Molecular cloning and characterization of multispecific organic anion transporter 4 expressed in the placenta

A cDNA encoding a novel multispecific organic anion transporter, OAT4, was isolated from a human kidney cDNA library. The OAT4 cDNA consisted of 2210 base pairs that encoded a 550-amino acid residue protein with 12 putative membrane-spanning domains. The amino acid sequence of OAT4 showed 38 to 44% identity to those of other members of the OAT family. Northern blot analysis revealed that OAT4 mRNA is abundantly expressed in the placenta as well as in the kidney. When expressed in Xenopus oocytes, OAT4 mediated the high affinity transport of estrone sulfate (K(m) = 1.01 microM) and dehydroepiandrosterone sulfate (K(m) = 0.63 microM) in a sodium-independent manner. OAT4 also mediated the transport of ochratoxin A. OAT4-mediated transport of estrone sulfate was inhibited by several sulfate conjugates, such as p-nitrophenyl sulfate, alpha-naphthyl sulfate, beta-estradiol sulfate, and 4-methylumbelliferyl sulfate. By contrast, glucuronide conjugates showed little or no inhibitory effect on the OAT4-mediated transport of estrone sulfate. OAT4 interacted with chemically heterogeneous anionic compounds, such as nonsteroidal anti-inflammatory drugs, diuretics, sulfobromophthalein, penicillin G, and bile salts, whereas tetraethylammonium, an organic cation, did not. OAT4 is the first member of the multispecific organic anion transporter family, which is expressed abundantly in the placenta. OAT4 might be responsible for the elimination and detoxification of harmful anionic substances from the fetus.     

Functional differences in steroid sulfate uptake of organic anion transporter 4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1) in human placenta

Human trophoblasts depend on the supply of external precursors such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16alpha-OH-DHEA-S for synthesis of estrogens. Recently, we have characterized the uptake of DHEA-S by isolated mononucleated trophoblasts and identified different transporter polypeptides involved in this process. Immunohistochemistry of 1st and 3rd trimester placenta detected organic anion transporter 4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1, former name OATP-B) in cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast, indicating that both transporter polypeptides are involved in placental uptake of foetal derived steroid sulfates. In the present study we have characterized and compared the kinetics of DHEA-S and estrone sulfate (E(1)S) uptake by these transporters stably expressed in FlpIn -HEK293 cells using the Flp recombinase-mediated site-specific recombination. Uptake of E(1)S by OAT4- and OATP2B1-transfected cells was highly increased compared to the non-transfected cells. In contrast, DHEA-S uptake was only highly increased in OAT4 (40 times), but only weakly enhanced in OATP2B1 cells. The uptake of DHEA-S and E(1)S by OAT4 was partly Na(+)-dependent (about 50%), whereas uptake of DHEA-S by OATP2B1 was Na(+)-independent. Kinetic analysis of the initial uptake rates of E(1)S by OAT4 and OATP2B1 gave very similar values for K(m) (about 20microM) and V(max) (about 600pmol/(minxmg protein)). In contrast, the affinity of DHEA-S towards OATP2B1 was about 10 times lower (K(m)>200microM) then for OAT4 (K(m)=29microM). Our results suggest different physiological roles of the two transporter polypeptides in placental uptake of foetal derived steroid sulfates. OATP2B1 seems not to be involved in de novo synthesis of placental estrogens but may contribute to the clearance of estrogen sulfates from foetal circulation.     

Functional expression of pig renal organic anion transporter 3 (pOAT3)

With the cloning of pig renal organic anion transporter 1 (pOAT1) (Biochimie 84 (2002) 1219) we set up a model system for comparative studies of cloned and natively isolated membrane located transport proteins. Meanwhile, another transport protein involved in p-aminohippurate (PAH) uptake on the basolateral side of the proximal tubule cells was identified, designated organic anion transporter 3 (OAT3). To explore the contribution of pOAT1 to the PAH clearance in comparison to OAT3, it was the aim of this study to extend our model by cloning of the pig ortholog of OAT3. Sequence comparisons of human organic anion transporter 3 (hOAT3) with the expressed sequence tag (EST) database revealed a clone and partial sequence of the pig renal organic anion transporter 3 (pOAT3) ortholog. Sequencing of the entire open reading frame resulted in a protein of 543 amino acid residues encoded by 1632 base pairs (EMBL Acc. No. AJ587003). It showed high homologies of 81%, 80%, 76%, and 77% to the human, rabbit, rat, and mouse OAT3, respectively. A functional characterization of pOAT3 in Xenopus laevis oocytes yielded an apparent Km (Kt) for [3H]estrone sulfate of 7.8 +/- 1.3 microM. Moreover, pOAT3 mediated [3H]estrone sulfate uptake was almost abolished by 0.5 mM of glutarate, dehydroepiandosterone sulfate, or probenecid consistent with the hallmarks of OAT3 function.     

