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Identification and Functional Characterization of Genetic Variants of Human Organic Cation Transporters in a Korean Population

Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, Korea (H.-J.K., I.-S.S., H.J.S., W.-Y.K., C.-H.L., J.-C.S., S.S.L., J.-G.S.); and Institute of Clinical Pharmacology, Xiang-Ya School of Medicine, Central South University, Changsha, China (H.-H.Z.)

(Received October 27, 2006; accepted January 11, 2007)

	Abstract

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Genetic variants of three human organic cation transporter genes (hOCTs) were extensively explored in a Korean population. The functional changes of hOCT2 variants were evaluated in vitro, and those genetic polymorphisms of hOCTs were compared among different ethnic populations. From direct DNA sequencing, 7 of 13 coding variants were nonsynonymous single-nucleotide polymorphisms (SNPs), including four variants from hOCT1 (F160L, P283L, P341L, and M408V) and three from hOCT2 (T199I, T201M, and A270S), whereas 6 were synonymous SNPs. The linkage disequilibrium analysis presented for three independent LD blocks for each hOCT gene showed no significant linkage among all three hOCT genes. The transporter activities of MDCK cells that overexpress the hOCT2-T199I, -T201M, and -A270S variants showed significantly decreased uptake of [³H]methyl-4-phenylpyridinium acetate (MPP⁺) or [¹⁴C]tetraethylammonium compared with those cells that overexpress wild-type hOCT2, and the estimated kinetic parameters of these variants for [³H]MPP⁺ uptake in oocytes showed a 2- to 5-fold increase in K_m values and a 10- to 20-fold decrease in V_max values. The allele frequencies of the five functional variants hOCT1-P283L, -P341L, and hOCT2-T199I, -T201M, and -A270S were 1.3, 17, 0.7, 0.7, and 11%, respectively, in a Korean population; the frequency distributions of these variants were not significantly different from those of Chinese and Vietnamese populations. These findings suggest that genetic variants of hOCTs are not linked among three genes in a Korean population, and several of the hOCT genetic variants cause decreased transport activity in vitro compared with the wild type, although the clinical relevance of these variants remains to be evaluated.

Knockout mouse models have been generated for the Oct1, Oct2, and Oct3 genes to elucidate the in vivo function of the OCT transporters. Oct1-, Oct2-, and Oct3-deficient mice are viable and display no obvious phenotypic abnormalities (Jonker et al., 2001, 2003; Zwart et al., 2001; Wang et al., 2002, 2003; Jonker and Schinkel, 2004). However, Oct1 gene knockout mice show dramatically reduced hepatic uptake of TEA and metformin (Jonker et al., 2001; Wang et al., 2002, 2003). In Oct1/2 double-knockout mice, the renal secretion of TEA is completely abolished, and the plasma levels of TEA are substantially increased (Jonker et al., 2003). Considering the differences in tissue distribution between mice and humans, a combined deficiency of Oct1 and Oct2 better reflects the effect of OCT2 deficiency on kidney function (Jonker and Schinkel, 2004). The accumulation of MPP⁺ in the heart and fetus is significantly reduced in Oct3-deficient mice compared with wild-type mice (Zwart et al., 2001; Jonker and Schinkel, 2004).

Several groups have recently reported polymorphic variations in the hOCT families. Twenty-five genetic polymorphisms of hOCT1 were identified in 57 white subjects (Kerb et al., 2002), and three (R61C, C88R, and G401S) of eight nonsynonymous variants showed reduced transport activities. Shu et al. (2003) have reported 15 protein-altering variants of hOCT1 from diverse ethnic backgrounds, and three variants (F160L, P341L, and M408V) were identified in all the ethnic groups. Four SNPs (R61C, G220V, G401S, and G465R) show reduced transport function, whereas S14F exhibits increased transport function. Three nonsynonymous SNPs (P283L, R287G, and P341L) of hOCT1 found in a Japanese population show reduced transport activity (Takeuchi et al., 2003; Sakata et al., 2004). Two SNPs (M165I and R400C) of the eight genetic variants of hOCT2, each with an allele frequency of more than 1% in an African American population, display significantly reduced transport activity (Leabman et al., 2002). The variant (A270S) with the highest allele frequency has a subtle effect on hOCT2 function (Leabman et al., 2002). Fujita et al. (2006) have also reported that A270S has a higher K_i value for tetrabutylammonium inhibition of MPP⁺ uptake (274 µM) than that of wild-type hOCT2 (148 µM). Several genomic variants, such as R120R and A411A, have been identified in hOCT3 without amino acid substitution (Lazar et al., 2003).

Because the genetic variants associated with impaired transport function in vitro may have an influence on substrate disposition in vivo (Kerb et al., 2002; Shu et al., 2003), it is of importance to identify the functional polymorphisms and ethnic diversity of the three hOCT genes, to understand interindividual differences in the disposition and distribution of organic cations. However, little information is available regarding genetic polymorphisms of the hOCT genes in the Korean population or on the ethnic differences among Asian groups regarding genetic polymorphisms related to impaired functional activity of hOCT transporters. Therefore, in this study, we evaluated extensively genetic polymorphisms of the three hOCT genes in Korean subjects and investigated the functional properties of the nonsynonymous SNPs. Moreover, we performed ethnic comparisons of the major functional SNPs of the hOCT transporter genes.

