Human organic anion transporter hOAT1 forms homooligomers

Human organic anion transporter hOAT1 belongs to a superfamily of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. To gain insight into the regulation of hOAT1, detailed information on its structural assembly is essential. In the present study, we investigate the quaternary structure of hOAT1 using combined approaches of chemical cross-linking, gel filtration chromatography, co-immunoprecipitation, cell surface biotinylation, and metabolic labeling. Chemical cross-linking of intact membrane proteins from LLC-PK1 cells stably expressing hOAT1 converted quantitatively hOAT1 monomer to putative trimer and higher order of oligomer, indicating that hOAT1 is present in the membrane as multimeric complexes. When co-expressed in LLC-PK1 cells, FLAG-tagged hOAT1 co-immunoprecipitated with myc-tagged hOAT1. The hOAT1 oligomer was also detected in gel filtration chromatography of total membranes from hOAT1-expressing LLC-PK1 cells. Cell surface biotinylation with membrane-impermeable reagents and metabolic labeling with [(35)S]methionine followed by immunoprecipitation showed that the oligomeric hOAT1 did not contain any other proteins. Taken together, this is the first study demonstrating that hOAT1 exists in the plasma membrane as a homooligomer, possibly trimer, and higher order of oligomer.     

Development and characterization of an immobilized human organic cation transporter based liquid chromatographic stationary phase

Membranes from a stably transfected cell line that expresses the human organic cation 1 transporter (hOCT1) have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form the hOCT1(+)-IAM stationary phase. Membranes from the parent cell line that does not express the hOCT1 were also immobilized to create the hOCT1(-)-IAM stationary phase. Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [(3)H]-methyl phenyl pyridinium ([(3)H]-MPP(+)) as the marker ligand and MPP(+), verapamil, quinidine, quinine, nicotine, dopamine and vinblastin as the displacers. The K(d) values calculated from the chromatographic studies correlated with previously reported K(i) values (r(2)=0.9987; p<0.001). The data indicate that the hOCT1(+)-IAM column can be used for the on-line determination of binding affinities to the hOCT1 and that these affinities are comparable to those obtained using cellular uptake studies. In addition, the chromatographic method was able to identify a previously undetected high affinity binding site for MPP(+) and to determine that hOCT1 bound (R)-verapamil to a greater extent than (S)-verapamil.     

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.     

Stereoselective binding of chiral ligands to single nucleotide polymorphisms of the human organic cation transporter-1 determined using cellular membrane affinity chromatography

Membranes from stably transfected cell lines that express two point mutations of the human organic cation transporter-1 (hOCT1), R488 M and G465R, have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form two cellular membrane affinity chromatography (CMAC) columns, CMAC(hOCT1_G465R) and CMAC(hOCT1_R488M). Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [³H] MMP⁺ (1-methyl-4-phenylpyridinium) as the marker ligand and various displacers, including the single enantiomers of verapamil, fenoterol, and isoproterenol. The chromatographic data obtained were used to refine a previously developed pharmacophore for hOCT1.     

The chloride dependence of the human organic anion transporter 1 (hOAT1) is blunted by mutation of a single amino acid

Organic anion transporter 1 (OAT1) is key for the secretion of organic anions in renal proximal tubules. These organic anions comprise endogenous as well as exogenous compounds including frequently used drugs of various chemical structures. The molecular basis for the polyspecificity of OAT1 is not known. Here we mutated a conserved positively charged arginine residue (Arg(466)) in the 11(th) transmembrane helix of human OAT1. The replacement by the positively charged lysine (R466K) did not impair expression of hOAT1 at the plasma membrane of Xenopus laevis oocytes but decreased the transport of p-aminohippurate (PAH) considerably. Extracellular glutarate inhibited and intracellular glutarate trans-stimulated wild type and mutated OAT1, suggesting for the mutant R466K an unimpaired interaction with dicarboxylates. However, when Arg(466) was replaced by the negatively charged aspartate (R466D), glutarate no longer interacted with the mutant. PAH uptake by wild type hOAT1 was stimulated in the presence of chloride, whereas the R466K mutant was chloride-insensitive. Likewise, the uptake of labeled glutarate or ochratoxin A was chloride-dependent in the wild type but not in R466K. Kinetic experiments revealed that chloride did not alter the apparent K(m) for PAH but influenced V(max) in wild type OAT1-expressing oocytes. In R466K mutants the apparent K(m) for PAH was similar to that of the wild type, but V(max) was not changed by chloride removal. We conclude that Arg(466) influences the binding of glutarate, but not interaction with PAH, and interacts with chloride, which is a major determinant in substrate translocation.     

