Introduction

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Multispecific organic anion and cation transporters play an important role in the removal of xenobiotics and endobiotics from the body. Recent expression cloning and molecular cloning studies have identified several members of a multispecific organic ion transporter family (Koepsell et al., 1999). A unique feature of the members of this gene family is their ability to transport a wide variety of structurally diverse organic anions or organic cations. The transporters identified thus far as the members of this family include the organic cation transporters OCT1 (Grundemann et al., 1994), OCT2 (Okuda et al., 1996), OCT3 (Kekuda et al., 1998), OCTN1 (Tamai et al., 1997), and OCTN2 (Wu et al., 1998), and the organic anion transporters OAT1 (Sekine et al., 1997; Sweet et al., 1997), OAT2 (Sekine et al., 1998), and OAT3 (Kusuhara et al., 1999). The homologs of several of these members have been cloned from different species.

The organic anion transporters are expressed primarily in the kidney (OAT1 and OAT3), liver (OAT2 and OAT3), and brain (OAT3). OAT1 interacts with a variety of anions such as p-aminohippurate (PAH), dicarboxylates, prostaglandin E₂, urate, and -lactam antibiotics (Sekine et al., 1997; Sweet et al., 1997). OAT2 also exhibits a similar broad specificity for several organic anions (Sekine et al., 1998). The most recently cloned OAT3 interacts with PAH, ochratoxin A, estrone sulfate, probenecid, -lactam antibiotics, and diuretics (Kusuhara et al., 1999). In general, the organic anion transporters do not interact with organic cations and the organic cation transporters do not interact with organic anions. The three organic anion transporters thus far identified differ considerably in their transport mechanism. OAT1 is an anion exchanger whose functional characteristics are identical with those of the anion exchanger described in the kidney basolateral membrane (Pritchard and Miller, 1993; Ullrich, 1994). In contrast to OAT1, the other two organic anion transporters OAT2 and OAT3 do not function as anion exchangers. The exact transport mechanism of OAT2 and OAT3 is not known. Several studies have established a functional link between OAT1 and the Na⁺-coupled dicarboxylate transporter in the kidney (Pritchard and Miller, 1993; Ullrich, 1994). Under physiological conditions, a high-affinity Na⁺-coupled dicarboxylate transporter in the kidney basolateral membrane transports dicarboxylates such as -ketoglutarate and glutarate actively into the tubular epithelial cells from the blood. This process establishes a cell-to-blood concentration gradient for these dicarboxylates. OAT1, an anion exchanger, uses this gradient for dicarboxylates as the driving force to actively transport several organic anions into the tubular cells in exchange for the dicarboxylates. The organic anions thus entering the cells across the basolateral membrane are subsequently secreted across the brush border membrane into the tubular lumen. The transporter responsible for the secretory process across the brush border membrane has not been identified at the molecular level.

Caenorhabditis elegans provides a simple model system to study the function of various genes expressed in higher organisms. This nematode is exposed to various xenobiotics in the soil produced by microorganisms and plants and also xenobiotics that arise from industrial pollution. Therefore, this organism must possess transport mechanisms to eliminate these xenobiotics. Recently, we reported the cloning of the first organic cation transporter from C. elegans (Wu et al., 1999). Here we report the cloning of the first organic anion transporter from this organism. This transporter, designated CeOAT1, is an anion exchanger and is thus functionally similar to the mammalian OAT1.

