Identification of a Novel Voltage-driven Organic Anion Transporter Present at Apical Membrane of Renal Proximal Tubule
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m303210200
ISSN1083-351X
AutoresPromsuk Jutabha, Yoshikatsu Kanai, Makoto Hosoyamada, Arthit Chairoungdua, Do Kyung Kim, Yuji Iribe, Ellappan Babu, Ju Young Kim, Naohiko Anzai, Varanuj Chatsudthipong, Hitoshi Endou,
Tópico(s)Ion Transport and Channel Regulation
ResumoA novel transport protein with the properties of voltage-driven organic anion transport was isolated from pig kidney cortex by expression cloning in Xenopus laevis oocytes. A cDNA library was constructed from size-fractionated poly(A)+ RNA and screened for p-aminohippurate (PAH) transport in high potassium medium. A 1856-base pair cDNA encoding a 467-amino acid peptide designated as OATV1 (voltage-driven organic anion transporter 1) was isolated. The predicted amino acid sequence of OATV1 exhibited 60–65% identity to those of human, rat, rabbit, and mouse sodium-dependent phosphate cotransporter type 1 (NPT1), although OATV1 did not transport phosphate. The homology of this transporter to known members of the organic anion transporter family (OAT family) was about 25–30%. OATV1-mediated PAH transport was affected by the changes in membrane potential. The transport was Na+-independent and enhanced at high concentrations of extracellular potassium and low concentrations of extracellular chloride. Under the voltage clamp condition, extracellularly applied PAH induced outward currents in oocytes expressing OATV1. The current showed steep voltage dependence, consistent with the voltage-driven transport of PAH by OATV1. The PAH transport was inhibited by various organic anions but not by organic cations, indicating the multispecific nature of OATV1 for anionic compounds. This transport protein is localized at the apical membrane of renal proximal tubule, consistent with the proposed localization of a voltage-driven organic anion transporter. Therefore, it is proposed that OATV1 plays an important role to excrete drugs, xenobiotics, and their metabolites driven by membrane voltage through the apical membrane of the tubular epithelial cells into the urine. A novel transport protein with the properties of voltage-driven organic anion transport was isolated from pig kidney cortex by expression cloning in Xenopus laevis oocytes. A cDNA library was constructed from size-fractionated poly(A)+ RNA and screened for p-aminohippurate (PAH) transport in high potassium medium. A 1856-base pair cDNA encoding a 467-amino acid peptide designated as OATV1 (voltage-driven organic anion transporter 1) was isolated. The predicted amino acid sequence of OATV1 exhibited 60–65% identity to those of human, rat, rabbit, and mouse sodium-dependent phosphate cotransporter type 1 (NPT1), although OATV1 did not transport phosphate. The homology of this transporter to known members of the organic anion transporter family (OAT family) was about 25–30%. OATV1-mediated PAH transport was affected by the changes in membrane potential. The transport was Na+-independent and enhanced at high concentrations of extracellular potassium and low concentrations of extracellular chloride. Under the voltage clamp condition, extracellularly applied PAH induced outward currents in oocytes expressing OATV1. The current showed steep voltage dependence, consistent with the voltage-driven transport of PAH by OATV1. The PAH transport was inhibited by various organic anions but not by organic cations, indicating the multispecific nature of OATV1 for anionic compounds. This transport protein is localized at the apical membrane of renal proximal tubule, consistent with the proposed localization of a voltage-driven organic anion transporter. Therefore, it is proposed that OATV1 plays an important role to excrete drugs, xenobiotics, and their metabolites driven by membrane voltage through the apical membrane of the tubular epithelial cells into the urine. In kidney, the secretion of organic anions takes place in the renal proximal tubular epithelial cells via at least two steps. The first is the transport of organic anions from the peritubular plasma across the basolateral membrane into the proximal tubular cells. p-Aminohippurate (PAH) 1The abbreviations used are: PAH, p-aminohippurate; OAT, organic anion transporter; OATV1, voltage-driven organic anion transporter 1; MES, 4-morpholineethanesulfonic acid. has been used as a prototypical substrate for the renal organic anion transport systems (1Moller J.V. Sheikh M.I. Pharmacol. Rev. 1983; 34: 315-358Google Scholar, 2Pritchard J.B. Miller D.S. Physiol. Rev. 1993; 73: 765-796Crossref PubMed Scopus (463) Google Scholar). The transport of PAH across the basolateral membrane of proximal tubular cells against the electrochemical gradient (3Burg M.B. Orloff J. Am. J. Physiol. 