	Materials and Methods

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Reagents. [³H]MPP⁺ (3.21 TBq/mmol), and [¹⁴C]-TEA (185 MBq/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Cell culture reagents, which included Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, and trypsin, were purchased from Invitrogen (Carlsbad, CA). Lipofectamine 2000 was purchased from Invitrogen.

Subjects. Korean subjects (n = 150) were recruited for this hOCT geno-typing study. Among these 150 subjects, 50 subjects were used to identify SNPs by full DNA sequencing. All of the participants were healthy according to their medical histories, physical examinations, and routine laboratory tests. All of the subjects provided written informed consent before participating in the present study, which was approved by the Institutional Review Board of Busan Paik Hospital (Busan, Korea). Genomic DNA samples from 100 Vietnamese and 100 Chinese subjects, which were stored in the DNA repository of PharmacoGenomics Research Center, Inje University (Busan, Korea), were used for the hOCT genotyping study. The racial background of the Vietnamese subjects was Viet Kinh, a major population group in Vietnam, and all the Chinese subjects were Han. The 100 DNA samples from German white persons were kindly provided by Ulrich M. Zanger of the Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology (Stuttgart, Germany).

DNA Purification and Direct Sequencing. For direct sequencing of the hOCT1, hOCT2, and hOCT3 genes, genomic DNA samples were isolated from the whole blood cells of 150 healthy Korean subjects using the QIAGEN DNA Extraction Kit (QIAGEN, Valencia, CA) according to the manufacturer's protocol. Specific primers were designed using the Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), to amplify 11 exons and the proximal promoter regions of each of the hOCT1, hOCT2, and hOCT3 genes (Table 1). PCR was performed in a reaction volume of 25 µlinthe presence of 150 ng of genomic DNA, 1x PCR buffer, 0.2 mM dNTPs, a 0.2 µM concentration of each primer, and1Uof Taq polymerase (Roche Applied Science, Penzberg, Germany). PCR was performed in the GeneAmp PCR 9700 (Applied Biosystems, Foster City, CA), with an initial denaturation step of 94°C for 5 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 54 to 69°C for 1 min, and extension at 72°C for 1 min. A final termination of elongation step was performed at 72°C for 5 min. The sequences of all the primers and details of the annealing temperatures are listed in Table 1. Amplicons were sequenced using an automated sequencer with the BigDye Terminator Sequencing Kit (Applied Biosystems).

Pyrosequencing Analysis. The primers used for pyrosequencing the hOCT1 and hOCT2 variants are summarized in Table 1. Biotinylated PCR products were immobilized onto streptavidin-coated beads (Streptavidin Sepharose High Performance; GE Healthcare, Little Chalfont, Buckinghamshire, UK) after strand separation for use in the PSQ 96 Sample Preparation Kit (pyrosequencing; GE Healthcare). In brief, the mixture of Sepharose bead slurry (3 µl), binding buffer (37 µl), PCR product (15 µl), and distilled water (25 µl) was incubated at room temperature for 10 min, and then centrifuged at 1400 rpm. The beads were transferred to a filter plate and the liquid was removed by vacuum filtration (Multiscreen Resist Vacuum Manifold; Millipore Inc., Billerica, MA). The DNA strands were separated in denaturation solution (0.5 M NaOH) for 5 s. The immobilized template was washed with 10 mM Tris-acetate washing buffer, pH 7.6, transferred to a PSQ 96 plate, and resuspended in 20 mM Tris-acetate annealing buffer, pH 7.6, that contained the sequencing primer. The sequencing primer was annealed at 80 to 90°C for 3 min. The sequence was analyzed using the PSQ 96 system with the SNP Reagent Kit (GE Healthcare).

Plasmids. To construct the hOCT2 expression plasmid (pcDNA3.1-hOCT2), we amplified hOCT2 cDNA (GenBank accession no. NM_003058 [GenBank] ) by PCR using the primer pair of 5'-GCCGGTACCATGCCCACCACCGTGGAC-3' and 5'-GCCGGTACCTTAGTTCAATGGAATGTCTAGTTTCTG-3' (underlined is the recognition site for KpnI) and the template of pEXO-hOCT2 (kind gift from Kathleen M. Giacomini, University of California at San Francisco). The isolated cDNAs were subcloned into the KpnI site of pcDN-A3.1(–).

To obtain mutant plasmids of hOCT2-T199I, -T201M, and -A270S, mutagenesis reactions were carried out on the DNA template of pcDNA3.1-hOCT2 using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The mutations, as well as the fidelity of the remaining DNA, were confirmed by sequencing. The double-stranded oligonucleotides used for site-directed mutagenesis of the hOCT2 gene were as follows: T199I: forward, 5'-GTTCTCATGGCCATTTCCCCAATCTATACGTGGATG-3'; reverse, 5'-CATCCACGTATAGATTGGGGAAATGGCCATGAGAAC-3'; T201M: forward, 5'-GGCCATTTCCCCAACCTATATGTGGATGTTAATTTTTCGC-3'; reverse, 5'-GCGAAAAATTAACATCCACATATAGGTTGGGGAAATGGCC-3'; and A270S: forward 5'-GTGGTTGCAGTTCACAGTTTCTCTGCCCAACTTCTTCTTC-3'; reverse, 5'-GAAGAAGAAGTTGGGCAGAGAAACTGTGAACTGCAACCAC-3'.