	Experimental Procedures

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Materials. PAH (p-[glycyl-2-³H(N)]aminohippuric acid; specific radioactivity, 40 Ci/mmol), TEA ([ethyl-1-¹⁴C]tetraethylammonium bromide; specific radioactivity, 55 mCi/mmol), [³H]MPP⁺ (1-methyl-4-phenylpyridinium ion; specific radioactivity, 60 Ci/mmol), [³H]MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; specific radioactivity, 80 Ci/mmol), [³H]choline (specific radioactivity, 85 Ci/mmol), and [³H]cimetidine (specific radioacitivity, 18.2 Ci/mmol) were obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Unlabeled anions and cations were obtained from Research Biochemicals International (Natick, MA) or Sigma Chemical Co. (St. Louis, MO). Cell culture media and Lipofectin were from Life Technologies (Rockville, MD). Restriction enzymes were from Promega (Madison, WI). Magna nylon transfer membranes were purchased from Micron Separations, Inc. (Westboro, MA). HeLa cells were obtained from American Type Culture Collection, Inc. (Rockville, MD). The Ready-to-Go oligolabeling kit used in the preparation of cDNA probes for library screening was from Amersham Pharmacia Biotech (Piscataway, NJ).

DNA Sequencing. Sequencing by the dideoxy chain termination method was performed by Taq DyeDeoxy terminator cycle sequencing with an automated Perkin-Elmer Applied Biosystems 377 Prism DNA sequencer (Perkin-Elmer, Norwalk, CT).

Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Probe Preparation. Total RNA was isolated from C. elegans with Trizol reagent (Life Technologies) according to manufacturer's protocol. Poly(A)⁺ RNA was then prepared by affinity chromatography on an oligo dT-cellulose column (Life Technologies). The integrity of the RNA sample was checked by formaldehyde-agarose gel electrophoresis.

Screening of cDNA Library. The SuperScript Plasmid System (Life Technologies) was used to establish the directional cDNA library using the poly(A)⁺ RNA isolated from C. elegans (Fei et al., 1998). The cDNA probe obtained by RT-PCR was labeled with [-³²P]dCTP using the Ready-to-Go oligolabeling kit (Pharmacia). The C. elegans cDNA library was screened with the probe under medium stringency conditions. Hybridization was carried out at 65°C for 20 h in a solution containing 5 × SSPE (1× SSPE = 0.15 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA), 5 × Denhardt's solution, 0.5% SDS, and 100 µg/ml of denatured salmon sperm DNA. Posthybridization washing was done as described earlier (Kekuda et al., 1998; Wu et al., 1999), which involved extensive washes with 3 × SSPE/0.5% SDS at room temperature. Positive clones were identified and the colonies purified by secondary screening.

Functional Expression of CeOAT1 in HeLa Cells. Functional analysis of the cloned cDNA was carried out by heterologous expression in human HeLa cells using the vaccinia virus expression technique (Blakely et al., 1992). The cloned cDNA is present in pSPORT vector under the control of T7 promoter. A recombinant vaccinia virus carrying the gene for T7 RNA polymerase mediates the expression of the cDNA in mammalian cells. Transport of [³H]PAH or other radiolabeled compounds in HeLa cells expressing the CeOAT1 cDNA was measured as described previously (Kekuda et al., 1998; Wu et al., 1999). The transport buffer was 25 mM HEPES/Tris (pH 7.5) supplemented with 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄ and 5 mM gluose. When Na⁺-free buffer was used, N-methyl D-glucamine chloride was used to replace NaCl in the transport buffer. Cells transfected with pSPORT vector alone were used to determine endogenous transport activity. The transport activity in cDNA-transfected cells was adjusted for the endogenous transport activity to calculate cDNA-specific activity.

Statistics. Uptake experiments were done in triplicate and repeated two or three times with separate transfections. Uptake values are given as means ± S.E. of these replicate values. Kinetic analysis was carried out by linear and nonlinear regression methods using the commercially available computer program Sigma Plot (Chicago, IL).