1969; 217: F1064-F1068Crossref PubMed Scopus (23) Google Scholar) occurs in exchange for intracellular dicarboxylates such as α-ketoglutarate (4Pritchard J.B. Am. J. Physiol. 1988; 255: F597-F604PubMed Google Scholar, 5Shimada H. Moewes B. Burckhardt G. Am. J. Physiol. 1987; 253: F795-F801PubMed Google Scholar). Two organic anion transporters, OAT1 (6Sekine T. Watanabe N. Hosoyamada M. Kanai Y. Endou H. J. Biol. Chem. 1997; 272: 18526-18529Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 7Sweet D.H. Wolff N.A. Pritchard J.B. J. Biol. Chem. 1997; 272: 30088-30095Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 8Sekine T Cha S.H. Endou H. Pflugers Arch. 2000; 440: 337-350Crossref PubMed Scopus (297) Google Scholar) and OAT3 (9Sweet D.H. Chan L.M. Walden R. Yang X.P. Miller D.S. Pritchard J.B. Am. J. Physiol. 2003; 284: F763-F769Crossref PubMed Scopus (197) Google Scholar), have been proposed to be responsible for this step. The second step is the exit of organic anions across the apical membrane at the tubular epithelial cells into the urine. Although this process is energetically downhill for organic anions, it has been believed that this process is also mediated by specific transporters. The luminal efflux system for PAH has been investigated mostly using brush border membrane vesicles. In dogs (10Aronson P.S. Annu. Rev. Physiol. 1989; 51: 419-441Crossref PubMed Scopus (90) Google Scholar, 11Blomstedt J.W. Aronson P.S. J. Clin. Invest. 1980; 65: 931-934Crossref PubMed Scopus (82) Google Scholar, 12Guggino S.E. Martin J.G. Aronson P.S. Am. J. Physiol. 1983; 244: F612-F621PubMed Google Scholar, 13Kahn A.M. Aronson P.S. Am. J. Physiol. 1983; 244: F56-F63PubMed Google Scholar) and rats (14Kahn A.M. Branham S. Weinman E.J. Am. J. Physiol. 1983; 245: F151-F158Crossref PubMed Google Scholar), anion exchange mechanisms have been demonstrated for the luminal efflux systems. They mediate probenecid-sensitive electroneutral exchange of anionic compounds including both organic (e.g. PAH, urate, and lactate) and inorganic (e.g. Cl–, HCO3- , OH–) anions. A distinct efflux system involving the voltage-driven transport was demonstrated in pig and rabbit (15Martinez F. Manganel M. Montrose-Rafizadeh C. Werner D. Roch-Ramel F. Am. J. Physiol. 1990; 258: F1145-F1153PubMed Google Scholar, 16Werner D. Martinez F. Roch-Ramel F. J. Pharmacol. Exp. Ther. 1990; 252: 792-799PubMed Google Scholar, 17Krick W. Wolff N.A. Burckhardt G. Pflugers Arch. 2000; 441: 125-132Crossref PubMed Scopus (26) Google Scholar). In bovine renal brush border membrane vesicles, a PAH/dicarboxylate exchange system was reported (18Schmitt C. Burckhardt G. Pflugers Arch. 1993; 423: 280-290Crossref PubMed Scopus (39) Google Scholar). PAH transport at the apical membrane in the OK kidney epithelial cell line was shown to be mediated by a voltage-driven transport system but not by an anion exchange system (19Habu Y. Yano I. Hashimoto Y. Saito H. Inui K. Pharmacol. Res. 2002; 19: 1822-1826Crossref Scopus (4) Google Scholar). In the physiological condition, a major component of the exit path of organic anions from renal proximal tubular cells has been proposed to be the facilitated diffusion along the electrochemical potential gradient (2Pritchard J.B. Miller D.S. Physiol. Rev. 1993; 73: 765-796Crossref PubMed Scopus (463) Google Scholar, 23Dantzler W.H. Biochim. Biophys. Acta. 2002; 1566: 169-181Crossref PubMed Scopus (55) Google Scholar). Voltage-driven organic anion transport plays an important role for this step. However, the molecular nature and precise functional properties of these efflux systems are still unknown. MRP2, an ABC (ATP-binding cassette) transporter, was suggested to be one of the exit paths of PAH in proximal tubular cells. Studies of membrane vesicles from MRP2-expressing HEK or Sf9 cells have indicated that MRP2 transports PAH (20Leier I. Hummel-Eisenbeiss J. Cui Y. Keppler D. Kidney Int. 2000; 57: 1636-1642Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 21Van Aubel R.A. Peters J.G. Masereeuw R. Van Os C.H. Russel F.G. Am. J. Physiol. 