Cellular Uptake Studies. MDCK cells were maintained in a humidified atmosphere of 5% CO₂ and 95% air and grown in DMEM that was supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 100 U/ml penicillin-streptomycin. The medium was changed every 2 days. After the culture had reached 90% confluence, the cells were trypsinized and seeded onto 12-well plates at a density of 3 x 10⁵ cells per well. At 80% confluence, the cell culture medium was substituted with serum-free medium, 2 h before transfection. Transient transfections of pcDNA3.1 and pcDNA3.1-hOCT2 wild-type and mutants were performed into MDCK cells using Lipofectamine 2000. After transfection, the cells were incubated for 48 h at 37°C. Uptake studies were performed after the transfected cells formed a monolayer. The medium was removed from the MDCK cell cultures, and the cells were washed with DMEM and preincubated for 1 h with serum-free DMEM at 37°C. The uptake was initiated by replacing the medium with 1 ml of serum-free DMEM that contained 200 nM [³H]MPP⁺ or [¹⁴C]TEA, and quenched by washing three times with 2 ml of ice-cold phosphate-buffered saline. The cells were lysed with 100 µl of cell lysis reagent (CCLR buffer; Promega, Madison, WI) that was composed of 100 mM potassium phosphate, pH 7.8, 1 mM EDTA, 7 mM 2-mercaptoethanol, 1% (v/v) Triton X-100, and 10% (v/v) glycerol. The radioactivity of the cell lysates was measured by a MicroBeta TriLux 96-well scintillation/luminescence detector (PerkinElmer Life and Analytical Sciences).

Western Blotting. The transfected MDCK cells were harvested by centrifugation at 6000 rpm for 3 min at 4°C in an Eppendorf 5415R centrifuge. Cell pellets were swelled in one volume of radioimmunoprecipitation assay cell lysis buffer [150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 7.5] for 10 min. Aliquots that contained 30 µgof protein were separated by SDS-polyacrylamide gel electrophoresis ina4to 12% gradient gel (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5% nonfat milk and probed with anti-OCT2 (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-actin antibodies (Cell Signaling Technology, Danvers, MA). The membranes were then incubated with horseradish peroxidase-conjugated anti-goat IgG or anti-rabbit IgG (Santa Cruz Biotechnology) and visualized using the ECL system (Santa Cruz Biotechnology).

cRNA Synthesis and Transport Measurements using Xenopus laevis Oocytes. The cRNA synthesis and uptake experiments were performed as described previously (Kusuhara et al., 1999). The capped cRNAs were synthesized in vitro using T7 RNA polymerase with linear plasmid DNA. Defolliculated oocytes were injected with 50 ng of the capped cRNA and incubated at 18°C in Barth's solution [88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO₃)₂, 0.4 mM CaCl₂, 0.8 mM MgSO₂, 2.4 mM NaHCO₃, 10 mM HEPES, pH 7.4] that contained 50 µg/ml gentamicin and 2.5 mM pyruvate. After incubation for 2 days, uptake experiments were performed at room temperature in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂,5mM HEPES, pH 7.4). The uptake reaction was initiated by replacing the ND96 solution with one that contained the radiolabeled ligand, and terminated by the addition of ice-cold ND96 solution. After washing five times, oocytes were solubilized with 10% SDS and the radioactivity in the oocytes was analyzed.

Immunofluorescence Analysis. X. laevis oocytes were injected with the cRNAs for the hOCT2 wild-type and mutants. Two days after injection, the oocytes were fixed with paraformaldehyde and incubated with the anti-hOCT2 antibody (dilution 1:50; Santa Cruz Biotechnology), followed by fluorescein isothiocyanate-labeled rabbit anti-goat IgG (dilution 1:200; Santa Cruz Biotechnology). The sections were examined under a Carl Zeiss LSM 510 META confocal microscope (Carl Zeiss Inc., Jena, Germany).

Linkage Disequilibrium Analysis. The population genetic analysis program Haploview version 3.2 was used for the linkage disequilibrium (LD) analysis and Hardy-Weinberg equilibrium (HWE) test. The Haploview software calculates marker metrics and allows the visualization of haplotype block structures (Gabriel et al., 2002; Barrerr et al., 2005). First, we genotyped the SNPs of the hOCT1, hOCT2, and hOCT3 genes, and subsequently embedded them into the LD blocks for Korean subjects. The pairwise LD between all the SNP pairs was estimated by calculating the |D'| values using the haplotype blocks described by Gabriel et al. (2002). All the genotyped results were checked to make sure that they did not deviate from HWE by the p value cutoff at 0.05. To rule out genotyping errors and recently accrued hOCT1, hOCT2, and hOCT3 mutations, a minor allele frequency (MAF) of at least 10% was used. The allele frequencies, individual genotypes, and assays can be downloaded in bulk from our web site and can also be browsed graphically.

For pairwise comparisons of allele frequencies between different ethnic groups, the ² test was performed using the SAS program (SAS Institute, Cary, NC), and p < 0.05 was considered to be statistically significant.

Data Analysis. Cellular uptake was calculated as the cell/medium ratio based on the intracellular uptake per microgram of cell protein relative to the initial drug concentration. Statistical significance was analyzed using the unpaired t test, and p < 0.05 was considered to be statistically significant. The reproducibility of the results in the present study was confirmed using three separate experiments. The data are expressed as the mean ± S.D.

