Polymorphisms in OATP-C
2001; Elsevier BV; Volume: 276; Issue: 38 Linguagem: Inglês
10.1074/jbc.m103792200
ISSN1083-351X
AutoresRommel G. Tirona, Brenda F. Leake, Gracia Merino, Richard B. Kim,
Tópico(s)Pharmacogenetics and Drug Metabolism
ResumoThe human organic anion transporting polypeptide-C (OATP-C) (gene SLC21A6) is a liver-specific transporter importantly involved in the hepatocellular uptake of a variety of endogenous and foreign chemicals. In this study, we demonstrate the presence of multiple functionally relevant single-nucleotide polymorphisms (SNPs) in OATP-C in a population of African- and European-Americans. Moreover, examination of 14 nonsynonymous polymorphisms indicated that genotypic frequencies were dependent on race. Functional assessment of 16OATP-C alleles in vitro revealed that several variants exhibited markedly reduced uptake of the OATP-C substrates estrone sulfate and estradiol 17β-d-glucuronide. Specifically, alterations in transport were associated with SNPs that introduce amino acid changes within the transmembrane-spanning domains (T217C (Phe-73 → Leu), T245C (Val-82 → Ala), T521C (Val-174 → Ala), and T1058C (Ile-353 → Thr)) and also with those that modify extracellular loop 5 (A1294G (Asn-432 → Asp), A1385G (Asp-462 → Gly), and A1463C (Gly-488 → Ala)). Cell surface biotinylation experiments indicated that the altered transport activity of some OATP-C variants was due, in part, to decreased plasma membrane expression. Given the relatively high genotypic frequency of the T521C (14%) transition in European-Americans and the G1463C (9%) transversion in African-Americans, SNPs in OATP-C may represent a heretofore unrecognized factor influencing drug disposition. The human organic anion transporting polypeptide-C (OATP-C) (gene SLC21A6) is a liver-specific transporter importantly involved in the hepatocellular uptake of a variety of endogenous and foreign chemicals. In this study, we demonstrate the presence of multiple functionally relevant single-nucleotide polymorphisms (SNPs) in OATP-C in a population of African- and European-Americans. Moreover, examination of 14 nonsynonymous polymorphisms indicated that genotypic frequencies were dependent on race. Functional assessment of 16OATP-C alleles in vitro revealed that several variants exhibited markedly reduced uptake of the OATP-C substrates estrone sulfate and estradiol 17β-d-glucuronide. Specifically, alterations in transport were associated with SNPs that introduce amino acid changes within the transmembrane-spanning domains (T217C (Phe-73 → Leu), T245C (Val-82 → Ala), T521C (Val-174 → Ala), and T1058C (Ile-353 → Thr)) and also with those that modify extracellular loop 5 (A1294G (Asn-432 → Asp), A1385G (Asp-462 → Gly), and A1463C (Gly-488 → Ala)). Cell surface biotinylation experiments indicated that the altered transport activity of some OATP-C variants was due, in part, to decreased plasma membrane expression. Given the relatively high genotypic frequency of the T521C (14%) transition in European-Americans and the G1463C (9%) transversion in African-Americans, SNPs in OATP-C may represent a heretofore unrecognized factor influencing drug disposition. single-nucleotide polymorphism organic anion transporting polypeptide single-strand conformational polymorphism polymerase chain reaction restriction fragment length polymorphism cytochrome P450 Variations in the genes encoding drug metabolizing enzymes have long been recognized as important determinants of drug disposition and response. Indeed, certain single-nucleotide polymorphisms (SNPs)1 in cytochrome P450 (CYP) enzymes, such as CYP2C9, CYP2C19, and CYP2D6, are well known determinants of altered drug responsiveness and are often associated with drug toxicity or, in some cases, loss of therapeutic efficacy (1Evans W.E. Relling M.V. Science. 