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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Functional characterization of testis-specific rodent multidrug and toxic compound extrusion 2, a class III MATE-type polyspecific H⁺/organic cation exporter

Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan

Submitted 3 July 2007 ; accepted in final form 12 August 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Mammalian multidrug and toxic compound extrusion (MATE) proteins are classified into three subfamilies: classes I, II, and III. We previously showed that two of these families act as polyspecific H⁺-coupled transporters of organic cations (OCs) at final excretion steps in liver and kidney (Otsuka et al. Proc Natl Acad Sci USA 102: 17923–17928, 2005; Omote et al. Trends Pharmacol Sci 27: 587–593, 2006). Rodent MATE2 proteins are class III MATE transporters, the molecular nature, as well as transport properties, of which remain to be characterized. In the present study, we investigated the transport properties and localization of mouse MATE2 (mMATE2). On expression in human embryonic kidney (HEK)-293 cells, mMATE2 localized to the intracellular organelles and plasma membrane. mMATE2 mediated pH-dependent TEA transport with substrate specificity similar to, but distinct from, that of mMATE1, which prefers N-methylnicotinamide and guanidine as substrates. mMATE2 expressed in insect cells was solubilized and reconstituted with bacterial H⁺-ATPase into liposomes. The resultant proteoliposomes exhibited ATP-dependent uptake of TEA that was sensitive to carbonyl cyanide 3-chlorophenylhydrazone but unaffected by valinomycin in the presence of K⁺. Immunologic techniques using specific antibodies revealed that mMATE2 was specifically expressed in testicular Leydig cells. Thus mMATE2 appears to act as a polyspecific H⁺/OC exporter in Leydig cells. It is concluded that all classes of mammalian MATE proteins act as polyspecific and electroneutral transporters of organic cations.

organic cation transporter; Leydig cell; guanidine; N-methylnicotinamide

hMATE1 and mMATE1 share 78.1% amino acid sequence identity and are predominantly expressed in luminal membranes of renal tubular cells as well as bile canaliculi (3, 14). On expression in HEK-293 cells, these transporters localize to the plasma membrane and mediate electroneutral H⁺/organic cation (OC) exchange (3, 14). Subsequently, a rat MATE1 (rMATE1) and rabbit MATE1 (rbMATE1) with similar localization and transport properties were also identified (12, 20, 24). Hence, the substrate specificity, energetics, and localization of these transporters are very similar to those of an electroneutral H⁺/OC exchanger(s) characterized in renal brush border membrane vesicles, the molecular identity of which has been long anticipated (2, 5, 7, 21–23). MATE1 proteins are considered to be the principal transporters responsible for the elimination of various OCs at the final excretion step in liver and kidney (13, 14).

In contrast to MATE1 transporters, hMATE2 and mMATE2 exhibit only low mutual sequence identity (38. 1%) and different expression patterns (14). hMATE2 is predominantly expressed in kidney, but not in other organs, including liver, whereas mMATE2 is specifically expressed in testis (14). Since hMATE2-K, a splicing variant of hMATE2, and rabbit MATE2-K, a rabbit counterpart of hMATE2-K, were shown to mediate electroneutral H⁺/OC exchange at the luminal surface of renal tubular cells (8, 24), hMATE2 seems to be a renal-specific electroneutral OC exporter (13). It is uncertain whether the physiological function of mMATE2 is similar to that of hMATE2 (14).

Phylogenetic analysis has shown that mammalian MATE transporters can be classified into three subgroups: classes I, II, and III (13) (Fig. 1A). Class I includes hMATE1, mMATE1, rMATE1, and rbMATE1. Class II includes hMATE2. There are no rodent isoforms of hMATE2. mMATE2 is not a member of class II but, rather, is a member of class III, which includes dog MATE3, chimpanzee MATE3, and rMATE2, and explains the low sequence identity between hMATE2 and mMATE2 and their different localizations.