To estimate the kinetic parameters for the uptake of [³H]MPP⁺ by X. laevis oocytes overexpressing hOCT2 wild-type and variants, the hOCT2-mediated uptake rates were calculated by subtracting the transport rate of noninjected oocytes from that of hOCT2-expressing oocytes, followed by fitting to the Michaelis-Menten curve by an iterative nonlinear least-squares method using the WinNonlin version 5.1 (Pharsight, Mountain View, CA). Intrinsic clearance (CL_int) was obtained by dividing V_max values by K_m values.

	Results

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Identification of Genetic Variations of the hOCT Genes in Korean Subjects. To identify genetic polymorphisms of hOCT1, hOCT2, and hOCT3, we screened all 11 exons and approximately 2 kilobases of the 5'-flanking sequence of each transporter gene, as well as 50 to 100 base pairs of the flanking intronic sequences. The genetic variants and their frequencies for the hOCT1, hOCT2, and hOCT3 genes in 50 Korean subjects are summarized in Table 2. Sixteen variants were identified in the noncoding or intronic regions, and 13 were identified in coding regions. Six SNPs of the coding region were synonymous and seven were nonsynonymous. Among seven nonsynonymous SNPs, hOCT1-F160L, and hOCT1-M408V did not cause functional changes and hOCT1-P283L, hOCT1-P341L, and hOCT2-A270S were reported to show decreased transport activity. Fourteen novel SNPs including 11 SNPs in the 5'-UTR region, two SNPs in the noncoding region, and one SNP in the coding region were identified.

To reveal the genetic structures of the hOCT1, hOCT2, and hOCT3 loci in this Korean population, pairwise LD was analyzed using common variants of hOCT1, hOCT2, and hOCT3. All of the SNPs were in HWE (p > 0.05). The hOCT1, hOCT2, and hOCT3 genes are all located on chromosome 6q26–27 and were nonuniformly distributed across the 333-kb contiguous sequence (Fig. 1). To evaluate the impact of hOCT1, hOCT2, and hOCT3 variants on the block structure, we analyzed the values of |D'| and r² between 16 variant sites [MAF >10%, p < 0.05]. As shown in the LD block analysis (Fig. 1), there seem to be only three major haplotype blocks for each of the hOCT1, hOCT2, and hOCT3 loci: the first block spans the E5 + 45G>A and 1022A>G SNPs (hOCT1); the second block spans the 808G>T and E2 + 32C>G SNPs (hOCT2); and the third block spans from the –2269C>T SNP to the 360T>C SNP (hOCT3). LD analysis for hOCT variants with more than 10% frequency in Korean subjects showed no significant linkage between the hOCT genes, which suggests that the mutations in these three transporters are independent.

View larger version (61K): [in this window] [in a new window]   FIG. 1. Pairwise linkage disequilibrium (LD) among 16 SNPs of the hOCT1, hOCT2, and hOCT3 genes in a Korean population. The cut off p value of the HWE is 0.05. The minimum MAF is 10%.

View larger version (25K): [in this window] [in a new window]   FIG. 2. Functional characterization of nonsynonymous variants of hOCT2 in MDCK cells. MDCK cells were transiently transfected with 1.6 µg of pcDNA3.1-hOCT2, pcDNA3.1-hOCT2-T199I, -T201M, and -A270S. A and B, uptake of [3H]MPP+ or [14C]TEA by MDCK cells with over-expressed hOCT2 wild-type or variants. The cells were incubated for 5 min at 37°C after the addition of 200 nM [3H]MPP+ or [14C]TEA to the apical side. The uptake of [3H]MPP+ or [14C]TEA was determined by measuring radioactivity. Transient transfection of vector, hOCT2-WT, -T199I, -T201M, and -A270S into MDCK cells and uptake study using [3H]MPP+ and [14C]TEA were performed three times independently. Bars represent means ± S.D. **, p < 0.01, relative to the wild-type (WT) and according to the Student's t test. C, Western blot analysis was performed using the cell lysates after transient transfection of vector, hOCT2-WT, -T199I, -T201M, and -A270S into MDCK cells. The hOCT2 proteins have molecular masses in the 50- to 75-kDa range and are indicated by an arrow.

To determine the kinetics of uptake of [³H]MPP⁺ via the hOCT2 wild-type and SNP variants (T199I, T201M, and A270S) expressed in X. laevis oocytes, the concentration dependence of the uptake rates of [³H]MPP⁺ was studied within the range of 0.005 to 30 µM (Fig. 3). The uptake of [³H]MPP⁺ was increased linearly up to 60 min (data not shown) and the uptake rate of [³H]MPP⁺ showed concentration dependence with increased concentration of MPP⁺ (Fig. 3A). Kinetic parameters such as K_m, V_max, and intrinsic clearance (CL_int) were shown in Fig. 3B. The approximate V_max values for the hOCT2-T199I, -T201M, and -A270S SNPs were 22.5-, 21.7-, and 11.7-fold decreased, respectively, compared with that for hOCT2-WT. The approximate K_m values for the hOCT2-T199I, -T201M, and -A270S SNPs were 3.7-, 5.1-, and 2.0-fold increased, respectively, compared with that for hOCT2-WT; and the approximate CL_int (obtained by dividing V_max by K_m) for the hOCT2-T199I, -T201M, and -A270S SNPs were 83.7-, 110.4-, and 23.4-fold decreased, respectively, compared with that for hOCT2-WT. The localizations of the hOCT2 wild-type and variant proteins in X. laevis oocytes were revealed by immunofluorescence analysis. No expression was shown when empty vector was injected into oocytes (Fig. 4A). The hOCT2 wild-type protein (Fig. 4B) and variant proteins (Fig. 4, C–E) were highly expressed and localized in the plasma membrane.

