Involvement of organic anion transporters in the efflux of uremic toxins across the blood-brain barrier

Renal failure causes multiple physiological changes involving CNS dysfunction. In cases of uremia, there is close correlation between plasma levels of uremic toxins [e.g. 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), hippurate (HA) and indoleacetate (IA)] and the degree of uremic encephalopathy, suggesting that uremic toxins are involved in uremic encephalopathy. In order to evaluate the relevance of uremic toxins to CNS dysfunction, we investigated directional transport of uremic toxins across the blood-brain barrier (BBB) using in vivo integration plot analysis and the brain efflux index method. We observed saturable efflux transport of [(3)H]CMPF, [(14)C]HA and [(3)H]IA, which was inhibited by probenecid. For all uremic toxins evaluated, apparent efflux clearance across the BBB was greater than apparent influx clearance, suggesting that these toxins are predominantly transported from the brain to blood across the BBB. Saturable efflux transport of [(3)H]CMPF, [(14)C]HA and [(3)H]IA was completely inhibited by benzylpenicillin, which is a substrate of rat organic anion transporter 3 (rOat3). Taurocholate and digoxin, which are common substrates of rat organic anion transporting polypeptide (rOatp), partially inhibited the efflux of [(3)H]CMPF. Transport experiments using a Xenopus laevis oocyte expression system revealed that CMPF, HA and IA are substrates of rOat3, and that CMPF (but not HA or IA) is a substrate of rOap2. These results suggest that rOat3 mediates brain-to-blood transport of uremic toxins, and that rOatp2 is involved in efflux of CMPF. Thus, conditions typical of uremia can cause inhibition of brain-to-blood transport involving rOat3 and/or rOatp2, leading to accumulation of endogenous metabolites and drugs in the brain.     

Comparative studies on the effect of a protein-synthesis inhibitor on aluminium-induced secretion of organic acids from Fagopyrum esculentum Moench and Cassia tora L. roots

Aluminium (Al)-induced secretion of organic acids from plant roots is considered a mechanism of Al resistance, but the processes leading to the secretion of organic acids are still unknown. In the present study, a protein-synthesis inhibitor, cycloheximide (CHM), was used to investigate its effect on Al-induced organic acid secretion in a pattern I (rapid exudation of organic acids under Al stress) plant buckwheat (Fagopyrum esculentum Moench) and a pattern II (exudation of organic acids was delayed by several hours under Al stress) plant Cassia tora L. A dose-response experiment showed that the secretion of oxalate by buckwheat roots was not affected by CHM when added in the range from 0 to 50 microM, with or without exposure to 100 microm Al, but the secretion of citrate was completely inhibited by 30 microM CHM in C. tora. A time-course experiment showed that even prolonged exposure to 20 microM CHM did not affect oxalate secretion in buckwheat, but significantly inhibited citrate secretion in C. tora. However, citrate synthase (CS) activity in C. tora was not affected during 12 h exposure to 100 microM Al when compared with that in control roots, although CHM can inhibit CS activity effectively. These results indicated that CS activity was not related to Al-regulated citrate efflux in C. tora. The total protein was decreased by 14.0% and 32.3% in C. tora and buckwheat root tip, respectively, after 3-h treatment with 20 microM CHM. A 3-h pulse with 20 microM CHM completely inhibited citrate efflux in C. tora during the next 6-h exposure to Al, although a small amount of citrate was exuded after 9-h exposure. However, oxalate efflux in buckwheat was not influenced by a similar treatment. In buckwheat, a 3-h pulse with 100 microM Al maintained oxalate secretion at a high level during the next 9 h, with or without CHM treatment. Conversely, in C. tora a 6-h pulse with 100 microM Al induced significant secretion of citrate which was inhibited by the CHM. Taken together, these findings suggest that both de novo synthesis and activation of an anion channel are needed for Al-induced secretion of citrate in C. tora, but in buckwheat the plasma membrane protein responsible for oxalate secretion pre-exists.     