View larger version (40K): [in this window] [in a new window]   Fig. 1. Classification of mammalian multidrug and toxic compound extrusion (MATE)-type transporters. A: phylogenetic tree of mammalian MATE-type transporters clearly suggests 3 subclasses of MATE transporters: class I (red), class II (blue), and class III (green). B: multiple-sequence alignment of mammalian class III transporters. *, Conserved amino acid residues. Horizontal bars indicate putative transmembrane regions. Peptide regions used in production of antisera are enclosed in red (mMATE2) and blue (mMATE1) boxes.

	MATERIALS AND METHODS

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cDNA. cDNA of mMATE2 (GenBank accession no. NP_001028714) was cloned by RT-PCR from mouse testis RNA (14). The sequence of mMATE2 cDNA was confirmed by comparison with the mouse genome sequence (16).

mMATE2-expressing HEK-293 cells. mMATE2 cDNA was amplified by PCR using the primers 5'-CACCGAATTCATGGAGCCGGCCGAGGACA-3' and 5'-CGTACTCGAGTTAGCCACGGTCATTGAAA-3' and ligated into a pENTR/D-TOPO vector (Invitrogen). mMATE2 cDNA was transferred from the pENTR/D-TOPO vector to pcDNA3.1/nV5-DEST (Invitrogen). This plasmid, pcDNA3.1/nV5-DEST-mMATE2, was used to transfect HEK-293 cells by lipofection using TransIT reagent (Mirus). HEK-293 cells were grown in DMEM containing 10% fetal calf serum, penicillin, and streptomycin at 37°C under 5% CO₂, as described elsewhere (14). After 24 h, 1.5 x 10⁶ cells per 10-cm dish were transfected with 10 µg of pcDNA3.1/nV5-DEST-mMATE2. After 2 days of incubation, the cells were used for immunohistochemistry and transport assay (see below). About 15% of HEK-293 cells were transfected under these conditions.

Expression of mMATE2 in insect cells. Recombinant baculoviruses containing mMATE2 cDNA were constructed using the Bac-to-Bac baculovirus expression system (Invitrogen) according to the manufacturer's protocol. mMATE2 cDNA was transferred from the pENTR/ D-TOPO vector to a destination vector (pDEST10-mMATE2). DH10Bac cells carrying bacmid DNA were transformed with pDEST10-mMATE2. Recombinant bacmid was isolated from DH10Bac cells and used for transfection of Sf9 cells to generate recombinant baculoviruses. Sf9 cells (5 x 10⁶ cells per 10-cm dish) were grown in complete TNM-FH insect culture medium (GIBCO) supplemented with 10% fetal calf serum, 0.25 µg/ml amphotericin B (Fungizone), and 100 µg/ml penicillin-streptomycin at 27°C. Sf9 cells were infected with recombinant baculoviruses at a multiplicity of infection of 2 and cultured for 72 h. Then the cells were harvested for membrane preparation. mMATE1 was also expressed in Sf9 cells as described above.

Solubilization and reconstitution of mMATE2. Sf9 cells (1–2 x 10⁷) were suspended in a buffer containing 20 mM Tris·HCl (pH 8.0), 0.1 M potassium acetate, 10% glycerol, 0.5 mM dithiothreitol, 1 µg/ml pepstatin A, and 1 µg/ml leupeptin and disrupted by sonication with a tip sonifier (model UD200, Tomy). Debris was removed by centrifugation of cell lysates at 700 g for 10 min, and the resultant supernatant was centrifuged again at 160,000 g for 1 h. The pellet was suspended in buffer containing 20 mM MOPS-Tris (pH 7.0), 10% glycerol, 1 µg/ml pepstatin A, and 1 µg/ml leupeptin to give a protein concentration of 1.5 mg/ml. Then octylglucoside was added to the mixture to give a final concentration of 2%. The mixture was vigorously vortexed and centrifuged at 260,000 g for 30 min, and the supernatant (solubilized mMATE2 fraction) was carefully collected. Coreconstitution of solubilized mMATE2 fraction with bacterial F-type ATPase into liposomes was carried out by the freeze-thaw method, as previously described (6, 11). Briefly, 300 µg of solubilized mMATE2 fraction were mixed with 60 µg of F-type ATPase and 0.5 mg of asolectin liposomes. The mixture was frozen at –80°C, thawed rapidly, and diluted 60-fold with a buffer containing 20 mM MOPS-NaOH (pH 7.0), 0.5 mM dithiothreitol, 0.1 M potassium acetate, and 5 mM magnesium acetate. Reconstituted proteoliposomes were pelleted by centrifugation at 160,000 g for 1 h at 4°C and suspended in 0.4 ml of buffer containing 20 mM MOPS-NaOH (pH 7.0), 100 mM KCl, and 5 mM magnesium acetate. Bacterial F-type ATPase was prepared as described previously (11).