2000; 279: F713-F717PubMed Google Scholar) in an ATP-dependent manner. Recently, it was shown that the human sodium-dependent phosphate cotransporter type 1 (NPT1) present at the apical membrane of proximal tubules transports organic anions including PAH. The PAH transport by NPT1 expressed in HEK293 was Cl– sensitive (22Uchino H. Tamai I. Yamashita K. Minemoto Y. Sai Y. Yabuuchi H. Miyamoto K. Takeda E. Tsuji A. Biochem. Biophys. Res. Commun. 2000; 270: 254-259Crossref PubMed Scopus (133) Google Scholar). Although NPT1 is a candidate of the exit path for PAH at the apical membrane of proximal tubules, the reported properties of NPT1, in particular its K+-dependence, is not consistent with those of the voltage-driven PAH transporter at the apical membrane. In the present study, to identify the transporter responsible for the exit of PAH through the apical membrane, we have performed expression cloning using pig kidney cortex poly(A)+ RNA and identified a novel transporter present at the apical membrane of proximal tubules that mediates voltage-driven facilitated diffusion. Expression of Pig Kidney Cortex Poly(A) + RNA—Xenopus laevis oocyte expression studies and uptake measurements were performed as described elsewhere (24Kanai Y. Nussberger S. Romero M.F. Boron W.F. Hebert S.C. Hediger M.A. J. Biol. Chem. 1995; 270: 16561-16568Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 25Utsunomiya-Tate N. Endou H. Kanai Y. J. Biol. Chem. 1996; 271: 14883-14890Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Defolliculated oocytes were injected with poly(A)+ RNA (50 ng) obtained from kidney cortex of adult female pig (body weight 102 kg). Three days after injection and incubation of oocytes at 18 °C, the uptake experiments were performed in high potassium uptake solution (98 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm HEPES, pH 7.4) containing 20 μm [14C]PAH (1 μCi/ml). Preparation of Size-fractionated Pig Kidney Poly(A) + RNA and Expression Cloning—Four hundred micrograms of pig kidney poly(A)+ RNA was size-fractionated using preparative gel electrophoresis (Bio-Rad, model 491 Prep cell) (6Sekine T. Watanabe N. Hosoyamada M. Kanai Y. Endou H. J. Biol. Chem. 1997; 272: 18526-18529Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 26Romero M.F. Kanai Y. Gunshin H. Hediger M.A. Methods Enzymol. 1998; 296: 17-52Crossref PubMed Scopus (74) Google Scholar, 27Kanai Y. Segawa H. Miyamoto K. Uchino H. Takeda E. Endou H. J. Biol. Chem. 1998; 273: 23629-23632Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar, 28Kim D.K. Kanai Y. Chairoungdua A. Matsuo H. Cha S.H. Endou H. J. Biol. Chem. 2001; 276: 17221-17228Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The isolated poly(A)+ RNA fractions were examined for [14C]PAH (20 μm) transport activity by expression in X. laevis oocytes in the high potassium uptake solution. The positive fraction showing the greatest activity of [14C]PAH uptake was used to construct a directional cDNA library (27Kanai Y. Segawa H. Miyamoto K. Uchino H. Takeda E. Endou H. J. Biol. Chem. 1998; 273: 23629-23632Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar, 28Kim D.K. Kanai Y. Chairoungdua A. Matsuo H. Cha S.H. Endou H. J. Biol. Chem. 2001; 276: 17221-17228Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 29Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1198) Google Scholar). Screening of the cDNA library was performed as described previously (6Sekine T. Watanabe N. Hosoyamada M. Kanai Y. Endou H. J. Biol. Chem. 1997; 272: 18526-18529Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 27Kanai Y. Segawa H. Miyamoto K. Uchino H. Takeda E. Endou H. J. Biol. Chem. 1998; 273: 23629-23632Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar, 28Kim D.K. Kanai Y. Chairoungdua A. Matsuo H. Cha S.H. Endou H. J. Biol. Chem. 2001; 