1999; 286: 487-491Crossref PubMed Scopus (2163) Google Scholar,2Meyer U.A. Zanger U.M. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 269-296Crossref PubMed Scopus (496) Google Scholar). More recently, there has been an increasing appreciation of the role of drug transporters in drug disposition. For example, the product of the MDR1 gene, P-glycoprotein, is an efflux transporter involved in the oral drug absorption, distribution to certain tissues, and biliary and urinary excretion of many drugs. Moreover, P-glycoprotein has been shown to have broad and overlapping substrate specificities to CYP enzymes (3Kim R.B. Wandel C. Leake B. Cvetkovic M. Fromm M.F. Dempsey P.J. Roden M.M. Belas F. Chaudhary A.K. Roden D. Wood A.J.J. Wilkinson G.R. Pharm. Res. (N. Y.). 1999; 16: 408-414Crossref PubMed Google Scholar). Furthermore, polymorphisms in theMDR1 gene have been associated with alterations in digoxin (4Hoffmeyer S. Burk O. von Richter O. Arnold H.P. Brockmoller J. Johne A. Cascorbi I. Gerloff T. Roots I. Eichelbaum M. Brinkmann U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3473-3478Crossref PubMed Scopus (2384) Google Scholar) and fexofenadine (5Kim R.B. Leake B.F. Choo E.F. Dresser G.K. Kubba S.V. Schwarz U.I. Taylor A. Xie H.-G. McKinsey J. Zhou S. Lu-Bin L. Schuetz J.D. Schuetz E.G. Wilkinson G.R. Clin. Pharmacol. Ther. 2001; 70: 189-199Crossref PubMed Scopus (961) Google Scholar) disposition. Removal of drugs/solutes from the portal circulation involves uptake into hepatocytes as the obligatory first step. Whereas passive diffusion can be involved in the uptake of many uncharged or lipophilic compounds, carrier-mediated processes are often critical for the efficient hepatic uptake of solutes and many drugs. Among the transporters expressed at the basolateral membrane of hepatocytes, members of the organic anion transporting polypeptide (OATP) family (6Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (552) Google Scholar) have been shown capable of mediating the hepatic uptake of a variety of structurally divergent compounds (7Meier P.J. Eckhardt U. Schroeder A. Hagenbuch B. Stieger B. Hepatology. 1997; 26: 1667-1677Crossref PubMed Scopus (326) Google Scholar). Within this family, OATP-B (8Tamai I. Nezu J. Uchino H. Sai Y. Oku A. Shimane M. Tsuji A. Biochem. Biophys. Res. Commun. 2000; 273: 251-260Crossref PubMed Scopus (557) Google Scholar,9Kullak-Ublick G.A. Ismair M.G. Stieger B. Landmann L. Huber R. Pizzagalli F. Fattinger K. Meier P.J. Hagenbuch B. Gastroenterology. 2001; 120: 525-533Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar), OATP-C (8Tamai I. Nezu J. Uchino H. Sai Y. Oku A. Shimane M. Tsuji A. Biochem. Biophys. Res. Commun. 2000; 273: 251-260Crossref PubMed Scopus (557) Google Scholar, 10Abe T. Kakyo M. Tokui T. Nakagomi R. Nishio T. Nakai D. Nomura H. Unno M. Suzuki M. Naitoh T. Matsuno S. Yawo H. J. Biol. Chem. 1999; 274: 17159-17163Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 11Hsiang B. Zhu Y. Wang Z. Wu Y. Sasseville V. Yang W.-P. Kirchgessner T.G. J. Biol. Chem. 1999; 274: 37161-37168Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, 12König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar), and OATP8 (13König J. Cui Y. Nies A.T. Keppler D. J. Biol. Chem. 2000; 275: 23161-23168Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar) have now been established as the major OATPs located at the basolateral membrane of human hepatocytes. OATP-C (gene SLC21A6), also known as liver specific transporter-1 (LST-1) (10Abe T. Kakyo M. Tokui T. Nakagomi R. Nishio T. Nakai D. Nomura H. Unno M. Suzuki M. Naitoh T. Matsuno S. Yawo H. J. Biol. Chem. 1999; 274: 17159-17163Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar) or OATP2 (11Hsiang B. Zhu Y. Wang Z. Wu Y. Sasseville V. Yang W.