Determination of the external loops and the cellular orientation of the N- and the C-termini of the human organic anion transporter hOAT1

The OAT (organic anion transporter) family mediates the absorption, distribution and excretion of a diverse array of environmental toxins and clinically important drugs. OAT dysfunction significantly contributes to renal, hepatic, neurological and fetal toxicity and disease. As a first step to establish the topological model of hOAT1 (human OAT1), we investigated the external loops and the cellular orientation of the N- and the C-termini of this transporter. Combined approaches of immunofluorescence studies and site-directed chemical labelling were used for such purpose. Immunofluorescence microscopy of Myc-tagged hOAT1 expressed in cultured cells identified that both the N- and the C-termini of the transporter were located in the cytoplasm. Replacement of Lys59 in the predicted extracellular loop I with arginine resulted in a mutant (K59R), which was largely inaccessible for labelling by membrane-impermeable NHS (N-hydroxysuccinimido)-SS (dithio)-biotin present in the extracellular medium. This result suggests that loop I faces outside of the cell membrane. A single lysine residue introduced into putative extracellular loops III, V and VI of mutant K59R, which is devoid of extracellular lysine, reacted readily with membrane-impermeable NHS-SS-biotin, suggesting that these putative extracellular loops are in the extracellular domains of the protein. These studies provided the first experimental evidence on the extracellular loops and the cellular orientation of the N- and the C-termini of hOAT1.     

Characterization of ochratoxin A transport by human organic anion transporters.

The purpose of this study was to investigate the characteristics of ochratoxin A (OTA) transport by multispecific human organic anion transporters (hOAT1 and hOAT3, respectively) using the second segment of proximal tubule (S2) cells from mice stably expressing hOAT1 and hOAT3 (S2 hOAT1 and S2 hOAT3). S2 hOAT1 and S2 hOAT3 exhibited a time- and dose-dependent, and a saturable increase in uptake of [3H]-OTA, with apparent Km values of 0.42 microM (hOAT1) and 0.75 microM (hOAT3). These OTA uptakes were inhibited by several substrates for the OATs. Para-aminohippuric acid (PAH), probenecid, piroxicam, octanoate and citrinin inhibited [3H]-OTA uptake by hOAT1 and hOAT3 in a competitive manner (Ki = 4.29-3080 microM), with the following order of potency: probenecid > octanoate > PAH > piroxicam > citrinin for hOAT1; probenecid > piroxicam > octanoate> citrinin > PAH for hOAT3. These results indicate that hOAT1, as well as hOAT3, mediates a high-affinity transport of OTA on the basolateral side of the proximal tubule, but hOAT1- and hOAT3-mediated OTA transport are differently influenced by the substrates for the OATs. These pharmacological characteristics of hOAT1 and hOAT3 may be significantly related with the events in the development of OTA-induced nephrotoxicity in the human kidney.     

Molecular cloning and characterization of two novel human renal organic anion transporters (hOAT1 and hOAT3)

The cloned organic anion transporters from rat, mouse, and winter flounder (rOAT1, mOAT1, fROAT) mediate the coupled exchange of alpha-ketoglutarate with multiple organic anions, including p-aminohippurate (PAH). We have isolated two novel gene products from human kidney which bear significant homology to the known OATs and belong to the amphiphilic solute facilitator (ASF) family. The cDNAs, hOAT1 and hOAT3, encode for 550- and 568-amino-acid residue proteins, respectively. hOAT1 and hOAT3 mRNAs are expressed strongly in kidney and weakly in brain. Both genes map to chromosome 11 region q11.7. PAH uptake by Xenopus laevis oocytes injected with hOAT1 mRNA is increased 100-fold compared to water-injected oocytes. PAH uptake is chloride dependent and is not further increased by preincubation of oocytes in 5 mM glutarate. Uptake of PAH is inhibited by probenicid, alpha-ketoglutarate, bumetanide, furosemide, and losartan, but not by salicylate, urate, choline, amilioride, and hydrochlorothiazide.     