View larger version (21K): [in this window] [in a new window]   FIG. 3. Kinetic analysis of the hOCT2 variants. A, concentration dependence of [3H]MPP+ uptake by oocytes expressing the hOCT2 wild-type and variants (T199I, T201M, and A270S). The uptake of [3H]MPP+ was measured at the concentration indicated after 30 min of incubation. Each data point represents the means ± S.E. after eight independent experiments. The level of hOCT2-mediated transport was obtained by subtracting the transport rates of the noninjected oocytes from those of the hOCT2-expressing oocytes. B, kinetic parameters were obtained as approximations by fitting to the Michaelis-Menten curve by an iterative nonlinear least-squares method.

View larger version (56K): [in this window] [in a new window]   FIG. 4. Localization of hOCT2 wild-type and its variants at the plasma membrane of X. laevis oocytes. No positive staining for hOCT2 is observed in the control oocytes injected with water instead of cRNAs (A). Immunofluorescence detection with hOCT2 antibody shows that the wild-type protein (B), as well as the three mutant proteins [T199I (C), T201M (D), and A270S (E)], are expressed at the plasma membrane.

Ethnic Frequencies of Functional Variants of hOCTs. The occurrence of functional variants of hOCT1 (P283L and P341L) and hOCT2 (T199I, T201M, and A270S) was analyzed by pyrosequencing analysis of 150 Korean subjects and 100 subjects each of Vietnamese, Chinese, and German origin. The pyrosequencing-based genotypes of 450 DNA samples were completely concordant with the data obtained by the reference method of direct sequencing, which suggests that the simplex and duplex assay methods developed in this study are time-efficient and can be applied to the genotyping of five functional SNPs of the hOCT genes (Fig. 5).

View larger version (25K): [in this window] [in a new window]   FIG. 5. Genotyping of the functional SNPs of hOCT1 and hOCT2 by pyrosequencing. Duplex and simplex assay designs with the expected and observed pyrograms for the hOCT1 (P283L and P341L) and hOCT2 (T199I, T201M, and A270S) variants are shown. The choice of sequence to analyze was based on the cDNA position with attached sequencing primer, which determines the nucleotide dispensation order. For each genotype, a pyrogram is shown, together with a graph of the expected results. Representative pyrograms illustrate heterozygous subjects. The simplex pyrograms show the following genotypes: A, hOCT1-P283L (848C/T); B, hOCT1-P341L (1022A/G); and C, hOCT2-A270S (808G/T). The duplex pyrograms denote the following genotypes: D, hOCT2-T199I and -T201M [596C/T (a) and 602C/T (b)]. The box indicates the position of each SNP.

	Discussion

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To understand the potential effects of genetic variations on xenobiotic transport activity, we carried out a comprehensive genetic polymorphism analysis of the human organic cation transporters hOCT1, hOCT2, and hOCT3. Twenty nine genetic mutations of hOCT1, hOCT2, and hOCT3 were recognized in the Korean subjects, and seven nonsynonymous variants, hOCT1-F160L, -P283L, -P341L, and -M408V and hOCT2-T199I, -T201M, and -A270S, were identified. Among them, the functional consequences of nonsynonymous SNPs of hOCT1 were well defined (Table 2), whereas little is known about the functional activities of those of hOCT2 (T199I and T201M). Thus, we evaluated the functional activities of the nonsynonymous SNPs of hOCT2 using MDCK cells and X. laevis oocytes. MDCK cells that overexpress hOCT2-T199I, -T201M, and -A270S showed decreased uptake of [³H]MPP⁺ and [¹⁴C]TEA compared with the wild-type (Fig. 2). MDCK cells are originated from canine kidney epithelium and express endogenous OCT2 transporter (Shu et al., 2001), which was consistent with the Western blot results that showed basal expression in empty vector-transfected MDCK cells (Fig. 2C). This may be why uptake rates of [³H]MPP⁺ and [¹⁴C]TEA in empty vector-transfected MDCK cells were 36 and 44% of those in wild-type hOCT2 transfected cells, respectively. If the uptake rates of [³H]MPP⁺ and [¹⁴C]TEA mediated by endogenous OCT2 were subtracted from those of transfected hOCT2-WT and variants, the uptake rates of [³H]MPP⁺ in hOCT2 variants (T199I, T201M, and A270S) were decreased to 38, 46, and 59% of hOCT2-WT, respectively, and the uptake rates of [¹⁴C]TEA in hOCT2 variants were decreased to 36, 24, and 41% of hOCT2-WT, respectively. To rule out the involvement of endogenous OCT2 in kinetic parameters of hOCT2 WT and variants, we measured concentration-dependent uptake of [³H]MPP⁺ in X. laevis oocytes, which show no endogenous OCT2 expression. Kinetic analysis of the hOCT2 mutants confirmed the functional changes of these variants, as evidenced by increased values of approximate K_m, and decreased values of approximate V_max and CL_int of all three hOCT2 variants compared with the wild-type hOCT2. There has been some controversy regarding the functional activity of the A270S variant because of the slight (approximately 2-fold) change in the K_i value of this mutant (Leabman et al., 2002; Fujita et al., 2006), which is consistent with our results. However, transport function should be evaluated in terms of CL_int, as well as K_m and V_max. According to our results, the approximate CL_int of the A270S variant was decreased 23.4-fold, which suggests decreased transport activity for the A270S mutant. However, the protein expression level and localization of the hOCT2 variants in MDCK cells and X. laevis oocytes were not different from those of the wild-type (Figs. 2 and 4). These results, taken together, suggest that the reduced transport activities of the hOCT2 variants (T199I, T201M, and A270S) are not attributable to either protein expression or plasma membrane localization.

