Functional Analysis of Human Organic Cation Transporter OCT3 (SLC22A3 ) Polymorphisms

J Pharmacol Sci 113, 263 – 266 (2010) Journal of Pharmacological Sciences ©2010 The Japanese Pharmacological Society Short Communication Functional Analysis of Human Organic Cation Transporter OCT3 (SLC22A3 ) Polymorphisms

Takeshi Sakata1 , Naohiko Anzai1, *, Toru Kimura1 , Daisaku Miura1 , Toshiyuki Fukutomi1 , Michio Takeda1 , Hiroyuki Sakurai1 , and Hitoshi Endou1 1Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Tokyo 181-8611, Japan

Received November 30, 2009; Accepted May 7, 2010

Abstract. We analyzed the functional properties of five single nucleotide polymorphisms (SNPs) in organic cation transporter OCT3 gene (SLC22A3 ) resulting in the amino acid changes with a transient expression system. Three SNPs (A116S, T400I, and A439V) exhibited reduced uptake of both [3 H]histamine and [3 H]MPP +, although their protein expressions were detected in the plasma membrane of transfected cells. This study suggests that the OCT3 variants will contribute to inter- individual variations leading to the differences in cationic drug disposition as well as certain disease processes such as hypertension, allergic diseases, and neuropsychiatric diseases by the clearance of endogenous organic cations such as biogenic amines.

Keywords : organic cation transporter, single nucleotide polymorphism, histamine

Membrane transporters play an important role in the elimination of various cationic substrates including thera- metabolism of drugs and endogenous substrates. Re- peutically important agents as well as in the inactivation cently, it was reported that certain single nucleotide of biogenic amines such as catecholamines and histamine polymorphisms (SNPs) in transporters including ABC (7). In the public SNP database (NCBI dbSNP, http:// (ATP-binding cassette) transporters and SLC (solute www.ncbi.nlm.nih.gov/SNP/), five nonsynonymous carrier) transporters are related to their altered transport SNPs of SLC22A3 are reported, but functional analysis activities, which may have clinical implications (1). of these genetic variations has not been performed yet. In Thus, functional analysis of genetic polymorphism of this report, we characterized the functional properties of transporters is clinically important. the hOCT3 variants in transiently transfected cells. Organic cation transporters (OCTs) mediate sodium- Full-length cDNA of hOCT3 was obtained as described independent electrogenic transport of small organic cat- previously (8). The QuickChange Site-Directed Muta- ions with different molecular structures (2). These organic genesis Kit (Stratagene, La Jolla, CA, USA) was used to cations include clinically used drugs (e.g., metformin), introduce point mutations into hOCT3 cDNA in the ex- endogenous compounds (e.g., catecholamine), as well as pression vector according to the manufacturer’s instruc- toxic substances (e.g., MPP+ ). Recently, several function- tions. Complementary oligonucleotides used for muta- ally relevant genetic variations in human OCT1 (hOCT1, genesis are described in Table 1. All the final sequences SLC22A1 ) (3, 4) and hOCT2 (SLC22A2 ) (5) were were confirmed by DNA sequencing. reported. In contrast, there is no report concerning the HEK293 cells and COS cells were maintained in functional characterization of genetic variations of Dulbecco’s modified Eagle’s medium (DMEM) supple- hOCT3, although the coding region polymorphisms were mented with 10% fetal bovine serum (FBS), 1 mM so- already reported (6). dium pyruvate, 100 U/ml penicillin, and 100 mg/ml hOCT3 ( SLC22A3 ), also designated as extraneuronal streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C monoamine transporter (EMT), expressed in the kidney, and 5% CO2 . Transient transfection with Lipofectamine liver, and placenta, participates in the cellular uptake and 2000 (Invitrogen) was performed according to the manufacturer’s instructions. After transfection, the cells were *Corresponding author. [email protected] grown 36 – 48 h before the experiments. 3 Published online in J-STAGE on June 16, 2010 (in advance) Cellular uptake of [ H]histamine (2.05 Bq/mmol) and doi: 10.1254/jphs.09331SC [3 H]MPP + (2.96 TBq/mmol), purchased from American