Critical amino acid residues in transmembrane domain 1 of the human organic anion transporter hOAT1

Human organic anion transporter 1 (hOAT1) belongs to a superfamily of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. Previously we suggested that the predicted transmembrane domain 1 (TM1) of hOAT1 might be important for its function. In the present study, we examined the role of each residue within TM1 of hOAT1 in substrate recognition and transport. Alanine scanning was used to construct mutants of hOAT1, and the uptake of model substrate para-aminohippurate was studied in COS-7 cells expressing the mutant transporters. This approach led to the discovery of two critical amino acid residues, Leu-30 and Thr-36. A substitution of Leu-30 or Thr-36 with alanine resulted in a complete loss of transport activities. We then further characterized Leu-30 and Thr-36 by mutagenizing these residues to amino acids with different physicochemical properties. Leu-30 was replaced with amino acids with varying sizes of side chains, including glycine, valine, and isoleucine. We showed that progressively smaller side chains at position 30 increasingly impaired hOAT1 function mainly because of the impaired surface expression of the transporter. Thr-36, another critical amino acid in TM1, was replaced by serine and cysteine. Similar to the substitution of Thr-36 by alanine, substitution by serine and cysteine at this position abolished transport activity without affecting the surface expression of the transporter. The fact that Thr-36 cannot be substituted with serine and that the side chains of alanine, serine, and cysteine are smaller than that of threonine by a methyl group indicate that both the methyl group and the hydroxyl group of Thr-36 could be critical for hOAT1 activity. Together we conclude that Leu-30 and Thr-36 play distinct roles in hOAT1 function. Leu-30 is important in targeting the transporter to the plasma membrane. In contrast, Thr-36 is critical for substrate recognition. The present study provided the first molecular evidence that transmembrane domain 1 is a critical determinant of hOAT1 function and may provide important insights into the structure-function relationships of the organic anion transporter family.     

Immobilized receptor- and transporter-based liquid chromatographic phases for on-line pharmacological and biochemical studies: a mini-review

This review addresses the synthesis and characterization of two different types of receptor-based liquid chromatographic supports, one based upon a trans-membrane ligand gated ion channel receptor (the nicotinic acetylcholine receptor) and the other a soluble nuclear receptor (the estrogen receptor). In addition, studies with the P-glycoprotein transporter are also reported. The nicotinic receptor was immobilized via hydrophobic insertion into the interstitial spaces of an immobilized artificial membrane (IAM) stationary phase. the estrogen receptor was tethered to a hydrophilic stationary phase and the membranes containing the Pgp transporter were coated on the surface of the IAM stationary phase. The stationary phases were characterized using known ligands and substrates for the respective non-immobilized proteins. The results from zonal and frontal chromatographic experiments demonstrated that the stationary phases could be used to determine binding affinities (expressed as dissociation constants, Kd,'s) and to resolve mixtures of ligands according to their relative affinities. In addition. competitive ligand binding studies on the P-glycoprotein-based stationary phase have established that this phase can be used to identify and characterize competitive displacement and allosteric interactions. These studies demonstrate that immobilized-receptor phases can be used for on-line pharmacological studies and as rapid screens for the isolation and identification of lead drug candidates from complex biological or chemical mixtures.     