-P. Kirchgessner T.G. J. Biol. Chem. 1999; 274: 37161-37168Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, 12König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar), is involved in the hepatic uptake of a broad array of endogenous compounds, such as taurocholate (11Hsiang B. Zhu Y. Wang Z. Wu Y. Sasseville V. Yang W.-P. Kirchgessner T.G. J. Biol. Chem. 1999; 274: 37161-37168Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar), estrone sulfate (8Tamai I. Nezu J. Uchino H. Sai Y. Oku A. Shimane M. Tsuji A. Biochem. Biophys. Res. Commun. 2000; 273: 251-260Crossref PubMed Scopus (557) Google Scholar), estradiol 17β-d-glucuronide (10Abe T. Kakyo M. Tokui T. Nakagomi R. Nishio T. Nakai D. Nomura H. Unno M. Suzuki M. Naitoh T. Matsuno S. Yawo H. J. Biol. Chem. 1999; 274: 17159-17163Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar), leukotriene C4 (10Abe T. Kakyo M. Tokui T. Nakagomi R. Nishio T. Nakai D. Nomura H. Unno M. Suzuki M. Naitoh T. Matsuno S. Yawo H. J. Biol. Chem. 1999; 274: 17159-17163Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar), prostaglandin E2, and thyroid hormone (10Abe T. Kakyo M. Tokui T. Nakagomi R. Nishio T. Nakai D. Nomura H. Unno M. Suzuki M. Naitoh T. Matsuno S. Yawo H. J. Biol. Chem. 1999; 274: 17159-17163Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). Recently, OATP-C has also been shown to mediate the cellular uptake of bilirubin and its glucuronide conjugates (12König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar, 14Cui Y. König J. Leier I. Buchholz U. Keppler D. J. Biol. Chem. 2001; 276: 9626-9630Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). Furthermore, a variety of drugs and xenobiotics, including the organic anion bromosulfophthalein (9Kullak-Ublick G.A. Ismair M.G. Stieger B. Landmann L. Huber R. Pizzagalli F. Fattinger K. Meier P.J. Hagenbuch B. Gastroenterology. 2001; 120: 525-533Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 14Cui Y. König J. Leier I. Buchholz U. Keppler D. J. Biol. Chem. 2001; 276: 9626-9630Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar), the glycoside oubain (9Kullak-Ublick G.A. Ismair M.G. Stieger B. Landmann L. Huber R. Pizzagalli F. Fattinger K. Meier P.J. Hagenbuch B. Gastroenterology. 2001; 120: 525-533Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar), the peptidomimetic BQ-123 (9Kullak-Ublick G.A. Ismair M.G. Stieger B. Landmann L. Huber R. Pizzagalli F. Fattinger K. Meier P.J. Hagenbuch B. Gastroenterology. 2001; 120: 525-533Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar), and the HMG-CoA-reductase inhibitor pravastatin (11Hsiang B. Zhu Y. Wang Z. Wu Y. Sasseville V. Yang W.-P. Kirchgessner T.G. J. Biol. Chem. 1999; 274: 37161-37168Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar), have been identified as OATP-C substrates. Accordingly, given the extent of its substrate specificity for endogenous and exogenous compounds, it is conceivable that genetic polymorphisms in OATP-C may have significant pharmacologic, toxicologic, and pathologic consequences. In this report, we describe the identification of SNPs inOATP-C and their prevalence in European-American and African-American populations. We show that a number of frequently occurring variant alleles are associated with profound reduction in transport activity. Radiolabeled [3H]estrone sulfate (53 Ci/mmol, >97% purity), [3H]estradiol 17β-d-glucuronide (44 Ci/mmol, >97% purity), and [3H]taurocholate (3.4 Ci/mmol, >97% purity) were purchased from PerkinElmer Life Sciences. [3H]BQ-123 (42 Ci/mmol, >95% purity) was obtained fromAmersham Pharmacia Biotech. [3H]Methotrexate (20 Ci/mmol, 99% purity) was purchased from American Radiolabeled Chemicals (St. Louis, MO). All other chemical and reagents, unless