Differential gene expression of organic anion transporters in male and female rats.

Sex-related differential gene expression of organic anion transporters (rOAT1, rOAT2, and rOAT3) in rat brain, liver, and kidney was investigated. There were no sex differences in the expression of rOAT1 mRNA. rOAT2 mRNA was abundant in the liver and weakly expressed in the kidney of male rats; however, the OAT2 gene was strongly expressed in both organs of females. The abundance of rOAT2 mRNA markedly increased in castrated male rat kidney; however, treatment of castrated male rats with testosterone led to a decrease of rOAT2 mRNA. Expression of rOAT3 mRNA in intact female rats was found in the kidney and brain, whereas in males rOAT3 mRNA was also found in the liver. rOAT3 mRNA markedly decreased in the liver of castrated male rats but increased in testosterone-treated castrated male rats. Moreover, rOAT3 mRNA increased in the hypophysectomized female rat liver, indicating that rOAT3 is an inducible isoform. The present findings suggest that sex steroids play an important role in the expression and maintenance of OAT2/3 isoforms in the rat liver and kidney. Our results provide information on the differential gene expression of OAT isoforms with sex hormone dependency.     

Citrate transporters play a critical role in aluminium-stimulated citrate efflux in rice bean (Vigna umbellata) roots

BACKGROUND AND AIMS: Aluminium (Al) stimulates the efflux of citrate from apices of rice bean (Vigna umbellata) roots. This response is delayed at least 3 h when roots are exposed to 50 microm Al, indicating that some inducible processes leading to citrate efflux are involved. The physiological bases responsible for the delayed response were examined here. METHODS: The effects of several antagonists of anion channels and citrate carriers, and of the protein synthesis inhibitor, cycloheximide (CHM) on Al-stimulated citrate efflux and/or citrate content were examined by high-pressure liquid chromatography (HPLC) or an enzymatic method. KEY RESULTS: Both anion channel inhibitors and citrate carrier inhibitors can inhibit Al-stimulated citrate efflux, with anthracene-9-carboxylic acid (A-9-C, an anion channel inhibitor) and phenylisothiocyanate (PI, a citrate carrier inhibitor) the most effective inhibitors. A 6 h pulse of 50 microm Al induced a significant increase of citrate content in root apices and release of citrate. However, the increase in citrate content preceded the efflux. Furthermore, the release of citrate stimulated by the pulse treatment was inhibited by both A-9-C and PI, indicating the importance of the citrate carrier on the mitochondrial membrane and the anion channel on the plasma membrane for the Al-stimulated citrate efflux. CHM (20 microm) also significantly inhibited Al-stimulated citrate efflux, confirming that de novo protein synthesis is required for Al-stimulated citrate efflux. CONCLUSIONS: These results indicate that the activation of genes possibly encoding citrate transporters plays a critical role in Al-stimulated citrate efflux.     

Maize ZmALMT2 is a root anion transporter that mediates constitutive root malate efflux

Root efflux of organic acid anions underlies a major mechanism of plant aluminium (Al) tolerance on acid soils. This efflux is mediated by transporters of the Al-activated malate transporter (ALMT) or the multi-drug and toxin extrusion (MATE) families. ZmALMT2 was previously suggested to be involved in Al tolerance based on joint association-linkage mapping for maize Al tolerance. In the current study, we functionally characterized ZmALMT2 by heterologously expressing it in Xenopus laevis oocytes and transgenic Arabidopsis. In oocytes, ZmALMT2 mediated an Al-independent electrogenic transport product of organic and inorganic anion efflux. Ectopic overexpression of ZmALMT2 in an Al-hypersensitive Arabidopsis KO/KD line lacking the Al tolerance genes, AtALMT1 and AtMATE, resulted in Al-independent constitutive root malate efflux which partially restored the Al tolerance phenotype. The lack of correlation between ZmALMT2 expression and Al tolerance (e.g., expression not localized to the root tip, not up-regulated by Al, and higher in sensitive versus tolerance maize lines) also led us to question ZmALMT2's role in Al tolerance. The functional properties of the ZmALMT2 transporter presented here, along with the gene expression data, suggest that ZmALMT2 is not involved in maize Al tolerance but, rather, may play a role in mineral nutrient acquisition and transport.     