Transport assay. For HEK-293 cells expressing mMATE2, pH-dependent TEA uptake was measured by previously published procedures (3, 14, 19). Briefly, the cells were suspended in a buffer containing 25 mM tricine-NaOH (pH 8.0), 125 mM NaCl, 4.8 mM KCl, 5.6 mM d-glucose, 1.2 mM CaCl₂, 1.2 mM KH₂PO₄, and 1.2 mM MgSO₄. The assay was initiated by addition of 50 µM radiolabeled TEA (5 kBq/assay; PerkinElmer Life Science) at 37°C. At the times indicated, aliquots (200 µl) were taken and filtered through 0.45-µm HA membrane filters (Millipore). The filter was washed once with 5 ml of ice-cold assay medium, and the radioactivity remaining on the filter was counted. Reconstituted proteoliposomes (5 µg of protein) were suspended in a buffer containing 20 mM MOPS-NaOH (pH 7.0), 5 mM magnesium acetate, and 100 mM KCl. ATP, at a final concentration of 4 mM, was added to the assay medium, and the mixture was incubated further for 5 min. The assay was initiated by addition of 50 µM radiolabeled TEA (5 kBq/assay; PerkinElmer Life Science). At the times indicated, aliquots (130 µl) were taken, applied to a Sephadex G-50 (fine) spin column poured in the cylinder of a 1-ml disposable syringe, and immediately centrifuged at 180 g for 1 min for separation of the proteoliposomes from the assay medium (6, 11). The radioactivity and protein concentration in the eluate were measured.

Antibodies. Site-specific rabbit polyclonal antibodies against mMATE2 were produced by repeated injections of glutathione S-transferase-fusion polypeptides comprising amino acid residues M1–A46 of mMATE2, a region specific to mMATE2 (Fig. 1B): MEPAEDSLGATIQPPELVRVPRGRSLRILLGLRGALSPDVRREAAA. Preabsorbed antiserum was prepared on incubation of antiserum (50 µl) with antigenic peptide (2 mg) in an ice bath for 10 h. Antiserum against mMATE1 was prepared as described previously (3). Monoclonal antibodies against Rab5 and early endosome antibody (EEA)-1 for early endosomes were obtained from Transduction Laboratories. Alexa Fluor 568-labeled anti-mouse IgG and Alexa Fluor 488-labeled anti-rabbit IgG were purchased from Molecular Probes. Horseradish peroxidase-F(ab')₂ goat anti-rabbit IgG (H + L) was obtained from Zymed.

Western blot analysis. Total membrane fractions of ddY mice (0.1 g wet wt depending on the organ used) were isolated, suspended in ice-cold 20 mM MOPS-Tris (pH 7.0) containing 0.3 M sucrose, 5 mM EDTA, and protease inhibitors (pepstatin A and leupeptin at 5 µg/ml each), homogenized, and centrifuged at 800 g for 8 min at 4°C. The postnuclear supernatant was centrifuged at 100,000 g for 1 h at 4°C. The pellet was suspended in the same buffer. The membrane preparations were denatured at room temperature for 30 min in the presence of 1% SDS and 10% β-mercaptoethanol. Samples (50–200 µg of protein) were subjected to electrophoresis and Western blot analysis, as described previously (3, 14).