276: 17221-17228Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 29Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1198) Google Scholar). cRNA synthesized in vitro from pools of ∼500 clones was injected into Xenopus oocytes. A positive pool showing the [14C]PAH uptake activity in the high potassium uptake solution was sequentially subdivided and analyzed until a single clone (OATV1) was identified. The cDNA was sequenced in both directions by the dye terminator cycle sequencing method using an ABI PRISM 3100 Genetic Analyzer (PE Biosystems). Functional Characterization—Twenty-five nanograms of OATV1 cRNA synthesized in vitro using T7 RNA polymerase from the OATV1 cDNA in pSPORT1 linearized with NotI were injected into defolliculated Xenopus oocytes. Two or 3 days after injection the uptake of radiolabeled substrates was measured in various uptake solutions (differing in ion composition) to examine its dependence on sodium or chloride. In sodium-free solution, LiCl or choline-Cl were used to replace NaCl in the standard uptake solution (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm HEPES, pH 7.4). For the high potassium uptake solution, NaCl in the standard uptake solution was replaced by KCl. For Cl–-free medium, sodium gluconate, potassium gluconate, calcium gluconate, and MgSO4 were used to replace NaCl, KCl, CaCl2, and MgCl2, respectively. To prepare the uptake solution at pH 5.5 and 6.5, MES was used instead of HEPES for the buffer system (30Segawa H. Fukasawa Y. Miyamoto K. Takada E. Endou H. Kanai Y. J. Biol. Chem. 1999; 274: 19745-19751Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). The uptake measurements were performed for 60 min at room temperature (22–25 °C). The radioactivity was counted by liquid scintillation spectrometry, and the values are expressed as femtomole per oocyte per min. To examine the effects of the extracellular concentration of K+ and Cl– on the OATV1-mediated [14C]PAH uptake, the concentrations of K+ or Cl– were varied over the range of 0 to 100 mm (0, 5, 10, 25, 50, and 100 mm). The concentrations of K+ in the uptake solution were varied in Na+-free solution in which [KCl] plus [choline-Cl] was equal to 100 mm. The concentration of Cl– was varied in the high potassium uptake solution in which [KCl] plus [K-gluconate] was equal to 100 mm. Kinetic Study—Because the uptake of substrates in high potassium uptake solution was linear longer than 2 h, the uptake was measured for 30 min for kinetic studies. The concentrations of PAH, urate, and estrone sulfate were varied from 10 μm to 10 mm, from 10 μm to 5 mm, and from 1 to 500 μm, respectively. OATV1-mediated substrate uptake was calculated as the difference between the values of uptake into cRNA-injected oocytes and those of control oocytes (without cRNA injection). The K m values were determined with the Eadie-Hofstee equation. To measure the K i values for the transport, oocytes expressing OATV1 were incubated for 30 min in high potassium uptake solution with various concentrations of [14C]PAH with or without addition of inhibitors. The K i values were determined by double-reciprocal plot analysis in which 1/uptake rate of [14C]PAH was plotted against 1/[14C]PAH concentration. The K i values were calculated from the following equation when competitive inhibition was observed: K i = (K t[I])/(K a – K t), where K a, K t, and [I] are K m of PAH with inhibitor, K m of PAH without inhibitor, and concentration of inhibitor, respectively (31Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley-Interscience, New York1975Google Scholar). Efflux Measurement—Fifty nanoliters of [14C]PAH (2.5 nCi, ∼1 mm), [14C]urate (2.5 nCi, ∼1 mm), or [3H]estrone sulfate (25 nCi, ∼20 mm) was injected into oocytes with a fine-tipped glass micropipette as described elsewhere (32Fukasawa Y. Segawa H. Kim J.Y. Chairoungdua A. Kim D.K. Matsuo H. Cha S.H. Endou H. Kanai Y. J. Biol. Chem. 2000; 275: 9690-9969Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 33Kanai Y. Fukasawa Y. Cha S.H. Segawa H. Chairoungdua A. Kim D.K. Matsuo H. Kim J.Y. Miyamoto K. Takeda E. Endou H. J. Biol. Chem. 2000; 275: 20787-20793Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The individual oocytes were incubated in ice-cold standard