Functional expression of organic anion transporters in hepatic organoids reconstructed by rat small hepatocytes

Small hepatocytes (SHs) are hepatic progenitor cells with hepatic characteristics. They can proliferate to form colonies in culture and change their morphology from flat to rising/piled-up with bile canaliculi (BC), which results in maturation. In this study, we examined whether SHs could express hepatic transporters with polarity, whether the transporters could transport organic anion substrates into BC, and whether the secreted substances could be recovered from BC. Immunocytochemistry and RT-PCR were carried out. [(3)H]-labeled estrogen derivatives were used to measure the functions of the transporters in SHs isolated from normal and multidrug resistance-associated protein (Mrp) 2-deficient rats. The results showed that organic anion-transporting proteins (Oatps) 1 and 2, Na(+)-dependent taurocholate co-transporting polypeptide (Ntcp), Mrp2, and bile-salt export pump (Bsep) were well expressed in rising/piled-up cells and that their expression was correlated to that of hepatocyte nuclear factor 4alpha. Although small SHs expressed not Oatps and Mrp2 but Mrp3, rising/piled-up SHs expressed Oatp1 and 2 and Mrp2 proteins in the sinusoidal and BC membranes, respectively. On the other hand, breast cancer resistant protein (Bcrp) and Mrp3 expression decreased as SHs matured. The substrate transported via Oatps and Mrp2 was secreted into BC and it accumulated in both BC and cyst-like structures. The secreted substrate could be efficiently recovered from BC reconstructed by SHs derived from a normal rat, but not from an Mrp2-deficient rat. In conclusion, SHs can reconstitute hepatic organoids expressing functional organic anion transporters in culture. This culture system may be useful to analyze the metabolism and excretion mechanisms of drugs.     

The role of dileucine in the expression and function of human organic anion transporter 1 (hOAT1)

Human organic anion transporter hOAT1 plays a critical role in the body disposition of environmental toxins and clinically important drugs including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. In the current study, we investigated the role of dileucine (L6L7) at the amino terminus of hOAT1 in the expression and function of the transporter. We substituted L6L7 with alanine (A) simultaneously. The resulting mutant transporter L6A/L7A showed no transport activity due to its complete loss of expression at the cell surface. Such loss of surface expression of L6A/L7A was consistent with a complete loss of an 80 kDa mature form and a dramatic decrease in a 60 kDa immature form of the mutant transporter in the total cell lysates. Treatment of L6A/L7A-expressing cells with proteasomal inhibitor resulted in a significant increase in the immature form of hOAT1, but not its mature form, whereas treatment of these cells with lysosomal inhibitor had no effect on the expression of the mutant transporters, suggesting that the mutant transporter was degraded through proteasomal pathway. The accumulation of mutant transporter in the endoplasmic reticulum (ER) was confirmed by coimmunolocalization of L6L7 with calnexin, an ER marker. Furthermore, treatment of L6A/L7A-expressing cells with sodium 4-phenylbutyrate (4PBA) and glycerol, two chemical chaperones, could not promote the exit of the immature form of the mutant transporter from the ER. Our data suggest that L6L7 are critical for the stability and ER export of hOAT1.     