Role of glycosylation in the organic anion transporter OAT1

Organic anion transporters (OAT) play essential roles in the body disposition of clinically important anionic drugs, including antiviral drugs, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We reported previously (Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519-1524) that tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited organic anion transport in COS-7 cells expressing a mouse organic anion transporter (mOAT1), suggesting an important role of glycosylation in mOAT1 function. In the present study, we investigated the effect of disrupting putative glycosylation sites in mOAT1 as well as its human counterpart, hOAT1, by mutating asparagine to glutamine and assessing mutant transporters in HeLa cells. We showed that the putative glycosylation site Asp-39 in mOAT1 was not glycosylated but the corresponding site (Asp-39) in hOAT1 was glycosylated. Disrupting Asp-39 resulted in a complete loss of transport activity in both mOAT1 and hOAT1 without affecting their cell surface expression, suggesting that the loss of function is not because of deglycosylation of Asp-39 per se but rather is likely because of the change of this important amino acid critically involved in the substrate binding. Single replacement of asparagines at other sites had no effect on transport activity indicating that glycosylation at individual sites is not essential for OAT function. In contrast, a simultaneous replacement of all asparagines in both mOAT1 and hOAT1 impaired the trafficking of the transporters to the plasma membrane. In summary, we provided the evidence that 1) Asp-39 is crucially involved in substrate recognition of OAT1, 2) glycosylation at individual sites is not required for OAT1 function, and 3) glycosylation plays an important role in the targeting of OAT1 onto the plasma membrane. This study is the first molecular identification and characterization of glycosylation of OAT1 and may provide important insights into the structure-function relationships of the organic anion transporter family.     

Murine renal organic anion transporters mOAT1 and mOAT3 facilitate the transport of neuroactive tryptophan metabolites

Tryptophan metabolites such as kynurenate (KYNA), xanthurenate (XA), and quinolinate are considered to have an important impact on many physiological processes, especially brain function. Many of these metabolites are secreted with the urine. Because organic anion transporters (OATs) facilitate the renal secretion of weak organic acids, we investigated whether the secretion of bioactive tryptophan metabolites is mediated by OAT1 and OAT3, two prominent members of the OAT family. Immunohistochemical analyses of the mouse kidneys revealed the expression of OAT1 to be restricted to the proximal convoluted tubule (representing S1 and S2 segments), whereas OAT3 was detected in almost all parts of the nephron, including macula densa cells. In the mouse brain, OAT1 was found to be expressed in neurons of the cortex cerebri and hippocampus as well as in the ependymal cell layer of the choroid plexus. Six tryptophan metabolites, including the bioactive substances KYNA, XA, and the serotonin metabolite 5-hydroxyindol acetate inhibited [³H]p-aminohippurate (PAH) or 6-carboxyfluorescein (6-CF) uptake by 50–85%, demonstrating that these compounds interact with OAT1 as well as with OAT3. Half-maximal inhibition of mOAT1 occurred at 34 µM KYNA and 15 µM XA, and it occurred at 8 µM KYNA and 11.5 µM XA for mOAT3. Quinolinate showed a slight but significant inhibition of [³H]PAH uptake by mOAT1 and no alteration of 6-CF uptake by mOAT3. [¹⁴C]-Glutarate (GA) uptake was examined for both transporters and demonstrated differences in the transport rate for this substrate by a factor of 4. Trans-stimulation experiments with GA revealed that KYNA and XA are substrates for mOAT1. Our results support the idea that OAT1 and OAT3 are involved in the secretion of bioactive tryptophan metabolites from the body. Consequently, they are crucial for the regulation of central nervous system tryptophan metabolite concentration. kidneys; brain; macula densa; transforming growth factor; N-methyl-D-aspartate receptor     

Steroid sulfatase inhibitors: promising new tools for breast cancer therapy?