stated otherwise, were obtained from Sigma and were of the highest grade available. A 10-ml sample of blood was obtained from healthy subjects and DNA was isolated using the Qiamp system (Qiagen Inc., Valencia, CA). All subjects were unrelated, healthy, volunteer adults residing in middle-Tennessee who were judged to be healthy on the basis of medical history, physical examination, and laboratory tests indicative of normal cardiac, renal, and liver function. Race was defined by self-reporting (15Senior P.A. Bhopal R. Brit. Med. J. 1994; 309: 327-330Crossref PubMed Scopus (426) Google Scholar). The protocol was approved by the Vanderbilt University Institutional Review Board-Health Sciences, and informed consent was obtained. PCR was carried out using ∼200 ng of human genomic DNA, consisting of dNTPs (0.25 mm each), the specific primer pair (4 μm each), in PCR buffer II (PerkinElmer Life Sciences) with 2.5 mmMgCl2 and 2.5 unit of AmpliTaqTM DNA polymerase (PerkinElmer Life Sciences), in a final reaction volume of 50 μl. The complete oligonucleotide primer sequences are provided in TableI. After the PCR, nonisotopic SSCP analysis was used to identify the presence of OATP-C allelic variants (16Hongyo T. Buzard G.S. Calvert R.J. Weghost C.M. Nucleic Acids Res. 1993; 21: 3637-3642Crossref PubMed Scopus (337) Google Scholar). The gel run time was varied to yield optimal separation of the single strands; for the most part, run times of 4–6 h were sufficient. Variations in the single-strand mobility patterns were clearly visualized.Table ISummary of nonsynonymous polymorphisms in OATP-CExon1-aForward and reverse primers used in PCR of OATP-C exons.PositionGenotyping method1-bForward primers for wild-type (WT) and mutant (MT) variants used in AS-PCR. Reverse primer is found in the left column.Allelic frequency1-cEA, European-American; AA, African American;EA (n = 49)AA (n = 22)25′-TGCCTATTGACATTATATAGTCC-3′,T217CRFLP (HindIII)0.020.005′-GATAACCAGTGGTGTAAAGCAT-3′(Phe-73 → Leu)35′-CTTGGACTCTATTTGCATCCATTC-3′T245CAS-PCR0.020.005′-CAAGGTACTGATAGTGGCACAG-3′(Val-82 → Ala)WT 5′-ATTTGCTTGTGATTGT-3′MT 5′-ATTTGCTTGTGATTGC-3′45′-GCAAATAAAGGGGAATATTTCTC-3′,A388GRFLP (ClaI)0.300.741-160,p < 0.01 relative to EA using Fisher's exact test.5′-AGAGATGTAATTAAATGTATAC-3′(Asn-130 → Asp),C463ARFLP (HphI)0.160.021-150, p < 0.05;(Pro-155 → Thr),A467GRFLP (BstNI), sequencing0.020.00(Glu-156 → Gly)55′-GTTAAATTTGTAATAGAAATGC-3′T521CAS-PCR0.140.021-150, p < 0.05;5′-GTAGACAAAGGGAAAGTGATCATA-3′(Val-174 → Ala)WT 5′-CATACATGTGGATATATGT-3′MT 5′-CATACATGTGGATATATGC-3′85′-AATCTTACATGACTTACGTTCAC-3′T1058CRFLP (BsrI), sequencing0.020.005′-CCACTTGGAATACAGTATTTAG-3′(Ile-353 → Thr)95′-CAGAAAACTCATATATGATTACAAC-3′A1294GRFLP (BbsI), sequencing0.010.005′-CATATTATGCAATTGATATAGTG-3′(Asn-432 → Asp)105′-TCTGCTTTCACTTTACTTCTTCC-3′A1385GRFLP (Fnu4H1), sequencing0.010.005′-GAATAAGGAGAGGAAAGTAAAAAC-3′(Asp-462 → Gly),G1463CRFLP (Fnu4HI)0.000.091-150, p < 0.05;(Gly-488 → Ala)145′-GTTATTACACACAATTTAAACTG-3′,A1964GRFLP (EcoRV), sequencing0.020.005′-GTTTGGAAACACAGAAGCAGAAG-3′(Asp-655 → Gly),A2000GAS-PCR0.020.341-160,p < 0.01 relative to EA using Fisher's exact test.(Glu-667 → Gly)WT 5′-GAAGTGTCATGGATGA-3′,MT 5′-GAAGTGTCATGGATGG-3′1-a Forward and reverse primers used in PCR of OATP-C exons.1-b Forward primers for wild-type (WT) and mutant (MT) variants used in AS-PCR. Reverse primer is found in the left column.1-c EA, European-American; AA, African American;1-150 , p < 0.05;1-160 ,p < 0.01 relative to EA using Fisher's exact test. Open table in a new tab Human liver samples (Nashville Regional Organ Procurement Agency, Nashville, TN) from Caucasian donors without any history of underlying liver pathology or prior medications were utilized in the total RNA extraction, using Trizol® reagent (Life