Organic anion transporter 3 is involved in the brain-to-blood efflux transport of thiopurine nucleobase analogs

Thiopurines are used as antileukemic drugs. However, during chemotherapy CNS relapses occur due to the proliferation of leukemic cells in the CNS resulting from restricted drug distribution in the brain. The molecular mechanism for this limited cerebral distribution remains unclear. The purpose of this study was to identify the transporter responsible for the brain-to-blood transport of thiopurines across the blood-brain barrier (BBB) using the brain efflux index method. [14C]6-Mercaptopurine (6-MP) and [3H]6-thioguanine were eliminated from rat brain in a time-dependent manner. The elimination of [14C]6-MP was inhibited by substrates of rat organic anion transporters (rOATs), including indomethacin and benzylpenicillin. rOAT1 and rOAT3 exhibited 6-MP uptake, while benzylpenicillin inhibited rOAT3-mediated uptake, but not that by rOAT1. rOAT3-mediated [14C]6-MP uptake was also inhibited by other thiopurine derivatives. Although methotrexate inhibited rOAT3-mediated [14C]6-MP uptake, the Ki value was 17.5-fold greater than the estimated brain concentration of methotrexate in patients receiving chemotherapy. Accordingly, 6-MP would undergo efflux transport by OAT3 from the brain without any inhibitory effect from coadministered methotrexate in the chemotherapy. In conclusion, rOAT3 is involved in the brain-to-blood transport of thiopurines at the BBB and is one mechanism of limited cerebral distribution.     

Plasma membrane anion channels in higher plants and their putative functions in roots

Recent years have seen considerable progress in identifying anion channel activities in higher plant cells. This review outlines the functional properties of plasma membrane anion channels in plant cells and discusses their likely roles in root function. Plant anion channels can be grouped according to their voltage dependence and kinetics: (1) depolarization-activated anion channels which mediate either anion efflux (R and S types) or anion influx (outwardly rectifying type); (2) hyperpolarization-activated anion channels which mediate anion efflux, and (3) anion channels activated by light or membrane stretch. These types of anion channel are apparent in root cells where they may function in anion homeostasis, membrane stabilization, osmoregulation, boron tolerance and regulation of passive salt loading into the xylem vessels. In addition, roots possess anion channels exhibiting unique properties which are consistent with them having specialized functions in root physiology. Most notable are the organic anion selective channels, which are regulated by extracellular Al3+ or the phosphate status of the plant. Finally, although the molecular identities of plant anion channels remain elusive, the diverse electrophysiological properties of plant anion channels suggest that large and diverse multigene families probably encode these channels.     

Mutational analysis of the role of GXXXG motif in the function of human organic anion transporter 1 (hOAT1)

Human organic anion transporter hOAT1 plays a critical role in the body disposition of environmental toxins and clinically important drugs including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. hOAT1 has two GXXXG motifs in its transmembrane domains 2 and 5, a motif linked to the protein processing and oligomerization of other proteins. In the current study, we substituted glycine of these GXXXG motifs with alanine and evaluated the effect of such mutations on the expression and function of hOAT1. Mutations of GXXXG motif in the transmembrane domain 2 resulted in mutants G144A and G148A, both of which had no transport activity due to complete loss in the surface and total cell expression of the transporter protein. Treatment of G144A- and G148A-expressing cells with proteasomal inhibitor resulted in the recovery of ER-resident immature form of hOAT1, but not its surface-resident mature form, whereas treatment of these cells with lysosomal inhibitor had no effect on the expression of the mutant transporters. Mutations of GXXXG motif in the transmembrane domain 5 resulted in mutants G223A and G227A, among which only G227 had dramatic reduction of transport activity due to dramatic loss in the surface and total cell expression of the transporter. The reduction in the surface expression of G227 was consistent with the decrease in maximum transport velocity Vmax. Treatment of G227A-expressing cells with proteasomal inhibitor or lysosomal inhibitor resulted in partial recovery of both the immature form and the mature form of hOAT1 in the total cell extracts. However, such partial recovery of the mature form in total cell extracts did not lead to the partial recovery of surface expression and function of the transporter. Our data suggest that the GXXXG motifs in transmembrane domains 2 and 5 play critical roles in the stability of hOAT1.     