Immunohistochemistry. Indirect immunofluorescence microscopy was performed as described previously (3, 14). Briefly, 7- to 8-wk-old male C57BL/6 mice were anesthetized with ether and perfused intracardially with saline and then 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The organs were isolated, and frozen sections were prepared. Cultured cells on poly-L-lysine-coated coverslips were fixed with 4% paraformaldehyde in PBS for 30 min. The cells were washed with PBS and incubated for 20 min, and organ sections were incubated for 30 min in the same buffer containing 0.1% Triton X-100 and then in PBS containing 2% goat serum and 0.5% bovine serum albumin. The specimens were incubated with antibodies diluted to 1 µg/ml or 1,000-fold (anti-mMATE2 or other antibody) with PBS containing 0.5% bovine serum albumin for 1 h at ambient temperature. Samples were washed four times with PBS and then reacted with the secondary antibody or Alexa Fluor 568-labeled anti-mouse IgG (2 µg/ml) or Alexa Fluor 488-labeled anti-rabbit IgG (4 µg/ml) for 1 h at ambient temperature. Finally, immunoreactivity was examined under an Olympus BX60 microscope or an Olympus FV300 confocal laser microscope. All animal procedures and care were approved by the Institutional Animal Care and Use Committee and were carried out according to the guidelines of Okayama University.

Miscellaneous procedure. Protein concentration was assayed using bovine serum albumin as a standard (17).

	RESULTS

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mMATE2 acts as a polyspecific OC transporter. The cDNA for mMATE2 encodes a 573-amino acid protein with 44–95% sequence identity to proteins of the MATE3 subclass, but with 58, 45, and 26% sequence identity to hMATE1, a member of class 1; hMATE2, a member of class 2; and Vibrio parahaemolyticus NorM Na⁺/multidrug antiporter, a prototype of the MATE family (1, 10), respectively (Fig. 1). Similar to other mammalian MATE transporters, mMATE2 hypothetically has 12 transmembrane helices.

As the first step of the study, we expressed mMATE2 in HEK-293 cells with the intention of characterizing the transport properties of mMATE2. We generated a specific antiserum against mMATE2 (Fig. 1B). The antiserum recognized mMATE2 as a 60-kDa protein, but not mMATE1 (Fig. 2A). The slightly lower mobility of mMATE1 and mMATE2 is due to the histidine tag fused to the MATE proteins expressed in Sf9 cells. When mMATE2 was expressed in HEK-293 cells, the protein migrated on the SDS-polyacrylamide gels with a relative mobility corresponding to 52 kDa and localized not only to the plasma membrane, but also to intracellular organelles (Fig. 2, B and C). The immunoreactivity disappeared when the antiserum was preabsorbed (Fig. 2, B and C). Double labeling with Rab5 and EEA-1, markers for early endosomes, indicated that intracellular mMATE2 and these markers roughly colocalized, suggesting that mMATE2 was present in early endosomes and plasma membrane (Fig. 2D).