Inhibition of aromatase is currently well-established as the major treatment option of hormone-dependent breast cancer in postmenopausal women. However, despite the effects of aromatase inhibitors in both early and metastatic breast cancer, endocrine resistance may cause relapses of the disease and progression of metastasis. Thus, driven by the success of manipulating the steroidogenic enzyme aromatase, several alternative enzymes involved in steroid synthesis and metabolism have recently been investigated as possible drug targets. One of the most promising targets is the steroid sulfatase (STS) which converts steroid sulfates like estrone sulfate (E1S) and dehydroepiandrosterone sulfate (DHEAS) to estrone (E1) and dehydroepiandrosterone (DHEA), respectively. Estrone and DHEA may thereafter be used for the synthesis of more potent estrogens and androgens that may eventually fuel hormone-sensitive breast cancer cells. The present review summarizes the biology behind steroid sulfatase and its inhibition, the currently available information derived from basic and early clinical trials in breast cancer patients, as well as ongoing research. Article from the Special Issue on Targeted Inhibitors.     

STEROID SULFATASE IN BRAIN: COMPARISON OF SULFOHYDROLASE ACTIVITIES FOR VARIOUS STEROID SULFATES IN NORMAL AND PATHOLOGICAL BRAINS, INCLUDING THE VARIOUS FORMS OF METACHROMATIC LEUKODYSTROPHY

Abstract– The enzymatic hydrolysis by brain homogenate of the sulfate esters of estrone, pregnenolone, dehydroepiandrosterone, testosterone, cholesterol and p-nitrophenol was studied. With homogenate of young rat brain, the pH optima of estrone sulfatase ⁴ 4 The term steroid sulfatase is used as a general name for the enzyme(s) which hydrolyzes the sulfate ester of a steroid. Simplified terms, such as estrone sulfatase, instead of the more formal terms, such as estrone sulfate sulfohydrolase, have been used throughout.
and arysulfatase C (p-nitrophenyl sulfate as substrate) were 8.2 and all other steroid sulfatases had pH optima at 6.6. Apparent K_ms for these steroid sulfates were widely different. The highest K_m value was 32.2 μm for estrone sulfate and the lowest was 0.66 μm for testosterone sulfate; the K_m for p-nitrophenyl sulfate was 30 fold higher than for estrone sulfate. Specific activity was also highest with estrone sulfatase and lowest with testosterone sulfatase; specific activity with aryl sulfatase C was over 3 fold higher than with estrone sulfatase. Estrone sulfatase activity was inhibited noncompetitively by sulfate esters of dehydroepiandrosterone, pregnenolone, and cholesterol; on the other hand, other steroid sulfatases were inhibited by these latter three sulfates competitively. Developmental changes of these sulfohydrolase activities in rat brain were almost identical with the exception of testosterone sulfatase activity; the latter sulfatase had a peak activity at 30 days old, while all other sulfatase had a peak at 20 days old. Thermal stability of all these activities was identical. Testosterone sulfatase activity in neurological mouse mutants, jimpy, msd, and quaking mice, was less than one half of littermate controls, while other steroid sulfatase levels in these mutants' brain were normal. All sulfatase activities were diminished in the brain of a metachromatic leukodystrophy patient with multiple sulfatase deficiency. The brains of classical metachromatic leukodystrophy patients contained normal levels of all steroid sulfatases and arylsulfatase C, with the single exception of testosterone sulfatase which level was less than 50% of control.     

Evidence for rat organic anion transporter 3 association with caveolin-1 in rat kidney

The rat organic anion transporter 3 (rOAT3) has recently been identified as the third isoform of the OAT family. The mechanisms that regulate rOAT3's functions remain to be elucidated. rOAT3 contributes for moving a number of negatively charged organic compounds between cells and their extracellular milieu. Caveolin (Cav) also plays a role as a membrane transporter. To address the relationship of these two proteins, we investigated the protein-protein interaction between rOAT3 and Cav-1. The rOAT3 mRNA and protein expression were observed in the rat kidney, and the expressions of Cav-1 mRNA and protein were also detected in the kidney. Confocal microscopy of the immuno-cytochemistry experiments using primary cultured renal proximal tubular cells showed that rOAT3 and Cav-1 were co-localized at the plasma membrane. This finding was confirmed by Western blot analysis using isolated caveolae-enriched membrane fractions from the rat kidney and immuno-precipitation experimentation. When rOAT3's synthesized cRNA of rOAT3 along with the antisense oligo deoxynucleotide ofXenopusCav-1 were co-injected intoXenopusoocytes, the [(3)H] estrone sulfate uptake was significantly decreased. These findings suggest that rOAT3 and caveolin-1 share a cellular expression in the plasma membrane and Cav-1 up-regulates the organic anionic compound uptake via rOAT3 under normal physiological conditions.     