Immobilized enzyme reactors based upon the flavoenzymes monoamine oxidase A and B

Monoamine oxidase (MAO) catalyzes the oxidative deamination of amines. The enzyme exists in two forms, MAO-A and MAO-B, which differ in substrate specificity and sensitivity to various inhibitors. Membrane fractions containing either expressed MAO-A or MAO-B have been non-covalently immobilized in the hydrophobic interface of an immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The MAO-containing stationary phases were packed into glass columns to create on-line immobilized enzyme reactors (IMERs) that retained the enzymatic activity of the MAO. The resulting MAO-IMERs were coupled through a switching valve to analytical high performance liquid chromatographic columns. The multi-dimensional chromatographic system was used to characterize the MAO-A (MAO-A-IMER) and MAO-B (MAO-B-IMER) forms of the enzyme including the enzyme kinetic constants associated with enzyme/substrate and enzyme/inhibitor interactions as well as the determination of IC(50) values. The results of the study demonstrate that the MAO-A-IMER and the MAO-B-IMER can be used for the on-line screening of substances for MAO-A and MAO-B substrate/inhibitor properties.     

Quantitative structure-retention relationships in affinity high-performance liquid chromatography

In this report the affinity high-performance liquid chromatography data, which were determined on silica-based human serum albumin, alpha1-acid glycoprotein, keratin, collagen, melanin, amylose tris(3,5-dimethylphenylcarbamate), and basic fatty acid binding protein columns, are discussed. Using a quantitative structure-retention relationship (QSRR) approach the affinity data were interpreted in terms of structural requirements of specific binding sites on biomacromolecules. The unique chromatographic properties of immobilized artificial membrane and cholesterol stationary phases were also analyzed from the point of view of mimicking biological processes. It has been demonstrated that chemometric processing of appropriately designed sets of chromatographic data derived in systems comprising biomolecules provides information of relevance for molecular pharmacology and rational drug design.     

Liver X receptor activation downregulates organic anion transporter 1 (OAT1) in the renal proximal tubule

Liver X receptors (LXRs) play an important role in the regulation of cholesterol by regulating several transporters. In this study, we investigated the role of LXRs in the regulation of human organic anion transporter 1 (hOAT1), a major transporter localized in the basolateral membrane of the renal proximal tubule. Exposure of renal S2 cells expressing hOAT1 to LXR agonists (TO901317 and GW3965) and their endogenous ligand [22(R)-hydroxycholesterol] led to the inhibition of hOAT1-mediated [(14)C]PAH uptake. This inhibition was abolished by coincubation of the above agonists with 22(S)-hydroxycholesterol, an LXR antagonist. Moreover, it was found that the effect of LXR agonists was not mediated by changes in intracellular cholesterol levels. Interestingly, the inhibitory effect of LXRs was enhanced in the presence of 9-cis retinoic acid, a retinoic X receptor agonist. Kinetic analysis revealed that LXR activation decreased the maximum rate of PAH transport (J(max)) but had no effect on the affinity of the transporter (K(t)). This result correlated well with data from Western blot analysis, which showed the decrease in hOAT1 expression following LXR activation. Similarly, TO901317 inhibited [(14)C]PAH uptake by the renal cortical slices as well as decreasing mOAT1 protein expression in mouse kidney. Our findings indicated for the first time that hOAT1 was downregulated by LXR activation in the renal proximal tubule.     

Interactions of benzylpenicillin and non-steroidal anti-inflammatory drugs with the sodium-dependent dicarboxylate transporter NaDC-3.

Sodium-dependent dicarboxylate transporters located in the basolateral membrane (NaDC-3) of renal proximal tubule cells maintain the driving force for exchange of organic anions and drugs against alpha-ketoglutarate via organic anion transporters OAT1 and OAT3. So far, information on direct interaction of drugs with the cloned NaDC-3 was missing. Here we tested the interaction of non-steroidal anti-inflammatory drugs (NSAIDs) and benzylpenicillin with NaDC-3 cloned from winter flounder (fNaDC-3) and human (hNaDC-3) kidneys. Flufenamate and benzylpenicillin inhibited [14C]succinate uptake in oocytes expressing fNaDC-3. Flufenamate elicited Na(+)-dependent currents in oocytes expressing fNaDC-3 with a reversal potential around -60 mV. Raising extracellular K+ concentration depolarized fNaDC3-expressing oocytes more in the presence of flufenamate than in its absence, an effect not seen with water-injected control oocytes. These findings suggest that flufenamate via interaction with fNaDC-3 increased the K+ conductance. Acetylsalicylate, indomethacin, and salicylate showed small potential-dependent inward currents in fNaDC-3 but not in hNaDC-3 expressing oocytes. Benzylpenicillin induced voltage-dependent inward currents which were Na(+)-dependent in oocytes expressing fNaDC-3. The currents were, however, much smaller than those induced by succinate, reflecting probably a low fit of the monovalent benzylpenicillin to the dicarboxylate binding site. The data show hitherto unknown effects of monovalent anionic drugs on a transporter for divalent di- and tricarboxylates.     

