oocytes and [3H]PAH transport was measured after 60 min in the presence of several organic anions and cations, and at reduced temperature. Data are presented as percent of control uptake. Values are mean ± S.E. for 4–6 animals (10 oocytes/treatment/animal). ** denotes p ≤ 0.01, and *** denotes p ≤ 0.001. BCG, bromcresol green.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Transport was also unaffected by the P-glycoprotein inhibitor cyclosporin A (CSA; 10 μm). Unlike urate and CSA, which gave values essentially identical to control, the cation TEA appeared to inhibit slightly, albeit at a high concentration (5 mm). Lower concentrations of TEA were not inhibitory and, at concentrations from 0.05 to 1 mm, actually stimulated uptake 20–40% (data not shown). To establish whether these modest effects might indicate that TEA was a substrate for ROAT1, the uptake of 200 μm [14C]TEA by ROAT1 cRNA and water-injected oocytes was measured. No difference in uptake between the two groups was observed (data not shown).The ability of α-KG to cis-inhibit PAH uptake (Fig. 8) is potentially diagnostic in that the basolateral dicarboxylate/organic anion exchanger should be inhibited by external α-KG, whereas luminal PAH carriers should not be inhibited by α-KG. Glutarate is also an effective counterion for this exchanger (3Pritchard J.B. Am. J. Physiol. 1988; 255: F597-F604PubMed Google Scholar). When ROAT1 cRNA-injected oocytes were incubated in OR-2 containing 0–1 mmglutarate, a clear, dose-dependent inhibition of PAH uptake was observed, with significant inhibition at 200 μmglutarate and above (Fig. 9).Figure 9Effect of external glutarate on PAH uptake. Two days after injection, oocytes were incubated for 60 min in OR-2 with 50 μm [3H]PAH and 0–1 mm glutarate. Uptake is expressed as percent of uptake when no compound was present, i.e. 0 mm glutarate. Data are mean ± S.E. values for 4 animals (10 oocytes/treatment/animal). * denotes p ≤ 0.05, and *** denotes p ≤ 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)trans-StimulationIf ROAT1 is the basolateral dicarboxylate/organic anion exchanger, increasing the intracellular concentration of α-KG (or glutarate) should induce trans-stimulation of PAH uptake (see Fig. 1). For this determination, glutarate is the preferred counterion since, in contrast to α-KG, it is not extensively metabolized (34Pritchard J.B. J. Pharmacol. Exp. Ther. 1990; 255: 969-975PubMed Google Scholar). Preliminary experiments showed substantial accumulation of 1 mm[14C]glutarate within uninjected Xenopusoocytes over time (170 pmol/oocyte after 90 min; data not shown). Incubating the oocytes in 1 mm glutarate for 90 min before exposure to PAH (i.e. preloading) significantly stimulated PAH uptake in ROAT1-expressing oocytes, as compared with non-preloaded oocytes (Fig. 10). Moreover, glutarate preloading had no effect on water-injected oocytes. Thus, glutarate induced trans-stimulation of ROAT1-mediated PAH uptake.Figure 10trans-Stimulation of PAH uptake.Water-injected or ROAT1 cRNA-injected oocytes, either non-preloaded (control) or preloaded by a 90-min incubation in 1 mm glutarate, were washed with glutarate-free medium and exposed to 50 μm [3H]PAH for 60 min in the absence (No Inhibitor) or presence (+ Probenecid) of 1 mm probenecid. Data are mean ± S.E. values from a representative animal (10 oocytes/treatment). The experiment was repeated four times and data calculated as percent of control uptake. Uptake by preloaded, cRNA-injected oocytes was 204 ± 19% (p ≤ 0.01). * denotes p ≤ 0.05, using paired Student's t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The specificity of trans-stimulation was assessed by comparing the effects of glutarate with the poorly metabolized dicarboxylate methylsuccinate, which cannot substitute for α-KG or glutarate on the α-KG/PAH exchanger (35Chatsudthipong V. Dantzler W.H. Am. J. Physiol. 1992; 263: F384-F391Crossref PubMed Google Scholar, 36Fritzsch G. Haase W. Rumrich G. Fasold H. Ullrich K.J. Pflugers Arch. 1984; 400: 250-256Crossref PubMed Scopus (41) Google Scholar). As shown in Fig.11, increasing the preloading concentration of glutarate from 0 to 5 mm increased glutarate's stimulatory effect on PAH uptake in a dose-dependent fashion, reaching an impressive 250% increase over non-preloaded oocytes at 5 mm. In contrast, methylsuccinate failed to stimulate PAH uptake over the same concentration range. Indeed, at 5 mm, methylsuccinate inhibited PAH uptake by 70% (p ≤ 0.01).Figure 11Dose response of glutaratetrans-stimulation. Oocytes injected with ROAT1 cRNA were preloaded for 90 min in 0–5 mm glutarate or methylsuccinate, washed briefly in dicarboxylate-free medium, and incubated with 50 μm [3H]PAH for 60 min. Uptake is expressed as percent of uptake with no preloading,i.e. 0 mm glutarate or methylsuccinate. Data are mean ± S.E. values for 4–6 animals (10 oocytes/treatment/animal). * denotes p ≤ 0.05, and ** denotes p ≤ 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)KineticsPAH uptake by ROAT1-injected oocytes increased steadily with time and was linear for about 1 h (data not shown). Using 10 min to approximate initial rate, the kinetics of ROAT1-mediated PAH transport were assessed by incubating oocytes expressing ROAT1 in medium containing 0.05–1 mm PAH (Fig.12 A). A double-reciprocal plot of the saturation data was constructed and linear regression analysis was performed to obtain estimates for K m of 70 μm and for V max of 6 pmol/oocyte/10 min (Fig. 12 B). It should be noted that, in the oocyte expression assay system, V maxreflects the degree of cRNA expression rather than a true measure of maximal uptake rate for a transporter in its native tissue.Figure 12Kinetic analysis of ROAT1-mediated PAH uptake. ROAT1 cRNA-injected oocytes were exposed to increasing concentrations of PAH for 10 min. A, saturation analysis. Total PAH uptake was corrected for diffusion by subtracting a diffusion correction factor of 9.18 ± 0.85 pmol/μm PAH. This factor was obtained by plotting the PAH uptake at 1 mm and 3 mm for three animals and calculating the mean change in uptake. B, double-reciprocal plot of the diffusion corrected data with linear regression analysis performed.K m was estimated to be 70 μm. The data shown are mean values ± S.E. from 3–4 animals (10 oocytes/treatment/animal).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONMany toxic anions, whether of endogenous or environmental origin, are eliminated from the body by the organic anion secretory system of the renal proximal tubule. This system has been actively investigated for more than 100 years, in part because of its effectiveness, which can clear the renal plasma of a good substrate like PAH in a single pass, and in part because of its critical role in protecting against the toxic effects of anionic xenobiotics through their rapid excretion via the urine (1Pritchard J.B. Miller D.S. Physiol. Rev. 1993; 73: 765-796Crossref PubMed Scopus (462) Google Scholar). Recent emphasis has been on the specificity of the basolateral carrier, which accepts a remarkably broad spectrum of agents, requiring only a hydrophobic backbone and negative or partial negative charges optimally separated by 6–7 Å (37Fritzsch G. Rumrich G. Ullrich K.J.