uptake solution (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm HEPES, pH 7.4) for 5 min and then transferred to the standard uptake solution kept at room temperature and incubated at room temperature for 2, 5, 15, 30, 60, 90, 120, or 150 min. Then, the incubation solution was collected to determine the efflux of substrates from the oocytes into bath solution at the end of the incubation period. The radioactivity remaining in the oocytes was also measured. The efflux value was expressed as % radioactivity calculated from the radioactivity in the bathing solution × 100%/(the radioactivity in oocyte plus the radioactivity in bathing solution). The dependence of [14C]PAH efflux on inorganic ions was determined by comparing the [14C]PAH uptake in the standard uptake solution and that in the solutions in which NaCl was replaced by KCl, sodium gluconate, potassium gluconate, LiCl, or choline-Cl as mentioned above. The pH dependence of [14C]PAH efflux was measured in the standard uptake solution with varied pH to 5.5, 6.5, 7.4, and 8.5. The efflux was measured for 15 min over which the linear efflux value was obtained. To examine the trans-stimulation of the efflux of PAH, [14C]PAH-injected oocytes were incubated in the standard uptake solution with or without non-radiolabeled PAH (0.01, 0.1, 1, 5, or 10 mm) for 15 min at room temperature. For the uptake and efflux measurements in the present study, 8–10 oocytes were used for each data point. The values are expressed as mean ± S.E. The reproducibility of the results was confirmed by three separate experiments with different batches of oocytes. The results from representative experiments are shown in figures. Electrophysiological Measurements—The electrical currents induced by 5 mm PAH in both control and OATV1-cRNA-injected X. laevis oocytes were recorded during voltage clamp at –60 to +40 mV in the standard uptake solution (24Kanai Y. Nussberger S. Romero M.F. Boron W.F. Hebert S.C. Hediger M.A. J. Biol. Chem. 1995; 270: 16561-16568Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 34Kanai Y. Stelzner M. Nussberger S. Khawaja S. Hebert S.C. Smith C.P. Hediger M.A. J. Biol. Chem. 1994; 269: 20599-20606Abstract Full Text PDF PubMed Google Scholar, 35Sekine T. Cha S.H. Hosoyamada M. Kanai Y. Watanabe N. Furuta Y. Fukuda K. Igarashi T. Endou H. Am. J. Physiol. 1998; 275: F298-F305PubMed Google Scholar). At each step (20 mV interval) of voltage clamp, the baseline electrical current was first determined and then 5 mm PAH was added to the bath medium for 2 min. The solution was then changed back to the standard uptake solution and the record was continued until the electrical current was returned to the baseline level. Northern Analysis—Poly(A)+ RNA (4 μg/lane) isolated from pig tissues was electrophoresed on 1% agarose, 2.2 m formaldehyde gel and transferred to nitrocellulose filter (Schleicher & Schuell). The filter was hybridized with 500-bp OATV1 cDNA labeled with [32P]dCTP at 42 °C overnight and washed finally in 0.1× SSC, 0.1% SDS at 65 °C (25Utsunomiya-Tate N. Endou H. Kanai Y. J. Biol. Chem. 1996; 271: 14883-14890Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Anti-peptide Antibody—A rabbit polyclonal antibody against a keyhole limpet hemocyanin-conjugated synthetic peptide, EVQDWAKER-QNTYL, corresponding to 14 amino acid residues near the carboxyl terminus (454–467 of the amino acid sequence) of OATV1 was generated and affinity purified as described elsewhere (36Hisano S. Haga H. Miyamoto K. Takeda E. Fukui Y. Brain Res. 1996; 710: 299-302Crossref PubMed Scopus (33) Google Scholar, 37Chairoungdua A. Segawa H. Kim J.Y. Miyamoto K. Haga H. Fukui Y. Mizoguchi K. Ito H. Endou H. Kanai Y. J. Biol. Chem. 1999; 274: 28845-28848Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Western Blot Analysis—Pig kidney epithelial membranes were prepared as described previously (38Thorens B. Sarkar H.K. Kaback H.R. Lodish H.F. Cell. 1988; 55: 281-290Abstract Full Text PDF PubMed Scopus (661) Google Scholar) and subjected to SDS-polyacrylamide gel electrophoresis (39Taketani Y. Segawa H. Chikamori M. Morita K. Tanaka K. Kido S. Yamamoto H. Iemori Y. Tatsumi S. Tsugawa N. Okano T. Kobayashi T. Miyamoto K. Takeda E. J. Biol. Chem. 1998; 273: 