Identification and characterization of single nucleotide polymorphisms of SLC22A11 (hOAT4) in Korean women osteoporosis patients

Single nucleotide polymorphisms (SNPs) are the most common form of human genetic variation. Non-synonymous SNPs (nsSNPs) change an amino acid. Organic anion transporters (OATs) play an important role in eliminating or reabsorbing endogenous and exogenous organic anionic compounds. Among OATs, hOAT4 mediates high affinity transport of estrone sulfate and dehydroepiandrosterone sulfate. The rapid bone loss that occurs in post-menopausal women is mainly due to a net decrease of estrogen. In the present study we searched for SNPs within the exon regions of hOAT4 in Korean women osteoporosis patients. Fifty healthy subjects and 50 subjects with osteoporosis were screened for genetic polymorphism in the coding region of SLC22A11 (hOAT4) using GC-clamp PCR and denaturing gradient gel electrophoresis (DGGE). We found three SNPs in the hOAT4 gene. Two were in the osteoporosis group (C483A and G832A) and one in the normal group (C847T). One of the SNPs, G832A, is an nsSNP that changes the 278th amino acid from glutamic acid to lysine (E278K). Uptake of [3H] estrone sulfate by oocytes injected with the hOAT4 E278K mutant was reduced compared with wild-type hOAT4. Km values for wild type and E278K were 0.7 microM and 1.2 microM, and Vmax values were 1.8 and 0.47 pmol/oocyte/h, respectively. The present study demonstrates that hOAT4 variants can causing inter-individual variation in anionic drug uptake and, therefore, could be used as markers for certain diseases including osteoporosis.     

Cell free expression and functional reconstitution of eukaryotic drug transporters

Polyspecific organic cation and anion transporters of the SLC22 protein family are critically involved in absorption and excretion of drugs. To elucidate transport mechanisms, functional and biophysical characterization of purified transporters is required and tertiary structures must be determined. Here, we synthesized rat organic cation transporters OCT1 and OCT2 and rat organic anion transporter OAT1 in a cell free system in the absence of detergent. We solubilized the precipitates with 2% 1-myristoyl-2-hydroxy- sn-glycero-3-[phospho- rac-(1-glycerol)] (LMPG), purified the transporters in the presence of 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or octyl glucoside, and reconstituted them into proteoliposomes. From 1 mL reaction vessels 0.13-0.36 mg of transporter proteins was purified. Thus, from five to ten 1 mL reaction vessels sufficient protein for crystallization was obtained. In the presence of 1% LMPG and 0.5% CHAPS, OCT1 and OAT1 formed homo-oligomers but no hetero-oligomers. After reconstitution of OCT1, OCT2, and OAT1 into proteoliposomes, similar Michaelis-Menten K m values were measured for uptake of 1-methyl-4-phenylpyridinium and p-aminohippurate (PAH (-)) by the organic cation and anion transporters, respectively, as after expression of the transporters in cells. Using the reconstituted system, evidence was obtained that OAT1 operates as obligatory and electroneutral PAH (-)/dicarboxylate antiporter and contains a low-affinity chloride binding site that stimulates turnover. PAH (-) uptake was observed only with alpha-ketoglutarate (KG (2-)) on the trans side, and trans-KG (2-) increased the PAH (-) concentration in voltage-clamped proteoliposomes transiently above equilibrium. The V max of PAH (-)/KG (2-) antiport was increased by Cl (-) in a manner independent of gradients, and PAH (-)/KG (2-) antiport was independent of membrane potential in the absence or presence of Cl (-).     