	Results

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Structural Features of CeOAT1. Database searches of the C. elegans genomic DNA sequence for genes homologous to the members of the mammalian organic cation/anion transporter family identified a putative candidate. The location of this gene corresponds to the CEL T01B11.5 gene on chromosome III. To clone this C. elegans organic cation/anion transporter, we obtained an RT-PCR product using C. elegans poly(A)⁺ RNA and primers designed on the basis of the predicted exonic sequences of the gene. The size of the RT-PCR product was ~0.45 kbp and its identity was confirmed by sequencing. This cDNA was used as the probe to screen a C. elegans cDNA library (Fei et al., 1998) to isolate the full-length clone for structural and functional analysis. The nucleotide sequence (GenBank accession no. AF152095) and the deduced amino acid sequence of CeOAT1 are given in Fig. 1. The cDNA is 1703 bp long with a 1581-bp long open reading frame (including the termination codon). The 5' and 3' untranslated regions are 15 bp and 107 bp long, respectively. The 3' untranslated region contains the poly(A)⁺ tail and the polyadenylation signal (AATAA). The predicted protein consists of 526 amino acids. Hydropathy analysis of the amino acid sequence predicts 12 transmembrane domains. When modeled similar to the known members of the mammalian organic cation/anion transporter superfamily, both the amino terminus and the carboxyl terminus of the CeOAT1 protein are located on the cytoplasmic side of the membrane. There is a long extracellular loop consisting of 57 amino acids between the transmembrane domains 1 and 2. This loop contains one potential site for N-linked glycosylation (Asn-96). The predicted molecular mass of the protein is 59 kDa.

View larger version (50K): [in this window] [in a new window]   Fig. 1.   Nucleotide sequence of CeOAT1 and predicted amino acid sequence.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Exon-intron organization of the C. elegans OAT1 gene and its organizational relationship to CeOAT1 cDNA. Black boxes represent the protein coding regions of the exons and gray boxes represent the 5'-untranslated region in exon 2 and 3'-untranslated region in exon 11. The location of exon 1 remains unknown.

Functional Characteristics of CeOAT1. To define the functional identity of CeOAT1, the cDNA was expressed in HeLa cells using the vaccinia virus expression technique and the ability of the expressed protein to transport organic cations and organic anions was assessed. Cells transfected with pSPORT vector alone without the cDNA insert were used as control. Because it was not possible to predict whether the cloned cDNA codes for an organic cation transporter or an organic anion transporter based on the amino acid sequence homology between CeOAT1 and mammalian organic cation and anion transporters, we first tested several organic cations as potential substrates for CeOAT1. None of the cations tested (choline, cimetidine, MPP⁺, MPTP, and TEA) was found to be a substrate for CeOAT1. The transport of these organic cations in cells transfected with CeOAT1 cDNA was similar to that in cells transfected with pSPORT vector alone (Fig. 3). However, the transport of the organic anion PAH was increased about 3.5-fold in CeOAT1-expressing cells, compared with control cells.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Transport of PAH and organic cations by CeOAT1. HeLa cells were transfected with either pSPORT vector alone or CeOAT1 cDNA. Transport of radiolabeled PAH and organic cations (choline, cimetidine, MPP+, MPTP, and TEA) was measured in these cells at pH 7.5 in the presence of Na+. The incubation time was 30 min and the concentration of PAH and organic cations was 10 µM. Transport measured in control cells transfected with vector alone was taken as 100% in each case. This value was, in pmol/106 cells/30 min, 578 ±6 (choline), 41.8 ± 0.4 (cimetidine), 184 ± 2 (MPP+), 79 ± 2 (MPTP), 5.8 ± 0.1 (TEA), and 8.4 ± 0.6 (PAH).