263 264 T Sakata et al

Table 1. Summary of nonsynonymous polymorphisms of hOCT3 in the NCBI database refSNP ID Sequence Protein residue Oligonucleotides for mutagenesis Average allele frequency

ttcct gggca For: 5′-ggtcttcctgggcatgcaacccgaccactac-3′ C 1.000 ssj0008476 C/T T44M gcagc ccgac Rev: 5′-gtagtggtcgggttgcatgcccaggaagacc-3′ T

cccac tcgcc For: 5′-ggacccactcgcctcattccccaaccgttc-3′ G 0.993 rs8187717 G/T A116S ccttc cccaa Rev: 5′-gaacggttggggaatgaggcgagtgggtcc-3′ T 0.007

atctt actaa For: 5′-cttgatcttactaatcattgagcgccttgg-3′ T 0.002 rs8187725 C/T T400I cattg agcgc Rev: 5′-ccaaggcgctcaatgattagtaagatcaag-3′ C 0.998

accac agtgg For: 5′-gaggaccacagtcgttacattgggaag-3′ rs12212246 C/T A439V Not available tacat tggga Rev: 5′-cttcccaatgtaacgactgtggtcctc-3′

gctct gttca For: 5′-ggagtttcgctctgtagcagtctgtgtgattttg-3′ rs9365165 C/T G475S Not available gtctg gttca Rev: 5′-caaaatcacacagactgctacagagcgaaactcc-3′

Radiolabeled Chemicals (St. Louis, MO, USA), were Burlingame, CA, USA). Fluorescence was visualized measured in hOCT3 (or its mutants)–transfected HEK293 with a laser confocal microscope. Contrast and bright- cells grown on poly-D -lysine-coated 24-well plates. The ness settings were chosen so that all pixels were within cells were incubated in serum-free Hanks’ balanced salt the linear range. solution (HBSS) containing the following: 125 mM Na In the public SNP database (NCBI dbSNP), we found gluconate, 4.8 mM K gluconate, 1.2 mM KH2 PO 4, 1.2 five nonsynonymous nucleotide polymorphisms of mM MgSO 4, 1.3 mM Ca gluconate, 5.6 mM glucose, and hOCT3 (SLC22A3 ): T44M, A116S, T400I, A439V, and 25 mM HEPES (pH 7.4) for 10 min. The uptake study G475S. As shown in Fig. 1A, T44 is located near the was started by adding HBSS containing [3 H]histamine 3′-end of the first transmembrane domain (TMD); A116 (200 nM) or [ 3H]MPP + (30 nM) to the plate. After 2 min, is in the middle of the long extracellular loop between the the cells were washed twice in ice-cold HBSS and then first and second TMDs; T400 is located near the 3′-end lysed in 0.1 N NaOH for 20 min, followed by measure- of the eighth TMD; A439 is located near the 5′-end of the ment of radioactivity by scintillation counting. tenth TMD; G475 is located near the 5′-end of the elev- COS cells transfected with vectors containing wild- enth TMD. Among these, T44 is conserved from hOCT1 type hOCT3 and mutants were washed with PBS con- to hOCT3 as well as hOCTN1 and hOCTN2, and T400 ++ taining 1 mM MgCl2 and 0.1 mM CaCl2 (PBS ), fixed is conserved from OCT1 to OCT3, but A116, A439, and in cold methanol for 7 min, and washed 3 times with G475 are found only for OCT3 (2). PBS ++. Fixed cells were permeabilized in permeabiliza- To examine whether hOCT3 polymorphisms found in tion buffer (0.1% BSA and 1% Triton X-100 in PBS++ ) the SNP database affect functional activities, we con- for 15 min and blocked in goat serum dilution buffer structed site-directed mutants and expressed them in (GSDB) (10% goat serum, 1% Triton X-100, and 10 mM HEK293 cells. As shown in Fig. 1, B and C, the uptakes glycine in PBS++ ) for 30 min. Cells were incubated with of representative OCT3 substrates, [3 H]histamine and primary antibodies diluted in GSDB buffer overnight at [3 H]MPP +, were largely reduced for A116S and A439V 4°C. An anti-Na,K-ATPase monoclonal antibody di- and moderately for T400I. The uptake of [3 H]histamine rected against an epitope between residues 338 to 724 of for G475S was slightly decreased. In contrast, the T44M the chicken Na,K-ATPase (9) and anti-OCT3 polyclonal mutant showed no significant changes in the uptake. antibody (Transgenic Inc., Kumamoto) were used for Plasma membrane localizations of hOCT3 variants primary antibodies. Cells were washed 3 times with were examined by immunofluorescent analysis. Figure 2 permeabilization buffer and then incubated with Alexa shows the staining of hOCT3 proteins in COS cells Fluor 488 anti-rabbit IgG, Alexa Fluor 546 anti-mouse transfected with cDNAs of hOCT3 variants. Wild-type IgG (Invitrogen), and 1 μ g/ml DAPI (Roche, Mannheim, and mutant hOCT3 proteins were localized in the plasma Germany) diluted in GSDB buffer for 1 h, after which membrane, detected by anti-Na,K-ATPase monoclonal they were washed 3 times in PBS++ and once in water. antibody, as well as in the cytoplasm, confirming that Cells were mounted in Vectashield (Vector laboratories, altered transport activities are not likely to be induced by Transport Activities of SLC22A3 SNPs 265