Characterization of an organic anion transport system in a placental cell line

Transporters within the placenta play a crucial role in the distribution of nutrients and xenobiotics across the maternal-fetal interface. An organic anion transport system was identified on the apical membrane of the rat placenta cell line HRP-1, a model for the placenta barrier. The apical uptake of 3H-labeled organic anion estrone sulfate in HRP-1 cells was saturable (Km = 4.67 microM), temperature and Na+ dependent, Li+ tolerant, and pH sensitive. The substrate specificity of the transport system includes various steroid sulfates, such as beta-estradiol 3,17-disulfate, 17 beta-estradiol 3-sulfate, and dehydroepiandrosterone 3-sulfate (DHEAS) but does not include taurocholate, p-aminohippuric acid (PAH), and tetraethylammonium. Preincubation of HRP-1 cells with 8-bromo-cAMP (a cAMP analog) and forskolin (an adenylyl cyclase activator) acutely stimulated the apical transport activity. This stimulation was further enhanced in the presence of IBMX (a phosphodiesterase inhibitor). Together these data show that the apical membrane of HRP-1 cells expresses an organic anion transport system that is regulated by cellular cAMP levels. This transport system appears to be different from the known taurocholate-transporting organic anion-transporting polypeptides and PAH-transporting organic anion transporters, both of which also mediate the transport of estrone sulfate and DHEAS.     

Transport of organic anions across the basolateral membrane of proximal tubule cells

Renal proximal tubules secrete diverse organic anions (OA) including widely prescribed anionic drugs. Here, we review the molecular properties of cloned transporters involved in uptake of OA from blood into proximal tubule cells and provide extensive lists of substrates handled by these transport systems. Where tested, transporters have been immunolocalized to the basolateral cell membrane. The sulfate anion transporter 1 (sat-1) cloned from human, rat and mouse, transported oxalate and sulfate. Drugs found earlier to interact with sulfate transport in vivo have not yet been tested with sat-1. The Na⁺-dicarboxylate cotransporter 3 (NaDC-3) was cloned from human, rat, mouse and flounder, and transported three Na⁺ with one divalent di- or tricarboxylate, such as citric acid cycle intermediates and the heavy metal chelator 2,3-dimercaptosuccinate (succimer). The organic anion transporter 1 (OAT1) cloned from several species was shown to exchange extracellular OA against intracellular α-ketoglutarate. OAT1 translocated, e.g., anti-inflammatory drugs, antiviral drugs, β-lactam antibiotics, loop diuretics, ochratoxin A, and p-aminohippurate. Several OA, including probenecid, inhibited OAT1. Human, rat and mouse OAT2 transported selected anti-inflammatory and antiviral drugs, methotrexate, ochratoxin A, and, with high affinities, prostaglandins E₂ and F_2α. OAT3 cloned from human, rat and mouse showed a substrate specificity overlapping with that of OAT1. In addition, OAT3 interacted with sulfated steroid hormones such as estrone-3-sulfate. The driving forces for OAT2 and OAT3, the relative contributions of all OA transporters to, and the impact of transporter regulation by protein kinases on renal drug excretion in vivo must be determined in future experiments. Electronic Publication     

Steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities in mouse tissues

The metabolism of estrone sulfate and dehydroisoandrosterone sulfate to the free, unconjugated steroids, estrone and dehydroisoandrosterone, was demonstrated in more than thirty different tissues from male and female BALB/c mice. The activity of steroid sulfatase, when expressed per mg tissue, was greatest in both the pituitary gland and the adrenal glands. The pituitary gland, however, had the lowest capacity for hydrolysis of steroid sulfates while the liver had the greatest capacity. 17 beta-Hydroxysteroid oxidoreductase activity also was demonstrated in all mouse tissues by the formation of estradiol-17 beta when using estrone sulfate as the substrate. The highest apparent activity for 17 beta-hydroxysteroid oxidoreductase was found in lung tissue, and the greatest capacity to form estradiol-17 beta from estrone sulfate was found in liver, lungs, kidneys and testes. This study demonstrates that the majority of mouse tissues have steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities.     