Functional Coupling between CeOAT1 and Mammalian NaDC3. Because CeOAT1 was found to transport the organic anion PAH, we analyzed whether CeOAT1 functions as an anion exchanger. In mammalian systems, OAT1 is an anion exchanger and is functionally coupled to a high-affinity Na⁺-coupled dicarboxylate transporter (Sekine et al., 1997; Sweet et al., 1997). OAT2 and OAT3 transport organic anions but do not function as anion exchangers even though the exact operational mechanism of these transporters is not known (Sekine et al., 1998; Kusuhara et al., 1999). There is no information available on Na⁺-coupled dicarboxylate transporters in C. elegans. Therefore, we used the Na⁺-coupled dicarboxylate transporter cloned from rat placenta (Kekuda et al., 1999) for assessing the anion exchange function of CeOAT1. We expressed CeOAT1 and rat (r)NaDC3 either individually or together in HeLa cells and measured PAH transport. In addition, we tested the influence of exogenously added -ketoglutarate (a dicarboxylate) and Na⁺ on PAH transport in cells expressing CeOAT1 and rNADC3 individually or together. As shown in Fig. 4A, when measured in the presence of Na⁺, the transport of PAH was increased about 4-fold as a result of expression of CeOAT1. Expression of rNaDC3 alone did not influence PAH transport, demonstrating that PAH is not a substrate for rNaDC3. When CeOAT1 and rNaDC3 were coexpressed, the transport of PAH was increased about 9-fold. This increase was significantly higher than the increase observed when CeOAT1 was expressed alone without rNaDC3. We then assessed the influence of 20 µM -ketoglutarate, a dicarboxylate substrate for rNaDC3, on CeOAT1-mediated PAH transport (Fig. 4B). Addition of this dicarboxylate did not have any noticeable effect on PAH transport in cells transfected with empty vector, CeOAT1 cDNA, or rNaDC3 cDNA. However, PAH transport in cells coexpressing CeOAT1 and rNaDC3 increased significantly in the presence of exogenously added -ketoglutarate (464 ± 5 versus 624 ± 17 pmol/10⁶ cells/30 min in the absence and presence of -ketoglutarate, respectively). There was a 13-fold stimulation of PAH transport in the presence of -ketoglutarate in cells coexpressing CeOAT1 and rNaDC3, compared with PAH transport in cells transfected with empty vector. This stimulation was significantly higher than the corresponding stimulation (9-fold) observed in the absence of -ketoglutarate. We then assessed the role of Na⁺ in the transport process. In the absence of Na⁺ and -ketoglutarate, expression of CeOAT1 increased PAH transport about 4-fold (Fig. 4C), a value similar to that observed in the presence of Na⁺. However, the transport of PAH mediated by CeOAT1 was not increased any further by coexpression of rNaDC3. These data show that PAH transport by CeOAT1 is not Na⁺-dependent and that Na⁺ is necessary for rNaDC3-induced stimulation of PAH transport by CeOAT1. When these transport measurements were made in the absence of Na⁺ but in the presence of -ketoglutarate, the dicarboxylate did not have any noticeable effect on CeOAT1-mediated PAH transport in cells expressing CeOAT1 alone or together with rNaDC3 (Fig. 4D). These data show that the stimulation of PAH transport caused by -ketoglutarate in cells coexpressing CeOAT1 and rNaDC3 is a Na⁺-dependent phenomenon. The stimulation of CeOAT1-mediated PAH transport observed even in the absence of exogenous -ketoglutarate when rNaDC3 was coexpressed with CeOAT1 is most likely due to the endogenous dicarboxylates. These dicarboxylates may leak out of the cells into the culture medium normally and expression of rNaDC3 in the cells may mediate the reuptake of these dicarboxylates and maintain the cell-to-medium concentration gradient for these exchange anions to energize CeOAT1. The role of endogenous dicarboxylates in facilitating the CeOAT1-mediated PAH transport is also evident from the findings that the expression of CeOAT1 alone caused a 3- to 4-fold increase in PAH transport, whether measured in the presence or absence of Na⁺. This increase was observed in the absence of exogenously added -ketoglutarate. The participation of endogenous dicarboxylates in the CeOAT1-mediated PAH transport under these experimental conditions may also explain why the increase in CeOAT1-mediated PAH transport was relatively small (~35%), in response to exogenously added -ketoglugarate in cells coexpressing CeOAT1 and rNaDC3.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Functional link between CeOAT1 and rat NaDC3. HeLa cells were transfected with vector alone (P + P), CeOAT1 cDNA (P + CeOAT1), rNaDC3 cDNA (P + NaDC3), or CeOAT1 cDNA plus rNaDC3 cDNA (CeOAT1 + NaDC3). Transport of PAH (50 µM) was measured in these cells (pH 7.5, 30-min incubation) in the presence (A and B) or absence (C and D) of Na+ and in the presence (B and D) or absence (A and C) of 20 µM -ketoglutarate.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Dose-response relationship for the influence of -ketoglutarate on CeOAT1-mediated PAH transport. CeOAT1 and rat NaDC3 were coexpressed in HeLa cells and transport of PAH (50 µM) was measured in the presence of Na+ (pH 7.5, 30-min incubation). The concentration of -ketoglutarate in the transport buffer was varied over the range of 0 to 100 µM.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Comparison of the stimulatory effects of various organic anions on CeOAT1-mediated PAH transport. CeOAT1 and rat NaDC3 were coexpressed in HeLa cells and transport of PAH (50 µM) was measured in the presence of Na+ (pH 7.5, 30-min incubation). The concentration of organic anions in the transport buffer was 20 µM. Values are percentage of stimulation of PAH transport in the presence of the indicated organic anions, compared with control PAH transport measured without the addition of any of these organic anions.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Substrate specificity of CeOAT1. CeOAT1 and rat NaDC3 were coexpressed in HeLa cells and the transport of [3H]PAH (50 µM) was measured in these cells in the presence of Na+ and 20 µM -ketoglutarate (pH 7.5, 30-min incubation). Unlabeled competitors were added to the transport buffer at a concentration of 2.5 mM. Results are given as percentage of control uptake in the absence of competitors (100% = 453 ± 8 pmol/106 cells/30 min).