Technologies, Inc.). Direct cloning of OATP-C full-length cDNA was obtained by reverse transcription-PCR using the primer pair 5′-GATATCTATATTTCAACCATGGACC-3′ and 5′-TTAACAATGTGTTTCACTATCTGCCCCAG-3′. Four independent OATP-Cclones from each liver were fully sequenced. SNPs were deemed to be authentic and present if the polymorphism was observed in at least two independent clones. A number of full-length cDNA clones derived from human liver samples contained various OATP-C mutations, making them suitable for in vitro expression studies. However, site-directed mutagenesis was utilized to create additional variant sites known to be present among European- and African-American subjects. Point mutations were introduced into the wild-type (OATP-C*1a) using the QuickChangeTMsite-directed mutagenesis kit (Stratagene, La Jolla, CA). Oligonucleotides containing the Val-174 → Ala, Asn-432 → Asp, and Asp-655 → Gly mutations were used to create OATP-C*5,OATP-C*7, and OATP-C*9 expression vectors, respectively. Two mutant sites, Arg-152 → Lys and Asp-241 → Asn, had to be introduced sequentially in order create theOATP-C*1c expression vector. The presence of the mutations was verified by full sequencing. Haplotypes could be defined when subjects homozygous at a single polymorphic site or multiple sites were identified. Additionally, when subjects homozygous for a SNP at one site were found to be heterozygous for another SNP at a different site, an allelic assignment was possible. Furthermore, the presence of SNPs in the liver-derived full-length cDNAs provided unequivocal verification of haplotype. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis was used to determine the genotypic frequencies of nine nonconservative polymorphisms. The primers (based on GenBankTM accession number AC022335) and restriction endonucleases used for PCR-RFLP analysis are summarized in Table I. Allele-specific PCR was performed to obtain genotypic frequencies for three polymorphisms (see Table Ifor primers). HeLa cells grown in 12-well plates (∼0.8 × 106 cells/well) were infected with vaccinia at a multiplicity of infection of 10 pfu/cell in serum-free Opti-MEM I medium (Life Technologies, Inc.) and allowed to adsorb for 30 min at 37 °C. Cells in each well were then transfected with 1 μg of wild-type or variant OATP-C cDNA packaged into pEFHis6-Topo vector (Invitrogen), along with LipofectinTM (Life Technologies, Inc.) and incubated at 37 °C for 16 h. The parental plasmid lacking any insert was used as control. Transport was then evaluated using labeled drug substrates as outlined previously (17Kim R.B. Leake B. Cvetkovic M. Roden M.M. Nadeau J. Walubo A. Wilkinson G.R. J. Pharmacol. Exp. Ther. 1999; 291: 1204-1209PubMed Google Scholar). In order to measure the estrone sulfate and estradiol 17β-d-glucuronide transport kinetics, radiolabeled drug uptake during the linear phase (first 3 min) was assessed in the presence of varying concentrations of unlabeled compound. Passive diffusion was determined by carrying out parallel experiments using the parental plasmid DNA lacking the transporter cDNA, and this value was then subtracted from the total uptake rate seen in the presence of the transporter cDNA. Michaelis-Menten-type nonlinear curve-fitting was carried out to obtain estimates of the maximal uptake rate (V max) and the concentration at which half the maximal uptake occurs (K m) (PrismTM, GraphPad, San Diego, CA). All experiments were carried out in duplicate on at least two to three separate experimental days. HeLa cells transfected with OATP-C cDNA were scraped off plates, and the resulting suspension was centrifuged at 21,000 × g for 3 min. The cell pellet was reconstituted with HED buffer (25 mm HEPES, 1.5 mm EDTA, 1 mm dithiothreitol, pH 7.4) containing