Steroid sulfatase inhibitor alters blood pressure and steroid profiles in hypertensive rats

Our hypothesis is that the steroid sulfatase gene (Sts) may indirectly contribute to the modulation of blood pressure (BP) in rats with genetic hypertension. The steroid sulfatase enzyme (STS) catalyzes the conversion of estrone sulfate, dehydroepiandrosterone sulfate, cholesterol sulfate and glucocorticoid sulfates to their active nonconjugated forms. This causes the elevation of biologically active steroids, such as glucocorticoids, mineralcorticoids as well as testosterone, which may lead to increased BP. The main objective was to examine the effects of a steroid sulfatase inhibitor on blood pressure and steroid levels in rats with hypertensive genetic backgrounds. Three treatment groups, 5-15 weeks of age were used: controls, estrone and STS inhibitor (estrone-3-O-sulfamate), (n=8 per group). BP was taken weekly by tail cuff, and serum testosterone (T), estrogens (E), and plasma corticosterone (C) levels were measured by radioimmunoassay. BP was significantly reduced by the STS inhibitor in the strains with genetically elevated BP. Also the inhibitor alone significantly reduced plasma corticosterone in all strains compared to estrone treatment with a concomitant as well as significant rise in estrogens and reduction in testosterone and body weight.     

Involvement of rat organic anion transporter 3 (rOAT3) in cephaloridine-induced nephrotoxicity: in comparison with rOAT1

This study was performed to elucidate the possible involvement of organic anion transporter 3 (OAT3) in cephaloridine (CER)-induced nephrotoxicity and compare the substrate specificity between rOAT3 and rat OAT1 (rOAT1) for various cephalosporin antibiotics, using proximal tubule cells stably expressing rOAT3 (S2 rOAT3) and rOAT1 (S2 rOAT1). S2 rOAT3 exhibited a CER uptake and a higher susceptibility to CER cytotoxicity than did mock, which was recovered by probenecid. Various cephalosporin antibiotics significantly inhibited both estrone sulfate uptake in S2 rOAT3 and para-aminohippuric acid uptake in S2 rOAT1. The Ki values of CER, cefoperazone, cephalothin and cefazolin for rOAT3- and rOAT1-mediated organic anion transport ranged from 0.048 to 1.14 mM and from 0.48 to 1.32 mM, respectively. These results suggest that rOAT3, at least in part, mediates CER uptake and CER-induced nephrotoxicity as rOAT1. There was some difference of affinity between rOAT3 and rOAT1 for cephalosporin antibiotics.     

Biology of a novel organic solute and steroid transporter, OSTalpha-OSTbeta

Using a comparative approach, recent studies have identified and functionally characterized a new type of organic solute and steroid transporter (OST) from skate, mouse, rat, and human genomes. In contrast to all other organic anion transporters identified to date, transport activity requires the coexpression of two distinct gene products, a predicted 340-amino acid, seven-transmembrane (TM) domain protein (OSTalpha) and a putative 128-amino acid, single-TM domain ancillary polypeptide (OSTbeta). When OSTalpha and OSTbeta are coexpressed in Xenopus oocytes, they are able to mediate transport of estrone 3-sulfate, dehydroepiandrosterone 3- sulfate, taurocholate, digoxin, and prostaglandin E2, indicating a role in the disposition of key cellular metabolites or signaling molecules. OSTalpha and OSTbeta are expressed at relatively high levels in intestine, kidney, and liver, but they are also expressed at lower levels in many human tissues. Indirect immunofluorescence microscopy revealed that intestinal OSTalpha and OSTbeta proteins are localized to the baso-lateral membrane of mouse enterocytes. In MDCK cells, mouse Ostalpha-Ostbeta mediated the vectorial movement of taurocholate from the apical to the basolateral membrane, but not in the opposite direction, indicating basolateral efflux of bile acids. Overall, these findings indicate that OSTalpha-OSTbeta is a heteromeric transporter that is localized to the basolateral membrane of specific epithelial tissues and serves to regulate the export and disposition of bile acids and structurally related compounds from the cell. If confirmed, this model would have important implications for the body's handling of various steroid-derived molecules and may provide a new pharmacologic target for altering sterol homeostasis.     