Chromatographic properties of human leukocyte alpha-interferon A on sorbents with sepharose and silochrome matrices

Chromatography on immobilized monoclonal antibodies NK-2 from a bacterial strain-producer resulted in a pure human leukocyte alpha-interferon A (alpha-INF-A) homogeneous upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reverse phase high performance liquid chromatography. The chromatographic properties of partially purified alpha-INF-A on synthetic and commercial sorbents containing immobilized dyes, aromatic dipeptides, chelating and hydrophobic ligands as well as on porous glass have been investigated. In most cases, [125I]alpha-INF-A was used as an inner standard. The chromatographic behaviour of native and [125I]-labeled alpha-INF-A was practically the same. alpha-INF-A was most effectively chromatographed on porous glass, L-Trp-L-Trp-Sepharose 4B and Cu2+-chelate sorbents. In the latter case, the feasibility of substitution of the Sepharose matrix for the silochrome one has been demonstrated. It has been proposed that alpha-INF-A has a hydrophobic "pocket" with exposed aromatic amino acid residues which are capable of selective binding to aromatic dipeptides.     

Development of an immobilized GPR17 receptor stationary phase for binding determination using frontal affinity chromatography coupled to mass spectrometry

A liquid chromatographic stationary phase containing immobilized membranes from cells expressing the P2Y-like receptor GPR17 is described. Cellular membranes from 1321N1 cells transiently transfected with GPR17 vector [GPR17(+)] and from the same cell line transfected with the corresponding empty vector [GPR17(−)] were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6-mm-i.d. glass columns to create GPR17(+)-IAM and GPR17(−)-IAM stationary phases. Frontal chromatography experiments on both GPR17(+)-IAM and GPR17(−)-IAM demonstrated the presence of a specific interaction with GPR17 only in the former that was maximized by increasing the membrane/IAM ratio. GPR17(+)-IAM was used in frontal affinity chromatography experiments to calculate the dissociation constants (K_d) of three ligands—the antagonist cangrelor (formerly AR-C69931MX, a P2Y₁₂/P2Y₁₃ antagonist), MRS2179 (a P2Y₁ receptor antagonist), and the agonist UDP—all of which have been reported to also interact with GPR17. Immobilized GPR17 retained its ability to specifically bind the three analytes, as demonstrated by the agreement of the calculated K_d values with previously reported data. Preliminary ranking experiments suggest the application of GPR17(+)-IAM in ranking affinity studies for the selection of new potential candidates.     

A three-dimensional model of human organic anion transporter 1: aromatic amino acids required for substrate transport

Organic anion transporters (OATs) play a critical role in the handling of endogenous and exogenous organic anions by excretory and barrier tissues. Little is known about the OAT three-dimensional structure or substrate/protein interactions involved in transport. In this investigation, a theoretical three-dimensional model was generated for human OAT1 (hOAT1) based on fold recognition to the crystal structure of the glycerol 3-phosphate transporter (GlpT) from Escherichia coli. GlpT and hOAT1 share several sequence motifs as major facilitator superfamily members. The structural hOAT1 model shows that helices 5, 7, 8, 10, and 11 surround an electronegative putative active site ( approximately 830A(3)). The site opens to the cytoplasm and is surrounded by three residues not previously examined for function (Tyr(230) (domain 5) and Lys(431) and Phe(438) (domain 10)). Effects of these residues on p-aminohippurate (PAH) and cidofovir transport were assessed by point mutations in a Xenopus oocyte expression system. Membrane protein expression was severely limited for the Y230A mutant. For the K431A and F438A mutants, [(3)H]PAH uptake was less than 30% of wild-type hOAT1 uptake after protein expression correction. Reduced V(max) values for the F438A mutant confirmed lower protein expression. In addition, the F438A mutant exhibited an increased affinity for cidofovir but was not significantly different for PAH. Differences in handling of PAH and cidofovir were also observed for the Y230F mutant. Little uptake was determined for cidofovir, whereas PAH uptake was similar to wild-type hOAT1. Therefore, the hOAT1 structural model has identified two new residues, Tyr(230) and Phe(438), which are important for substrate/protein interactions.