14575-14581Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The separated proteins were transferred electrophoretically to a Hybond-P polyvinylidene difluoride transfer membrane (Amersham Biosciences). The membranes were treated with diluted affinity purified anti-OATV1 antibody (1:5,000) overnight at 4 °C. Thereafter, horseradish peroxidase-conjugated anti-rabbit IgG was used as the secondary antibody (Jackson ImmunoResearch Laboratories, Inc). The signals were detected using the ECL Plus system (Amersham Biosciences). The specificity of immunoreaction was confirmed by an absorption experiment in the presence of antigen peptide (100 μg/ml). Immunohistochemistry—Three-micrometer paraffin sections of pig kidney were processed for light microscopic immunohistochemical analysis as described previously (36Hisano S. Haga H. Miyamoto K. Takeda E. Fukui Y. Brain Res. 1996; 710: 299-302Crossref PubMed Scopus (33) Google Scholar). The kidney sections were incubated with affinity purified anti-OATV1 antibody (1:1,000) at 4 °C overnight and treated with Envision (+) rabbit peroxidase (DAKO) for 30 min. The immunoreactions were detected with diaminobenzidine (0.8 mm) (40Tojo A. Sekine T. Nakajima N. Hosoyamada M. Kanai Y. Kimura K. Endou H. J. Am. Soc. Nephrol. 1999; 10: 464-471PubMed Google Scholar). For absorption experiments, the serial kidney sections were treated with the primary antibody in the presence of antigen peptide (100 μg/ml). Screening of cDNA Library—Total poly(A)+ RNA was isolated from pig kidney cortex and size-fractionated. When expressed in Xenopus oocytes, the fractions corresponding to ∼1.7 to 2.2 kilobases exhibited the peak activity of the uptake of 20 μm [14C]PAH in high potassium uptake solution: 1.5–2.0-fold of the uptake by control water-injected oocytes. These poly(A)+ RNA fractions were used as a template for construction of a cDNA library. The library was screened for [14C]PAH uptake by expression in Xenopus oocytes to isolate a 1,856-base pair cDNA clone that encodes a protein designated as OATV1 (voltage-driven organic anion transporter 1). The OATV1 cDNA contains an open reading frame encoding a putative 467-amino acid protein. Twelve transmembrane domains were predicted on the OATV1 amino acid sequence based on SOSUI algorithm (41Hirokawa T. Boon-Chieng S. Mitaku S. Bioinformatics. 1998; 14: 378-379Crossref PubMed Scopus (1574) Google Scholar). The amino acid sequence of OATV1 exhibited the highest identity (60–65%) to that of sodium-dependent phosphate cotransporter type 1 (NPT1) of each species (rat, mouse, rabbit, and human). The homology to amino acid sequences of sodium-dependent phosphate cotransporter type 2 (NPT2) and other OATs is ∼25–30%. Tissue Distribution—The Northern blot using the OATV1 cDNA fragment as a probe showed a strong signal in pig kidney and liver (Fig. 1a). The OATV1 transcript was not detected in brain, lung, small intestine, and skeletal muscle. The major transcript detected in liver and kidney was ∼3.2 kb. In addition, a faint band at ∼1.8 kb was also detected in both kidney and liver. The 4.4-kb faint band was detected only in liver. In Western blot analysis, the antibody raised against the COOH terminus peptide of OATV1 recognized a band of 60 kDa in the membrane protein prepared from total pig kidney and the brush border membrane of pig kidney cortex (Fig. 1b). This band was consistent with a predicted molecular mass of OATV1 protein (51 kDa). The band disappeared in the presence of antigen peptide in the absorption test, confirming the specificity of the immunoreaction (Fig. 1b). Transport Activity of OAT V 1—Because the luminal facilitated transport system in pig and rabbit was reported to be markedly dependent on membrane potential (15Martinez F. Manganel M. Montrose-Rafizadeh C. Werner D. Roch-Ramel F. Am. J. Physiol. 1990; 258: F1145-F1153PubMed Google Scholar, 16Werner D. Martinez F. Roch-Ramel F. J. Pharmacol. Exp. Ther. 1990; 252: 792-799PubMed Google Scholar), we examined the effect of raising external K+ on the OATV1-mediated transport. The elevation of external K+ depolarizes the plasma membrane of Xenopus oocyte (42Grundemann D. Gorboulev V. Gambaryan S. Veyhl M. Koepsell H. Nature. 