protease inhibitors (Complete, Roche Molecular Biochemicals) and lysed by sonication. Samples were diluted with Laemmli buffer, and 3.75 μg of total cell protein were separated by SDS-PAGE on 12% gels. Following transfer onto nitrocellulose membranes, blots were probed with a rabbit polyclonal antibody (1:3000 dilution) raised against the C-terminal peptide of OATP-C (ESLNKNKHFVPSAGADSETHC). This antibody was custom-made by Research Genetics (Huntsville, AL). In order to normalize sample loading, blots were stripped and reprobed with anti-calnexin antibody (StressGen, Victoria, British Columbia, Canada). Bands were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech). HeLa cells were grown on 6-well plates and transfected with theOATP-CcDNAs using a similar protocol for transport experiments. Sixteen hours posttransfection, cells were washed with ice-cold phosphate-buffered saline Ca/Mg (138 mmNaCl2, 2.7 mm KCl, 1.5 mmKH2PO4, 9.6 mmNa2HPO4, 1 mm MgCl2, 0.1 mm CaCl2, pH 7.3) and then treated with a membrane-impermeable biotinylating agent (sulfo-NHS-SS-biotin, 1.5 mg/ml, Pierce) at 4 °C for 1 h. Subsequently, the cells were washed three times with ice-cold phosphate-buffered saline Ca/Mg containing 100 mm glycine and then incubated for 20 min at 4 °C with the same buffer to remove the remaining labeling agent. After washing with phosphate-buffered saline Ca/Mg, cells were disrupted with 700 μl of lysis buffer (10 mm Tris-base, 150 mm NaCl, 1 mm EDTA, 0.1% SDS, 1% Triton X-100, pH 7.4) containing protease inhibitors (Complete, Roche Molecular Biochemicals) at 4 °C for 1 h with constant agitation. Following centrifugation, 140 μl of streptavidin agarose beads (Pierce) were added to 600 μl of cell lysate and incubated for 1 h at room temperature. Beads were washed four times with ice-cold lysis buffer, and the biotinylated proteins were released by incubation of the beads with 2× Laemmli buffer for 30 min at room temperature. Samples of the total cell lysate (12.5 μl) and the biotinylated fractions (25 μl) were subjected to Western analysis for detection of immunodetectable OATP-C and the intracellular, endoplasmic reticulum-resident protein calnexin, as described above. Determination of the statistical differences between various group parameters was determined using either Student's ttest, Mann-Whitney U test, analysis of variance (using Tukey-Kramer multiple comparison test), or Fisher's exact test, as appropriate. A p value of C463A > T521C > A2000G > G1463C), and their genotypic frequencies appeared to be dependent on race (Table I). G388C and G1463C polymorphisms were more common in African-Americans than European-Americans; however, the converse was true for C463A and T521C mutations (p < 0.05). The most common polymorphisms in African-Americans were A388G (74%), A2000G (34%), and G1463C (9%), whereas the A388G (30%), C463A (16%), and T521C (14%) were predominant among European-Americans. Other nonsynonymous polymorphisms were observed at low genotypic frequencies (<2%) in this study population, and their racial dependence could not be established. With regards to the synonymous polymorphisms, G411A occurred at a higher frequency in African- than European-Americans (41% versus 18%,p < 0.05), whereas C571T was more common in European-Americans (54% versus 5%, p < 0.05). An array of expression plasmids comprising 16 OATP-C allelic variants (Fig. 1 B) was constructed. When expressed using the recombinant vaccinia system (VTF-7), OATP-C*1a was capable of transporting known substrates, including BQ-123, estrone sulfate, taurocholate, and estradiol 17β-d-glucuronide (Fig.3 A). Moreover, we found that methotrexate is a novel substrate for OATP-C (Fig. 3 A). To determine the relative transport efficiencies of the OATP-C variants, we focused