Detoxification of xenobiotics by plant cells: characterisation of vacuolar amphiphilic organic anion transporters

The transport activities of two primary ATP-dependent organic-anion transporters in the tonoplast of isolated barley (Hordeum vulgare L. cv. Klaxon) vacuoles have been characterised with N-ethylmaleimide glutathione (NEM-SG) and taurocholate as substrates. The transporters showed different sensitivities to organic anions and a variety of transport inhibitors and drugs. The vacuolar uptake of NEM-SG was inhibited by carbonylcyanide 4-trifluoromethoxyphenylhydrazone, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), S-(2,4-dinitrophenyl)glutathione, alkyl-S-glutathione derivatives and taurocholate but stimulated by probenecid. The uptake of taurocholate was inhibited by vinblastine, DIDS and probenecid. Both transporters were unaffected by verapamil. The kinetic properties of the transporters indicate a general preference for amphiphilic anions with some substrate overlap. These characteristics of the transporters are similar to those displayed by the multidrug resistance protein of mammalian drug-resistant cells. We suggest that these vacuolar transporters be described as plant multispecific organic anion transporters (pMOATs).Abbreviations Bm-S bimane S-glutathione - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - DNP-SG S-(2,4-dinitrophenyl)glutathione - FCCP carbonylcyanide 4-trifluoromethoxyphenylhydrazone - LTC₄ cysteinyl leukotriene - MDR multidrug transporter - MRP multidrug resistance protein - NEM-SG N-ethylmaleimide glutathione We thank Prof E. Martinoia for technical advice on the uptake experiments and Prof J. Palmer for helpful discussions and suggestions. M.B.-K. was partially sponsored by a grant from Stichting VSB Fonds, The Netherlands. IACR receives grant-aided support from the Biotechnology and Biological Science Research Council of the United Kingdom     

The organic anion transporter SLCO2A1 constitutes the core component of the Maxi‐Cl channel

The maxi‐anion channels (MACs) are expressed in cells from mammals to amphibians with ~60% exhibiting a phenotype called Maxi‐Cl. Maxi‐Cl serves as the most efficient pathway for regulated fluxes of inorganic and organic anions including ATP. However, its molecular entity has long been elusive. By subjecting proteins isolated from bleb membranes rich in Maxi‐Cl activity to LC‐MS/MS combined with targeted siRNA screening, CRISPR/Cas9‐mediated knockout, and heterologous overexpression, we identified the organic anion transporter SLCO2A1, known as a prostaglandin transporter (PGT), as a key component of Maxi‐Cl. Recombinant SLCO2A1 exhibited Maxi‐Cl activity in reconstituted proteoliposomes. When SLCO2A1, but not its two disease‐causing mutants, was heterologously expressed in cells which lack endogenous SLCO2A1 expression and Maxi‐Cl activity, Maxi‐Cl currents became activated. The charge‐neutralized mutant became weakly cation‐selective with exhibiting a smaller single‐channel conductance. Slco2a1 silencing in vitro and in vivo, respectively, suppressed the release of ATP from swollen C127 cells and from Langendorff‐perfused mouse hearts subjected to ischemia–reperfusion. These findings indicate that SLCO2A1 is an essential core component of the ATP‐conductive Maxi‐Cl channel.     

Possible involvement of organic anion and cation transporters in renal excretion of xanthine derivatives, 3-methylxanthine and enprofylline