View larger version (12K): [in this window] [in a new window]   Fig. 8.   Saturation kinetics of CeOAT1-mediated transport of PAH. CeOAT1 and rat NaDC3 were coexpressed in HeLa cells. Transport of PAH was measured in these cells in the presence of Na+ and 20 µM -ketoglutarate (pH 7.5, 30-min incubation). Transport measured in cells transfected with vector alone was used to adjust for endogenous transport activity. Results shown represent CeOAT1-specific transport activity. The concentration of PAH was varied over the range of 0.05-2 mM; Inset: Eadie-Hofstee plot. V, PAH transpsort in nmol/106 cells/30 min; S, PAH concentration in mM.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

This study represents the first report on the molecular and functional characterization of an organic anion transporter from C. elegans. CeOAT1 consists of 526 amino acids and exhibits considerably low homology to the members of the mammalian organic cation and anion transporter family. The function of CeOAT1 was characterized by using PAH as the anionic substrate. PAH is a prototypical organic anion that is widely used as a substrate for mammalian organic anion transporters. Among five different organic cations tested (choline, cimetidine, MPP⁺, MPTP, and TEA), none was recognized as a substrate by CeOAT1. All of the mammalian organic cation transporters thus far cloned interact with MPP⁺ and TEA (Koepsell et al., 1999). Similarly, the organic cation transporter cloned from C. elegans (CeOCT1) also interacts with all of the organic cations tested in the current study as potential substrates for CeOAT1 (Wu et al., 1999). These data show that CeOAT1 is an organic anion transporter and not an organic cation transporter.