1994; 372: 549-552Crossref PubMed Scopus (608) Google Scholar). As shown in Fig. 2, the uptake values of [14C]PAH, [14C]urate, [3H]estrone sulfate, and [3H]estradiol-17β-glucuronide by OATV1 expressing oocytes were significantly higher in high potassium solution (98 mmK+) compared with those incubated in standard uptake solution (2 mm K+), whereas control oocytes showed no difference between these conditions, suggesting that OATV1-mediated transport is dependent on membrane voltage. The uptake of [14C]PAH was saturable and followed Michaelis-Menten kinetics (Fig. 3a) with a K m value of 4.38 ± 0.96 mm (mean ± S.E. of three separate experiments) (Fig. 3b). K m values for [14C]urate and [3H]estrone sulfate were >5 and 0.212 mm, respectively (data not shown).Fig. 3Concentration dependence of OATV1-mediated [14C]PAH uptake. a, OATV1-mediated [14C]PAH uptake by oocytes expressing OATV1 was measured in a high potassium uptake solution (98 mm K+) and plotted against PAH concentration. The PAH uptake was saturable and fit to the Michaelis-Menten curve. b, Eadie-Hofstee plot analysis was performed on the PAH uptake. K m and V max values were 4.40 mm and 18.38 pmol/oocyte/min, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Characteristic of OAT V 1-mediated Organic Anion Transport—The PAH uptake by OATV1-expressing oocytes was also measured in various uptake solutions with different ionic contents. Replacement of Na+ with Li+ or choline+ did not affect [14C]PAH uptake (Fig. 4a), indicating that OATV1-mediated [14C]PAH transport was not dependent on Na+ in the uptake solution. The replacement of Na+ with K+ or Rb+ increased the OATV1-mediated transport (Figs. 2 and 4a). Fig. 4b further shows the dependence of OATV1-mediated [14C]PAH uptake on K+ concentration, indicating that raising extracellular K+ increased PAH transport in a concentration-dependent manner. Fig. 4a also shows the effect of the replacement of Cl– with other anions. PAH uptake was higher in OATV1-expressing oocytes incubated in potassium gluconate solution than that in KCl uptake solution. PAH uptake was also higher in sodium gluconate, NaBr, or NaF solution than that in NaCl solution. As shown in Fig. 4c, PAH transport was dependent on Cl– concentration. In the chloride-free solution and the solution containing 5 mm chloride, OATV1 expressing oocytes showed a significant elevation of PAH uptake (Fig. 4c). The PAH uptake did not show any remarkable pH dependence within the pH range of 5.5 and 8.5 (Fig. 4d). We also found that urate transport by OATV1 showed the same properties as those of PAH transport (data not shown). Voltage-dependent PAH Transport— Fig. 4 showed that PAH uptake by OATV1-expressing oocytes was affected by the changes of K+ or Cl– concentration in the uptake solution. It seemed that PAH uptake is dependent on membrane voltage change generated by the alteration of K+ and Cl– concentrations. To confirm the voltage dependence of PAH transport by OATV1, we performed electrophysiological measurement of PAH-induced currents under the voltage clamp condition. PAH application to the bath medium induced outward current in the oocytes expressing OATV1, whereas no significant current was elicited by 5 mm PAH in the control oocytes. The PAH-induced current was dependent on membrane voltage (Fig. 5). The electric currents induced by 5 mm PAH were increased when the holding potential was elevated from –60 to +40 mV as shown in Fig. 5. Characteristic of OAT V 1-mediated PAH Efflux—According to the voltage-driven transport of PAH, in the physiological condition PAH is supposed to be transported from the intracellular compartment to the extracellular compartment along the electrical gradient. As shown in Fig. 6a, OATV1-expressing oocytes preloaded with [14C]PAH showed time-dependent efflux of radioactivity when incubated in the standard uptake solution. The voltage dependence of PAH transport was also observed for the efflux experiments. The OATV1-expressing oocytes showed a reduction of PAH efflux from the oocytes to the extracellular medium when the oocytes were incubated in high potassium uptake solution, which generates inside positive potential (Fig. 6b). The effect of pH on [14C]PAH efflux was examined in the standard upt
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