Whether organic anion and cation transporters are involved in the renal excretion of xanthine derivatives, 3-methylxanthie and enprofylline, remains unclear. In this study, we have investigated the effects of typically predominant substrates for organic anion and cation transporters on the tubular secretion of 3-methylxanthine and enprofylline in rats. In the renal clearance experiments using typical substrates for organic anion transporters, probenecid and p-aminohippurate, probenecid (20 mg/kg), but not p-aminohippurate (100 mg/kg), significantly decreased the renal clearance and clearance ratio of 3-methylxanthine and enprofylline. The typical substrates for organic cation transport systems, tetraethylammonium (30.6 mg/kg) and cimetidine (50 or 100 mg/kg), significantly decreased the renal clearance and clearance ratio of 3-methylxanthine and enprofylline. These results suggest that the renal secretory transport of 3-methylxanthine and enprofylline are mediated by probenecid-, cimetidine- and tetraethylammonium-sensitive transport systems. Uric acid, an organic anion, significantly inhibited the renal secretion of 3-methylxanthine, but not enprofylline, suggesting that the renal tubular transport of 3-methylxanthine is also mediated via uric acid-sensitive transport system. These findings suggest the possibility that both organic anion and cation transporters are, at least, involved in the renal tubular transport of 3-methylxanthine and enprofylline in rats.     

Review. CLC-mediated anion transport in plant cells

Plants need nitrate for growth and store the major part of it in the central vacuole of cells from root and shoot tissues. Based on few studies on the two model plants Arabidopsis thaliana and rice, members of the large ChLoride Channel (CLC) family have been proposed to encode anion channels/transporters involved in nitrate homeostasis. Proteins from the Arabidopsis CLC family (AtClC, comprising seven members) are present in various membrane compartments including the vacuolar membrane (AtClCa), Golgi vesicles (AtClCd and AtClCf) or chloroplast membranes (AtClCe). Through a combination of electrophysiological and genetic approaches, AtClCa was shown to function as a 2NO3-/1H+ exchanger that is able to accumulate specifically nitrate into the vacuole, in agreement with the main phenotypic trait of knockout mutant plants that accumulate 50 per cent less nitrate than their wild-type counterparts. The set-up of a functional complementation assay relying on transient expression of AtClCa cDNA in the mutant background opens the way for studies on structure-function relationships of the AtClCa nitrate transporter. Such studies will reveal whether important structural determinants identified in bacterial or mammalian CLCs are also crucial for AtClCa transport activity and regulation.     

Role of blood-brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate,a uremic toxin: its involvement in neurotransmitter metabolite clearance from the brain

Renal impairment is associated with CNS dysfunctions and the accumulation of uremic toxins, such as indoxyl sulfate, in blood. To evaluate the relevance of indoxyl sulfate to CNS dysfunctions, we investigated the brain-to-blood transport of indoxyl sulfate at the blood-brain barrier (BBB) using the Brain Efflux Index method. [(3)H]Indoxyl sulfate undergoes efflux transport with an efflux transport rate of 1.08 x 10(-2)/min, and the process is saturable with a Km of 298 microm. This process is inhibited by para-aminohippuric acid, probenecid, benzylpenicillin, cimetidine and uremic toxinins, such as hippuric acid and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid. RT-PCR revealed that an OAT3 mRNA is expressed in conditionally immortalized rat brain capillary endothelial cell lines and rat brain capillary fraction. Xenopus oocytes expressing OAT3 were found to exhibit [(3)H]indoxyl sulfate uptake, which was significantly inhibited by neurotransmitter metabolites, such as homovanillic acid and 3-methoxy-4-hydroxymandelic acid, and by acyclovir, cefazolin, baclofen, 6-mercaptopurine, benzoic acid, and ketoprofen. These results suggest that OAT3 mediates the brain-to-blood transport of indoxyl sulfate, and is also involved in the efflux transport of neurotransmitter metabolites and drugs. Therefore, inhibition of the brain-to-blood transport involving OAT3 would occur in uremia and lead to the accumulation of neurotransmitter metabolites and drugs in the brain.     