The five functional variants of hOCT1 and hOCT2 are distributed throughout the loops (hOCT1-P283L and -P341L and hOCT2-T199I and -T201M) and transmembrane domains (hOCT2-A270S) of the protein, according to the presumed transmembrane topology of hOCT transporters (Leabman et al., 2002; Popp et al., 2005). Considering that several cysteine, proline, and arginine residues are believed to maintain the secondary structures of proteins and/or bind charged substrates (Burckhardt and Wolff, 2000), the reduced functional activities of hOCT1 (Takeuchi et al., 2003; Sakata et al., 2004) and hOCT2 caused by amino acid substitutions may be related to structural changes in the substrate recognition region and/or functional domain. The changes in approximate V_max values (11.7- to 22.5-fold change) of the hOCT2-T199I, -T201M, and -A270S mutants in the third extracellular loop and sixth transmembrane domain of the hOCT2 transporter were larger than the changes in the approximate K_m values (2.0–5.1-fold change) of the variants. The critical amino acids of Tyr222 and Thr226 in the substrate-binding pocket of hOCT1 are highly conserved among the hOCT subtypes of various species and are located in the fourth transmembrane domain (Popp et al., 2005). Therefore, the amino acid substitutions in hOCT2-T199I, -T201M, and -A270S mutants might have inhibitory effects on conformational changes of the transporter rather than on substrate binding.

The allele frequencies of the hOCT1 (P283L and P341L) and hOCT2 (T199I, T201M, and A270S) variants were analyzed in Korean, Vietnamese, Chinese, and white German subjects by pyrosequencing. The hOCT1-P283L, hOCT2-T199I, and hOCT2-T201M SNPs were detected very rarely, and showed no differences between Asians and white Germans, which indicates that it is not useful to genotype routinely these mutations for predictions of in vivo distribution and disposition of organic cations. On the other hand, the allele frequencies of hOCT1-P341L and hOCT2-A270S in the Korean population (17 and 11%, respectively) were similar to those in other Asian populations, and significantly higher than those in the white German population (2 and 2.5%, respectively). Therefore, impaired transport activities related to hOCT1-P341L and hOCT2-A270S SNPs may differ between Asians and white Germans, with consequent effects on the pharmacokinetics of certain substrates. The hOCT1-P341L variant was identified in a white German population in this study, whereas this mutation was not reported in a white and European-American population (Kerb et al., 2002; Shu et al., 2003). In addition, the hOCT2-A270S mutation revealed different allele frequencies between the white and white German populations [i.e., 15.7% for whites (Leabman et al., 2002) and 2.5% for our population]. The lower diversity of the white German population compared with the European-American and white pool may explain the different frequencies observed for hOCT1-P341L and hOCT2-A270S.

Contrary to those nonsynonymous SNPs of hOCT1 and hOCT2 genes, only three synonymous variants (R120R, G193G, and A411A) were detected from hOCT3 gene. The absence of a nonsynonymous mutation in the hOCT3 gene and the positive value of Tajima's D may reflect selective mechanisms against certain amino acid changes that have operated during the evolution of the human species and may also be due to demographic factors, such as population subdivision (Lazar et al., 2003). In addition, 13 of 29 hOCT SNPs were detected in the 5'-UTRs of the individual transporter genes, and several SNPs showed relatively high frequency (> 30%), which raises the possibility that the SNPs in the 5'-UTRs may affect the regulation of expression of these transporters. The hOCT1 gene has been reported to be transactivated by hepatocyte nuclear factor-4 (HNF-4), and the mRNA expression of organic cation transporters in liver, kidney, and duodenum is altered differently after targeted disruption of the transcription factor HNF-1 (Maher et al., 2006; Saborowski et al., 2006). Site-directed mutagenesis of HNF-4 binding site in the promoter region of the hOCT1 gene more strongly decreased luciferase activity of reporter construct hOCT1(–2620/+116)mut than that of wild-type reporter construct (Saborowski et al., 2006). Moreover, Shu et al. (2001) reported steroid hormone-mediated regulation of OCT2 in MDCK cells. These reports, taken together, suggested that transcription factors such as HNF-4 and nuclear receptors may be involved in the tissue-specific regulation of organic cation transporters. In silico analysis using the TRANSFAC database (http://www.cbil.upenn.edu/cgi-bin/tess) revealed that several SNPs in 5'-UTR region occurred in the binding sequence for transcription factors, such as GATA, Sp1, MyoD, YY1, and c-Myc, and the binding sequences for nuclear receptors such as GR, RXR-, and RAR-, which suggest that the SNPs in the 5'-UTR region may cause expressional changes of respective transporters. The functional consequences of 13 SNPs in the upstream regions as well as transcriptional regulation of hOCT transporters are under investigation.