View larger version (34K): [in this window] [in a new window]   Fig. 2. Expression of mouse MATE2 (mMATE2) in HEK-293 cells. A: immunologic specificity of anti-mMATE2 antibodies. Membrane fractions (10 µg of protein each) prepared from mMATE1- and mMATE2-expressing Sf9 cells were solubilized with SDS sample buffer and subjected to electrophoresis. After proteins were transferred to a nitrocellulose membrane, blots were incubated with antibodies to mMATE1 or mMATE2 (left). Blots with mMATE2 incubated with antiserum preabsorbed with antigenic peptide are shown at right. Immunologic reactivity was visualized by enhanced chemiluminescence. Positions of molecular weight markers are indicated. B: HEK-293 cells transfected with pcDNA3.1 (mock control) or pcDNA3.1/nV5-DEST-mMATE2 (mMATE2) were prepared, and membrane fraction (50 µg of protein) was analyzed by Western blotting with antiserum against mMATE2 or antiserum preabsorbed antibodies (preabsorbed). C: HEK-293 cells from the same experiment were immunostained with antiserum against mMATE2 or antiserum preabsorbed with antigenic peptide. Scale bar, 5 µm. Sections were also observed under Nomarski microscope, and merged images are shown. D: pcDNA3.1/nV5-DEST-mMATE2-transfected HEK-293 cells were doubly immunostained with antibodies against mMATE2 (a) and Rab5 (b) or mMATE2 (d) and EEA1 (e) and then observed. Superimposed (merged) images (c and f) are also shown. Scale bars, 5 µm.

mMATE2-expressing cells exhibited time-dependent transport activity of TEA (Fig. 3A), a typical substrate for H⁺-coupled OC exporter (2, 4, 5, 7, 13, 21–23). The transport activity of mMATE2 was saturable with respect to substrate concentration, with K_m and V_max for TEA of 710 µM and 400 pmol·min^–1·mg protein^–1, respectively (Fig. 3B). The transport was weakly pH dependent (Fig. 3C): transport activity was lower at pH 6.0–6.5, increased slightly with increasing extracellular pH, and was maximal at pH 8.5. The addition of 10 µM cyanide 3-chlorophenylhydrozone (CCCP), which dissipates the proton electrochemical gradient across the membranes, inhibited the uptake, whereas 1 µM valinomycin in the presence of 65 mM KCl, which causes membrane depolarization, did not have much effect (Fig. 3D). Neither Na⁺ nor Cl^– was required for the transport activity (Fig. 3E). The pharmacology of the cis inhibition of TEA transport by mMATE2 was similar to, but distinct from, that of hMATE1 (14) and mMATE1 (3) (Table 1). It is strongly inhibited by cimetidine, methylphenylpyridinium, and rhodamine 123 (Table 1), but not by organic anions such as p-aminohippurate and uric acid (data not shown). N-methylnicotinamide (NMN) and guanidine inhibited mMATE2-mediated TEA uptake (Table 1), whereas these compounds did not significantly affect TEA transport by hMATE1 and mMATE1 (3, 14). Testosterone, procainamide, and choline moderately inhibited TEA uptake (Table 1). Although this transport assay did not consider any contributions from intracellular mMATE2, these results are consistent with the idea that mMATE2 is an H⁺-coupled OC exporter.

View larger version (15K): [in this window] [in a new window]   Fig. 3. mMATE2 mediates electroneutral H+/TEA exchange. A: time course of TEA (50 µM) uptake at pH 8.0 by HEK-293 cells expressing mMATE2. Dashed line, net uptake of TEA 5 min after subtraction of the corresponding mock control cell value. B: dose dependence of TEA uptake at pH 8.0. C: pH dependence of TEA uptake. TEA uptake after 5 min was measured in HEK-293 cells expressing mMATE1 or control cells incubated at pH 6.0–9.0. D: TEA uptake at pH 8.0 in the absence or presence of 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or 1.0 µM valinomycin in buffer containing 65 mM KCl and 65 mM NaCl (control) requires a membrane potential or pH gradient. E: TEA uptake in buffer containing 130 mM KCl (Na+ free) or 250 mM sucrose requires NaCl. Assays were terminated after 5 min. Error bars represent standard deviation of 3 samples; 100% activity corresponds to 110 pmol/mg protein.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Reconstitution of mMATE2 with bacterial F-type ATPase. mMATE2 was expressed in insect Sf9 cells, solubilized from the membrane fraction, and reconstituted with bacterial F-type ATPase into asolectin liposomes. A: Western blot analysis; 10 µg of membrane fraction protein from mMATE2-expressing Sf9 cells, 20 µg of solubilized fraction protein, and 20 µg of reconstituted proteoliposomal protein were applied to the gel. B: time course of TEA uptake by reconstituted proteoliposomes in the presence (+ATP) or absence (–ATP) of ATP. TEA uptake by the proteoliposomes containing only F-type ATPase as sole protein source was also measured in the presence of ATP (–mMATE). C: effects of CCCP and valinomycin on ATP-dependent TEA uptake in reconstituted liposomes (n = 3).