Among the mammalian organic anion transporters, OAT1 is an anion exchanger, whereas OAT2 and OAT3 are not. The operational mechanism of OAT2 and OAT3 remains unknown at present. The current study shows that CeOAT1 is an anion exchanger. Therefore, CeOAT1 is functionally similar to mammalian OAT1. The anion exchange property of CeOAT1 was investigated in the present study by coexpressing this transporter with the rat Na⁺-coupled dicarboxylate transporter. The functional link between CeOAT1 and rNaDC3 is schematically represented in Fig. 9. We have shown previously that rat NaDC3 transports dicarboxylates such as -ketoglutarate in a Na⁺ gradient-dependent manner with a Na^+/dicarboxylate stoichiometry of 3:1 (Kekuda et al., 1999). When expressed heterologously in HeLa cells, rNaDC3 mediates Na⁺-dependent concentrative transport of -ketoglutarate (and other dicarboxylic substrates) in these cells. The resultant cell-to-medium concentration gradient for -ketoglutarate enchances CeOAT1-mediated PAH transport because the dicarboxylate provides the exchange anion for the entry of PAH. The results of the present study provide strong support for the functional coupling between CeOAT1 and rNaDC3. A similar mechanism operates in the mammalian kidney where OAT1 and a high-affinity Na⁺-coupled dicarboxylate transporter, both expressed in the basolateral membrane of the tubular epithelial cells, work together in the active uptake of PAH from the blood into the cells (Pritchard and Miller, 1993; Ullrich, 1994). It is very likely that the CeOAT1 operates in a similar manner in association with a dicarboxylate transporter natively expressed in this organism. The molecular identity and the functional characteristics of the C. elegans dicarboxylate transporter, however, remain unknown.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   A schematic model for functional link between CeOAT1 and rat NaDC3.

Because -ketoglutarate is an excellent substrate for rat NaDC3 and also because this dicarboxylate stimulates PAH transport via CeOAT1, it is likely that -ketoglutarate is an exchange anion for CeOAT1. Similarly, fumarate also appears to be accepted by CeOAT1 as an exchange anion. On the other hand, glutarate, succinate, and malate are also excellent substrates for rat NaDC3 (Kekuda et al., 1999), but these dicarboxylates exhibit relatively much lower stimulatory effect on PAH transport mediated by CeOAT1. This suggests that glutarate, succinate, and malate are poor substrates for CeOAT1 as exchange anions. This is interesting because glutarate as well as -ketoglutarate are accepted as substrates by mammalian OAT1, but succinate and fumarate are not (Shimada et al., 1987; Pritchard, 1988, 1990). However, CeOAT1 accepts fumarate but not glutarate as a substrate. Apparently, even though there is considerable similarity in the transport mechanism between CeOAT1 and mammalian OAT1, these two transporters differ significantly in their substrate specificity. Oxalate and lactate are not transported by rat NaDC3; therefore, the lack of stimulatory effects of these anions on CeOAT1-mediated PAH transport does not permit us to draw any conclusions regarding whether or not these anions are accepted as substrates by CeOAT1. It can be argued that even though -ketoglutarate as well as fumarate stimulate CeOAT1-mediated PAH transport in cells coexpressing CeOAT1 and rNaDC3, only fumarate is accepted as an exchange anion by CeOAT1 because of the possible intracellular conversion of -ketoglutarate to fumarate. This is, however, unlikely because succinate can also be converted to fumarate intracellularly, but this dicarboxylate, although an excellent substrate for rNaDC3, does not stimulate CeOAT1-mediated PAH transport. It is therefore likely that both -ketoglutarate and fumarate are accepted as exchange anions by CeOAT1 and that intracellular metabolism of dicarboxylates does not play any significant role in the observed effects of these compounds on CeOAT1-mediated PAH transport.

Both the CeOAT1 and mammalian OAT1 are multispecific, based on the broad specificity of organic anions that can compete with PAH for transport via these transporters. Penicillin-G, indomethacin, probenecid, and furosemide are excellent substrates for C. elegans and mammalian OAT1s. The proposed function of OAT1 in mammalian systems is in the elimination of various xenobiotics. This is supported by the expression of OAT1 in the kidney, an organ that plays an important role in xenobiotic elimination. C. elegans possesses an excretory system consisting of four cells that functions in the elimination of xenobiotics analogous to the function of the kidney in animals. It is likely that the OAT1 described here is expressed in this excretory system in C. elegans and functions in the elimination of organic anions. However, the physiological significance of the differences in the apparent substrate specificity between CeOAT1 and mammalian OAT1 in relation to the handling of endogenous and exogenous organic anions in the C. elegans versus mammalian organisms is not readily apparent.