Impaired organic anion transport in kidney and choroid plexus of organic anion transporter 3 (Oat3 (Slc22a8)) knockout mice

To begin to develop in vivo model systems for the assessment of the contributions of specific organic anion transporter (OAT) family members to detoxification, development, and disease, we carried out a targeted disruption of the murine organic anion transporter 3 (Oat3) gene. Surviving Oat3(-/-) animals appear healthy, are fertile, and do not exhibit any gross morphological tissue abnormalities. No Oat3 mRNA expression was detected in kidney, liver, or choroid plexus (CP) of Oat3(-/-) mice. A distinct phenotype manifested by a substantial loss of organic anion transport capacity in kidney and CP was identified. Uptake sensitive to inhibition by bromosulfophthalein or probenecid was observed for taurocholate, estrone sulfate, and para-aminohippurate in renal slices from wild-type mice, whereas in Oat3(-/-) animals transport of these substances was greatly reduced. No discernable differences in uptake were observed between hepatic slices from wild-type and Oat3(-/-) littermates, suggesting Oat3 does not play a major role in hepatic organic anion uptake. Cellular accumulation of fluorescein was reduced by approximately 75% in CP from Oat3(-/-) mice. However, capillary accumulation of fluorescein-methotrexate was unchanged, indicating the effects of Oat3 loss are restricted to the entry step and that Oat3 is localized to the apical membrane of CP. These data indicate a key role for Oat3 in systemic detoxification and in control of the organic anion distribution in cerebrospinal fluid.     

Characterization, in the guinea pig, of a hepatic cytosol protein binding dehydroepiandrosterone sulfate and estrone sulfate

A protein which binds dehydroepiandrosterone sulfate and estrone sulfate was detected in the cytosolic fraction of female Guinea-pig liver. It is characterized by a molecular mass of 14,400 Da, its affinity for DHEA sulfate (KD = 8.8 microM) and estrone sulfate (KD = 8.5 microM), and its lack of affinity for free steroids such as dehydroepiandrosterone or estrone. It is eluted by gel filtration on Sephadex G-50 simultaneously with the inhibitor of microsomal DHEA sulfatase recently described by some of us. This protein could be implicated in the intracellular transport or in the metabolism of sulfated steroids.     

Role of aromatic transmembrane residues of the organic anion transporter,rOAT3, in substrate recognition

Organic anion transporters (OATs, SLC21) are important in the excretion of endogenous and exogenous compounds in the kidney. The rat organic anion transporter, rOAT3, mediates the transport of organic anions such as p-aminohippurate (PAH) and estrone sulfate as well as the basic compound, cimetidine. In the present study, we examined the role of conserved transmembrane aromatic amino acid residues of rOAT3 in substrate recognition and transport. Alanine scanning followed by amino acid replacements was used to construct mutants of rOAT3. The uptake of model compounds was studied in Xenopus laevis oocytes expressing the mutant transporters. We observed that four mutants in transmembrane domain 7 (TMD 7), W334A, F335A, Y341A, and Y342Q, and one mutant in transmembrane domain 8 (TMD 8), F362S, exhibited a less than 2-fold enhanced uptake of PAH and cimetidine in comparison to wild-type rOAT3, which exhibited a 16-fold enhanced uptake of PAH and an 8-fold enhanced uptake of cimetidine. Estrone sulfate uptake in oocytes expressing any one of these five mutants remained at least 8-fold enhanced. The data suggest that the five residues, W334, F335, Y341, Y342, and F362, contribute differently to the transport of the small hydrophilic organic substrates PAH and cimetidine in comparison to the large hydrophobic organic substrate estrone sulfate. The effects of side chains of these five residues on transporter functions were also evaluated by constructing conservative mutations. We observed that the residues contribute to PAH and cimetidine transport in different ways: the -OH group of Y342, the indole ring of W334, and the aromatic rings of F335, Y341, and F362 are important for PAH and cimetidine transport by rOAT3. These data suggest that there is an aromatic pocket composed mainly of residues in TMD 7 in the translocation pathway of rOAT3, which is important for the transport of PAH and cimetidine. Aromatic residues in this pocket may interact directly with substrates of rOAT3 through hydrogen bonds and pi-pi interactions.