View larger version (14K): [in this window] [in a new window]   Fig. 5. Western blot analysis of mMATE2 in crude membrane fractions of various mouse tissues. A: samples (50 µg of protein from testis microsomal fraction and 200 µg of proteins from microsomal fraction of other organs) were subjected to SDS-PAGE, and the presence of mMATE2 was analyzed by Western blot with anti-mMATE2 antiserum. B: samples (50 µg of protein from testis microsomal fraction) were electrophoresed, and imunoreactivity was measured by Western blot with antiserum preabsorbed with antigenic peptide. Positions of molecular weight markers and standard mMATE2 are shown at left. Arrow indicates the position of mMATE2.

View larger version (50K): [in this window] [in a new window]   Fig. 6. mMATE2 is expressed in Leydig cells in testis. Cross section of testis was immunostained with antiserum against mMATE2 (A and B) or control serum (C). Sections were also observed under Nomarski microscope, and merged images are shown. Arrows, Leydig cells; T, seminiferous tubule. Scale bar, 5 µm.

	DISCUSSION

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We showed that mMATE2 mediates pH-dependent TEA antiport by the standard filtration technique with mMATE2-expressing HEK-293 cells. The transport properties, such as kinetics, pH dependence, independence of Na⁺, and cis inhibition profile, are similar to those of mMATE1 and hMATE1. Localization of mMATE2 in early endosomes is probably an artifact due to overexpression; thus there is latent TEA uptake ability when HEK-293 cells were used for transport assay. To analyze transport activity of mMATE more quantitatively, mMATE2 expressed in Sf9 cells could be solubilized and reconstituted with a bacterial ATP-dependent proton pump in proteoliposomes. Bacterial F-type ATPase is one of the most well-characterized electrogenic proton pumps. The addition of ATP led to the formation of an electrochemical gradient of protons across the proteoliposomal membrane. Accordingly, mMATE2-mediated uptake of TEA was triggered on the addition of ATP. The ATP-dependent uptake of TEA is sensitive to CCCP but unaffected by valinomycin in the presence of K⁺. Together, these results provide convincing evidence for the electroneutral H⁺/OC exchange by mMATE2.

Because of its site of expression, mMATE2 is significant compared with other characterized mammalian MATE transporters. As shown here, mMATE2 is specifically expressed in testicular Leydig cells. The rat counterpart (rMATE2) was also present in testicular Leydig cells, as revealed by immunohistochemical analysis with anti-mMATE2 antiserum (unpublished observations). Although the physiological function of mMATE2 in Leydig cells is unknown, a fascinating hypothesis is that mMATE2 could act as a testosterone exporter and, hence, is at least partially responsible for secretion of this substance from Leydig cells. Testosterone is a hydrophobic, cationic sex hormone produced in Leydig cells. The molecular mechanism of testosterone secretion from the cells is poorly characterized. Cis inhibition studies in mMATE2-expressing HEK-293 cells suggest that the transporter recognizes testosterone as a transport substrate (Table 1) and, thus, support the hypothesis. Direct measurement of testosterone transport by mMATE2-expressing HEK-293 cells is, however, very difficult because of the extremely hydrophobic nature of the hormone, which leads to very high background values in these types of studies.