on estrone sulfate and estradiol 17β-d-glucuronide, as they appeared to be the most efficiently transported substrates among the compounds tested. Under linear kinetic conditions, estrone sulfate transport activities of the originally reported OATP-Cs (*1a, *1b, and *1c), were not different from each other (Fig. 3 B). For estradiol 17β-d-glucuronide, transport activities with OATP-C*1a and *1b were comparable and slightly greater than *1c (Fig.3 C). With a number of other OATP-C variants (*2, *3, *5, *6, *9, *10, *12, and *13), estrone sulfate transport activities were significantly lower than the reference allele (*1a) (Fig.3 B). Similar results were seen for estradiol 17β-d-glucuronide transport, except that OATP-C*7 now had slightly lower transport activity, and *10 was indistinguishable from the reference allele (Fig. 3 C). For the majority of variants, the transport activity differences were not associated with alterations in total OATP-C protein expression because immunoblot analysis (Fig. 3 C) indicated a lack of relationship between the total amount of immunoreactive protein and estrone sulfate uptake rate. A clear exception was seen with OATP-C*2, the expression of which appears to be lower than that of the other variants, consistent with its lower transport activity. In OATP-C transfected HeLa cells, the polyclonal antibody reacts with a diffuse ∼71-kDa band that is absent in cells transfected with control vector (Fig. 3 C), showing that the antibody was specific. Treatment of the HeLa cell homogenates with N-glycosidase F resulted in the disappearance of the major 71-kDa band and the emergence of a single ∼64-kDa band (not shown), indicating that OATP-C was glycosylated in HeLa cells. With crude human liver homogenates, the OATP-C antibody reacts with proteins with apparent molecular masses of ∼82 and ∼64 kDa (not shown). For a more comprehensive characterization, the concentration-dependent uptake kinetics of estrone sulfate was examined for all 16 allelic variants, and the results are summarized in Table II. Some variants (e.g. OATPC*5, Fig.4 A) possessed significantly higher K m values, whereas others (e.g.OATP-C*6, Fig. 4 A) demonstrated lowerV max values than those obtained with OATP-C*1a. Likewise, for one variant (e.g. OATP-C*9), bothV max and K m values differed in comparison to the reference allele (Fig. 4 A). To determine whether reductions in transport activities for OATP-C variants are seen with other substrates than estrone sulfate, concentration-dependent uptake kinetics were also determined using estradiol 17β-d-glucuronide. In general, similar trends were observed for the kinetics of estradiol 17β-d-glucuronide among OATP-C variants compared with estrone sulfate (Fig. 4). Intrinsic clearances for estradiol 17β-d-glucuronide were lower for OATP-C*2, *3, *5, *6, and *9 variants when compared with *1a (Fig. 4 B), and for the most part, this was due to decreased V max. Upon association of the transport activities with the corresponding SNPs, we were able to deduce that that Phe-73 → Leu (OATP-C*2), Val-174 → Ala (OATP-C*5), Ile-353 → Thr (OATP-C*6), Gly-488 → Ala (OATP-C*9), and Asp-655 → Gly (OATP-C*10) mutations significantly reduced estrone sulfate and/or estradiol-17β-d-glucuronide transport activities of OATP-C. Intrinsic clearance values for allelic variants containing any one of these amino acid substitutions were markedly lower than OATP-C*1a and ranged from 7 to 53% of the reference allele (Table IIand Fig. 4). For the OATP-C*3 allele, it was not possible to establish whether Val-82 → Ala and/or Glu-156 → Gly mutations were important for the reduced OATP-C activity.Table IIKinetic parameters (mean ± S.E.) for [3H]estrone sulfate transport by OATP-C va
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