With the identification of functionally important genetic polymorphisms of the hOCT1 and hOCT2 genes, it will be of great interest to determine whether these polymorphisms also correlate with altered drug responses and sensitivities in patients (Jonker and Schinkel, 2004). According to previous reports (Leabman and Giacomini, 2003; Yin et al., 2006), the genetic component that contributes to variation in the renal clearance of metformin, which is a substrate of hOCT2 that is eliminated exclusively by transporter-mediated renal secretion (Kimura et al., 2005; Fujita et al., 2006), is estimated to be particularly high (93%). These findings suggest that variation in the renal clearance of metformin has a strong genetic component, and that genetic variations in hOCT2 may explain, to a large degree, this pharmacokinetic variability (Leabman and Giacomini, 2003). Further studies in humans are underway in our laboratory. In addition, the pyridinium metabolites (HPP⁺ and RHPP⁺) of haloperidol are known to distribute into the brain and cause side effects, such as neurodegeneration. HPP⁺ and RHPP⁺ are substrates for the hOCT family (Kang et al., 2006). In other words, the uptake of HPP⁺ is mediated by hOCT1, hOCT2, and hOCT3, whereas the uptake of RHPP⁺ is increased in hOCT1- and hOCT3- but not hOCT2-overexpressing MDCK cells. This suggests that functional genetic variants of the hOCT transporters may produce different brain distributions of HPP⁺ and RHPP⁺ and, consequently, different levels of neurotoxicity. Therefore, the information gained about the functional genetic polymorphisms and the ethnic diversity of the hOCT family will help us to understand interindividual drug responses and the pharmacokinetics of cationic drugs.

	Acknowledgments

 
The hOCT-containing plasmids were kindly provided by Dr. Kathleen M. Giacomini (University of California at San Francisco, CA, USA) and Dr. Hitoshi Endou (Fuji Biomedix Co., Tokyo, Japan).

	Footnotes

 
This study was supported by the Korea Science and Engineering Foundation (KOSEF) through the National Research Lab. Program funded by the Ministry of Science and Technology (M10300000370-06J0000-37010) and by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A030001).

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.013581.

ABBREVIATIONS: hOCT, human organic cation transporter; TEA, tetraethylammonium; HPP⁺, 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]pyridinium; MPP⁺, methyl-4-phenylpyridinium acetate; SNP, single nucleotide polymorphism; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; MDCK, Madin-Darby canine kidney; LD, linkage disequilibrium; HWE, Hardy-Weinberg equilibrium; MAF, minor allele frequency; UTR, untranslated region; HNF, hepatocyte nuclear factor.

Address correspondence to: Dr. Jae-Gook Shin, Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, 633-165 Gaegum 2-Dong, Jin-Gu, Busan 614-735, Korea. E-mail: phshinjg{at}inje.ac.kr

	References

Top Abstract Materials and Methods Results Discussion References

 


Barrerr JC, Fry B, Maller J, and Daly MJ (2005) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 15: 263–265.

Burckhardt G and Wolff NA (2000) Structure of renal organic anion and cation transporters. Am J Physiol 278: F853–F866.

Fujita T, Urban TJ, Leabman MK, Fujita K, and Giacomini KM (2006) Transport of drugs in the kidney by the human organic cation transporter, OCT2 and its genetic variants. J Pharm Sci 95: 25–36.[CrossRef][Medline]

Fukushima-Uesaka H, Maekawa K, Ozawa S, Komamura K, Ueno K, Shibakawa M, Kamakura S, Kitakaze M, Tomoike H, Saito Y, et al. (2004) Fourteen novel single nucleotide polymorphisms in the SLC22A2 gene encoding human organic cation transporter (OCT2). Drug Metab Pharmacokinet 19: 239–244.[CrossRef][Medline]

Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B, Higgins J, DeFelice M, Lochner A, Faggart M, et al. (2002) The structure of haplotype blocks in the human genome. Science (Wash DC) 296: 2225–2229.[Abstract/Free Full Text]

Gorboulev V, Ulzheimer JC, Akhoundova A, Ulzheimer-Teuber I, Karbach U, Quester S, Baumann C, Land F, Busch AE, and Koepsell H (1997) Cloning and characterization of two human polyspecific organic cation transporters. DNA Cell Biol 16: 871–881.[Medline]

Jonker JW and Schinkel AH (2004) Pharmacological and physiological functions of the polyspecific organic cation transporters: OCT1, 2, and 3 (SLC22A1–3). J Pharmacol Exp Ther 308: 2–9.[Abstract/Free Full Text]

Jonker JW, Wagenaar E, Mol CA, Buitelaar M, Koepsell H, Smit JW, and Schinkel AH (2001) Reduced hepatic uptake and intestinal excretion of organic cations in mice with a targeted disruption of the organic cation transporter1 (Oct1 [Slc22a1]) gene. Mol Cell Biol 21: 5471–5477.[Abstract/Free Full Text]

Jonker JW, Wagenaar E, Van Eiji S, and Schinkel AH (2003) Deficiency in the organic cation transporters 1 and 2 (Oct1 and Oct2 [Slc22a1 and Slc22a2]) in mice abolishes the renal secretion of organic cations. Mol Cell Biol 23: 7902–7908.[Abstract/Free Full Text]

Kang HJ, Lee SS, Lee CH, Shim JC, Shin HJ, Liu KH, Yoo MA, and Shin JG (2006) Neurotoxic pyridinium metabolites of haloperidol are substrates of human organic cation transporters. Drug Metab Dispos 34: 1145–1151.[Abstract/Free Full Text]

Kerb R, Brinkmann U, Chatskaia N, Gorbunov D, Gorboulev V, Mornhinweg E, Keil A, Eichelbaum M, and Koepsell H (2002) Identification of genetic variations of the human organic cation transporter hOCT1 and their functional consequences. Phamacogenetics 12: 591–595.