It is of interest to note that mMATE2 seems to recognize NMN and guanidine as substrates, in addition to TEA, choline, procainamide, cimetidine, and quinidine (Table 1). It is well established that an electroneutral H⁺/OC exchanger(s) located in renal brush border membrane transports NMN and guanidine (5, 7, 9, 18, 21–23). However, hMATE1 and mMATE1 exhibit only weak affinity to these substances (3, 14). We noticed that a small amount of mMATE2 is present in renal tubular cells (unpublished observation). Taken together, these observations suggest that mMATE2 may be responsible for excretion of NMN and guanidine and may play a role in excretion of OCs in combination with mMATE1 in renal tubular cells. Further studies on the localization of mMATE2 in renal tubular cells and its transport ability for guanidine and NMN are needed to clarify these issues.

The present results support the idea that members of all MATE subclasses of mammalian origin mediate similar polyspecific H⁺/OC exchange. Therefore, we can summarize the overall features of mammalian MATE transporters as follows. Class I is ubiquitously present throughout the body but is predominantly found in kidney and liver. Classes II and III have more specific expression patterns than class I and are expressed primarily in kidney and testes, respectively. The functions of these classes may be coordinated and may play a role in the elimination of metabolic OCs and xenobiotics at the final step of excretion in kidney. The physiological role(s) of class III does not seem to involve excretion of OCs, because rodent MATE2 is predominantly expressed in Leydig cells. Whether all mammals, especially humans, possess a counterpart to mMATE2 or in class III is unknown and awaits further study.

	GRANTS

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M. Hiasa and T. Matsumoto were supported by Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists. This work was supported in part by Japanese Ministry of Education, Science, Sport, and Culture Grant-in-Aid for Research 16017264 and the Smoking Research Foundation (to Y. Moriyama).

	ACKNOWLEDGMENTS

 
We thank Dr. M. Otsuka, S. Arioka, and K. Shimizu for help in the initial stage of the study.

	FOOTNOTES

 

Address for reprint requests and other correspondence: Y. Moriyama, Dept. of Membrane Biochemistry, Okayama Univ. Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, Japan (e-mail: moriyama{at}pharm.okayama-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^* M. Hiasa and T. Matsumoto contributed equally to this work.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
1. Brown MH, Paulsen IT, Skurray RA. The multidrug efflux protein NorM is a prototype of a new family of transporters. Mol Microbiol 31: 394–395, 1999.[CrossRef][Web of Science][Medline]

2. Hawk C, Dantzler WH. Tetraethylammonium transport by isolated perfused snake renal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 246: F476–F487, 1984.[Abstract/Free Full Text]

3. Hiasa M, Matsumoto T, Komatsu T, Moriyama Y. Wide variety of locations for rodent MATE1, a transporter protein that mediates the final excretion step for toxic organic cations. Am J Physiol Cell Physiol 291: C678–C686, 2006.[Abstract/Free Full Text]

4. Hvorup RN, Winnen B, Chang AB, Jiang Y, Zhou XF, Saier MH Jr. The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily. Eur J Biochem 270: 799–813, 2003.[Web of Science][Medline]

5. Inui KI, Masuda S, Saito H. Cellular and molecular aspects of drug transport in the kidney. Kidney Int 58: 944–958, 2000.[CrossRef][Web of Science][Medline]

6. Juge N, Yoshida Y, Yatsushiro S, Omote H, Moriyama Y. Vesicular glutamate transporter contains two independent transport machineries. J Biol Chem 281: 39499–39506, 2006.[Abstract/Free Full Text]

7. Koepsell H. Polyspecific organic cation transporters: their functions and interactions with drugs. Trends Pharmacol Sci 25: 375–381, 2004.[CrossRef][Medline]

8. Masuda S, Terada T, Yonezawa A, Tanihara Y, Kishimoto K, Katsura T, Ogawa O, Inui K. Identification and functional characterization of a new human kidney-specific H⁺/organic cation antiporter, kidney-specific multidrug and toxin extrusion 2. J Am Soc Nephrol 17: 2127–2135, 2006.[Abstract/Free Full Text]