Loading of nitrate into the xylem: apoplastic nitrate controls the voltage dependence of X-QUAC,the main anion conductance in xylem-parenchyma cells of barley roots

We report here that NO(3)(-) in the xylem exerts positive feedback on its loading into the xylem through a change in the voltage dependence of the Quickly Activating Anion Conductance, X-QUAC. Properties of this conductance were investigated on xylem-parenchyma protoplasts prepared from roots of Hordeum vulgare by applying the patch-clamp technique. Chord conductances were minimal around -40 mV and increased with plasma membrane depolarisation as well as with hyperpolarisation. Two gates with opposite voltage dependences were postulated. When 30 mM Cl- in the bath was replaced by NO(3)(-), a shift in the midpoint potential of the depolarisation-activated gate by about -60 mV from 43 to -16 mV occurred (K(m) = 3.4 mM). No such effect was seen when chloride was replaced by malate. Addition of 10 mM NO(3)(-)to the pipette solution and reduction of [Cl-] from 124 to 4 mM (to simulate cytoplasmic concentrations) did not interfere with the voltage dependence of X-QUAC activation, nor was it affected by changes in external [K+]. If only the NO(3)(-) effect on gating was considered, an increase of the NO(3)(-) concentration in the xylem sap to 5 mM would result in an enhancement of NO(3)(-) efflux by about 30%. Although the driving force for NO(3)(-) efflux would be reduced simultaneously, NO(3)(-) efflux into the xylem through X-QUAC would be maintained with high NO(3)(-) concentrations in the xylem sap; a situation which occurs for instance during the night.     

Identification of a sodium-dependent organic anion transporter from rat adrenal gland

In this study, a novel sodium-dependent organic anion transporter (Soat) was identified. Soat is expressed in rat brain, heart, kidney, lung, muscle, spleen, testis, adrenal gland, small intestine, and colon. The Soat protein consists of 370 amino acids and shows 42% and 31% overall amino acid sequence identity to the ileal sodium-dependent bile acid transporter (Isbt) and the Na(+)/taurocholate cotransporting polypeptide (Ntcp), respectively. Soat is predicted to have nine transmembrane domains, with an N-terminus outside the cell and an intracellular C-terminus. The Soat gene is localized on chromosome 14 and is coded by six exons mapped in region 14p22. When expressed in Xenopus laevis oocytes, Soat shows transport function for estrone-3-sulfate (Km = 31 microM, Vmax = 5557 fmol/oocyte/30 min) and dehydroepiandrosterone sulfate (Km = 30 microM, Vmax = 5682 fmol/oocyte/30 min). Soat does not transport taurocholate, estradiol-17beta-glucuronide, nor ouabain.     

Olfactory mucosa-expressed organic anion transporter, Oat6, manifests high affinity interactions with odorant organic anions

We have characterized the expression of organic anion transporter 6, Oat6 (slc22a20), in olfactory mucosa, as well as its interaction with several odorant organic anions. In situ hybridization reveals diffuse Oat6 expression throughout olfactory epithelium, yet olfactory neurons laser-capture microdissected from either the main olfactory epithelium (MOE) or the vomeronasal organ (VNO) did not express Oat6 mRNA. These data suggest that Oat6 is expressed in non-neuronal cells of olfactory tissue, such as epithelial and/or other supporting cells. We next investigated interaction of Oat6 with several small organic anions that have previously been identified as odortype components in mouse urine. We find that each of these compounds, propionate, 2- and 3-methylbutyrate, benzoate, heptanoate, and 2-ethylhexanoate, inhibits Oat6-mediated uptake of a labeled tracer, estrone sulfate, consistent with their being Oat6 substrates. Previously, we noted defects in the renal elimination of odortype and odortype-like molecules in Oat1 knockout mice. The finding that such molecules interact with Oat6 raises the possibility that odorants secreted into the urine through one OAT-mediated mechanism (Eraly et al., JBC 2006) are transported through the olfactory mucosa through another OAT-mediated mechanism. Oat6 might play a direct or indirect role in olfaction, such as modulation of the availability of odorant organic anions at the mucosal surface for presentation to olfactory neurons or facilitation of delivery to a distal site of chemosensation, among other possibilities that we discuss.