Kimura N, Masuda S, Tanihara Y, Ueo H, Okuda M, Katsura T, and Inui K (2005) (2005) Metformin is a superior substrate for renal organic cation transporter OCT2 rather than hepatic OCT1. Drug Metab Pharmacokinet 20: 379–386.[CrossRef][Medline]

Koepsell H (1999) Organic cation transporters in intestine, kidney, liver, and brain. Annu Rev Physiol 60: 243–266.

Kusuhara H, Sekime T, Utsunomiya-Tate N, Tsuda M, Kojima R, Cha SH, Sugiyama Y, Kamai Y, and Endou H (1999) Molecular cloning and characterization of a new multispecific organic anion transporter from rat brain. J Biol Chem 274: 13675–13680.[Abstract/Free Full Text]

Lazar A, Grundemann D, Berkels R, Taubert D, Zimmermann T, and Schomig E (2003) Genetic variability of the extraneuronal monoamine transporter EMT (SLC22A3). J Hum Genet 48: 226–230.[CrossRef][Medline]

Leabman MK and Giacomini KM (2003) Estimating the contribution of genes and environment to variation in renal drug clearance. Phamacogenetics 13: 581–584.

Leabman MK, Huang CC, Kawamoto M, Johns SJ, Stryke D, Ferrin TE, De Young J, Taylor T, Clark AG, Herskowitz I, et al. (2002) Pharmacogenetics of Membrane Transporters investigators: polymorphisms in a human kidney xenobiotic transporter, OCT2, exhibit altered function. Pharmacogenetics 12: 395–405.[CrossRef][Medline]

Maher JM, Slitt AL, Callaghan TN, Cheng X, Cheng C, Gonzalez FJ, and Klaassen CD (2006) Alterations in transporter expression in liver, kidney, and duodenum after targeted disruption of the transcription factor HNF1a. Biochem Pharmacol 72: 512–522.[CrossRef][Medline]

Popp C, Gorboulev V, Müller TD, Gorbunov D, Shatskaya N, and Koepsell H (2005) Amino acids critical for substrate affinity of rat organic cation transporter 1 line the substrate binding region in a model derived from the tertiary structure of lactose permease. Mol Pharmacol 67: 1600–1611.[Abstract/Free Full Text]

Saborowski M, Kullak-Ublick GA, and Eloranta JJ (2006) The human organic cation transporter-1 gene is transactivated by hepatocyte nuclear factor-4. J Pharmacol Exp Ther 317: 778–785.[Abstract/Free Full Text]

Sakata T, Anzai N, Shin HJ, Noshiro R, Hirata T, Yokoyama H, Kanai Y, and Endou H (2004) Novel single nucleotide polymorphisms of organic cation transporter 1 (SLC22A1) affecting transport functions. Biochem Biophys Res Commun 16: 789–793.

Shu Y, Bello CL, Mangravite LM, Feng B, and Giacomini KM (2001) Functional characterization and steroid hormone-mediated regulation of an organic cation transporter in Madin-Darby canine kidney cells. J Pharmacol Exp Ther 299: 392–398.[Abstract/Free Full Text]

Shu Y, Leabman MK, Feng B, Mangrabite LM, Huang CC, Stryke D, Kawamoto M, Johns SJ, De Young J, Carlson E, et al. (2003) Evolutionary conservation predicts function of variants of the human organic cation transporter, OCT1. Proc Natl Acad Sci USA 100: 5902–5907.[Abstract/Free Full Text]

Takeuchi A, Motohashi H, Okuda M, and Inui K (2003) Decreased function of genetic variants, Pro283Leu and Arg287Gly, in human organic cation transporter hOCT1. Drug Metab Pharmacokinet 18: 409–412.[CrossRef][Medline]

Wang DS, Jonker JW, Kato Y, Kusuhara H, Schinkel AH, and Sugiyama Y (2002) Involvement of organic cation transporter 1 in hepatic and intestinal distribution of metformin. J Pharmacol Exp Ther 302: 510–515.[Abstract/Free Full Text]

Wang DS, Kusuhara H, Kato Y, Jonker JW, Schinkel AH, and Sugiyama Y (2003) Involvement of organic cation transporter 1 in lactic acidosis caused by metformin. Mol Pharmacol 63: 844–848.[Abstract/Free Full Text]

Yin OO, Tomlinson B, and Chow MS (2006) Variability in renal clearance of substrates for renal transporters in Chinese subjects. J Clin Pharmcol 46: 157–163.

Zhang L, Dresser MJ, Gray AT, Yost SC, Terashita S, and Giacomini KM (1997) Cloning and functional expression of a human liver organic cation transporter. Mol Pharmacol 51: 913–921.[Abstract/Free Full Text]

Zwart R, Verhaagh S, Buitelaar M, Popp-Snijders C, and Barlow DP (2001) Impaired activity of the extraneuronal monoamine transporter system known as uptake-2 in Orct3/Slc22a3-deficient mice. Mol Cell Biol 21: 4188–4196.[Abstract/Free Full Text]

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