9. Miyamoto Y, Tiruppathi C, Ganapathy V, Leibach FH. Multiple transport systems for organic cations in renal brush-border membrane vesicles. Am J Physiol Renal Fluid Electrolyte Physiol 256: F540–F548, 1989.[Abstract/Free Full Text]

10. Morita Y, Kodama K, Shiota S, Mine T, Kataoka A, Mizusima T, Tsuchiya T. NorM, a putative multidrug efflux protein of Vibrio parahaemolyticus and its homolog in Escherichia coli. Antimicrob Agents Chemother 42: 1778–1782, 1998.[Abstract/Free Full Text]

11. Moriyama Y, Iwamoto A, Hanada H, Maeda M, Futai M. One-step purification of Escherichia coli H⁺-ATPase (F₀F₁) and its reconstitution into liposomes with neurotransmitter transporters. J Biol Chem 266: 22141–22146, 1991.[Abstract/Free Full Text]

12. Ohta K, Inoue K, Hayashi Y, Yuasa H. Molecular identification and functional characterization of rat multidrug and toxin extrusion type transporter 1 as an organic cation/ H⁺ antiporter in the kidney. Drug Metab Dispos 34: 1868–1874, 2006.[Abstract/Free Full Text]

13. Omote H, Hiasa M, Matsumoto T, Otsuka M, Moriyama Y. The MATE proteins as fundamental transporters of metabolic and xenobiotic organic cations. Trends Pharmacol Sci 27: 587–593, 2006.[CrossRef][Medline]

14. Otsuka M, Matsumoto T, Morimoto R, Arioka S, Omote H, Moriyama Y. A human transporter protein that mediates the final excretion step for toxic organic cations. Proc Natl Acad Sci USA 102: 17923–17928, 2005.[Abstract/Free Full Text]

15. Putman M, van Veen HW, Koning WN. Molecular properties of bacterial multidrug transporters. Microbiol Mol Biol Rev 64: 672–693, 2000.[Abstract/Free Full Text]

16. Sambrook J, Russell DW. Molecular Cloning—A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.

17. Schaffner W, Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem 56: 502–514, 1973.[CrossRef][Web of Science][Medline]

18. Schaki C, Schild L, Overney J, Roch-Ramel F. Secretion of tetraethylammonium by proximal tubules of rabbit kidneys. Am J Physiol Renal Fluid Electrolyte Physiol 245: F238–F246, 1983.[Abstract/Free Full Text]

19. Tamai I, Yabuuchi H, Nezu J, Sai Y, Oku A, Shimane M, Tsuji A. Cloning and characterization of a novel human pH-dependent organic cation transporter, OCTN1. FEBS Lett 419: 107–111, 1997.[CrossRef][Web of Science][Medline]

20. Terada T, Masuda S, Asaka J, Tsuda M, Katsura T, Inui K. Molecular cloning, functional characterization and tissue distribution of rat H⁺/organic cation antiporter MATE1. Pharm Res 23: 1696–1701, 2006.[CrossRef][Web of Science][Medline]

21. Ullrich KJ. Affinity of drugs to the different renal transporters for organic anions and organic cations. In: Membrane Transporters as Drug Targets, edited by Amidon GL, Sadee W. New York: Kluwer Academic/Plenum, 1999.

22. Wright SH, Wunz TM. Transport of tetraethylammonium by rabbit renal brush-border and basolateral membrane vesicles. Am J Physiol Renal Fluid Electrolyte Physiol 252: F1040–F1050, 1987.

23. Wright SH, Dantzler WH. Molecular and cellular physiology of renal organic cation and anion transport. Physiol Rev 84: 987–1049, 2004.[Abstract/Free Full Text]

24. Zhang X, Cherrington NJ, Wright SH. Molecular identification and functional characterization of rabbit MATE1 and MATE2-K. Am J Physiol Renal Physiol 293: F360–F370, 2007.[Abstract/Free Full Text]

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