substrate drugs. For example, Zwart et al. reported that A G475S A116S Oct3/Slc22a3 (mouse homologue of hOCT3)-deficient T44M Extracellular mice exhibit decreased accumulation of the neurotoxin MPP + in the heart (10). In the same way, aforementioned Plasma functionally relevant hOCT3 variations are likely to ex- 1 2 3 4 5 6 7 8 9 10 11 12 membrane plain some of the inter-individual variations in cationic

NH Intracellular drug disposition. Thus, it would be interesting to know if + COOH there are changes in the MPP susceptibility of individu- T400I A439V als with altered hOCT3 function. To date, there is one report on phenotypic differences B suspected to be due to hOCT3 polymorphism. Aoyama 0.20 et al. found that some SNPs in hOCT3/SLC22A3 are related to the development of polysubstance abuse in Japa- 0.15 nese individuals with dependence on the amphetamine ** ** derivative methamphetamine (MAP) (11). It may be that 0.10 *** *** altered disposition of MAP affects one’s behavior to seek other addictive substances. Interestingly, it was reported 0.05 that OCT3 is involved in the disposition of MAP and behavioral changes induced by MAP in an animal model 0.00

H]Histamine (pmol/mg protein per 2 min) (12). Obviously, it is necessary to determine whether 3

[ control WT T44M A116S T400I A439V G475S MAP is a transport substrate of hOCT3 and whether the SNPs reported in this study affect transport activity of C 0.8 hOCT3. 0.7 Furthermore, removal of endogenous cations such as 0.6 catecholamine and histamine may be affected by the 0.5 transport function of OCT3 (13). For example, Schneider ** 0.4 et al. reported that pharmacologic modulation of hista- 0.3 *** mine transport via OCT3 might become important in the (pmol/mg protein per 2 min) + 0.2 *** control of basophil functions during allergic diseases 0.1 (14) and Vialou et al. demonstrated that the presence of H]MPP 3 [ 0.0 OCT3 is critical for salt-intake regulation that is closely control WT T44M A116S T400I A439V G475S related to blood pressure (15). Thus, it would be interest- Fig. 1. Functional characterization of hOCT3 variants. A: Sche- ing to see the effect of functionally relevant SNPs in matic representation of hOCT3. Transmembrane topology is based on hOCT3 on the disease process of allergy and hyperten- the previous report (2). Nonsynonymous nucleotide substitutions are sion. indicated by arrows. B: The uptake of [3 H]histamine (200 nM) at 2 From the point of the structure–function relationship, min in empty (control), hOCT3 wild-type (WT), and mutant (T44M, the reduction of transport function in T400I of hOCT3 A116S, T400I, A439V, and G475S) cDNA–transfected HEK293 cells was measured as described in experimental procedures. Each seems interesting because residue T400 is conserved value represents the mean ± S.D. of eight determinations. C: The up- from OCT1/Oct1 to OCT3/Oct3 (2). Functional analysis take of [3 H]MPP + (30 nM) at 2 min in empty, hOCT3 wild-type, and of the mutants that have the corresponding amino acid mutant cDNA–transfected HEK293 cells was measured as described replacement in hOCT1 and hOCT2 may provide infor- in experimental procedures. The data represent the mean ± S.D. for mation about the importance of this residue in the func- four determinations. **P < 0.01, ***P < 0.001, compared to the wild- tion of organic cation transport in hOCTs. Further study type by Student’s t -test. is necessary to clarify this structure–function relationship. In summary, we characterized the functional proper- the decreased expression levels of hOCT3 protein at the ties of the hOCT3 variants and found, for the first time, plasma membranes, but due to the decreased functional that three variants are associated with reduced transport activity of transporter per se. activity. This study suggests that the hOCT3 variants will Since membrane transporters and metabolic enzymes contribute to inter-individual variations in cationic drug are both involved in renal and hepatic clearance of drugs, disposition as well as certain disease processes such as alteration of drug transport activities in the tissues could hypertension and allergic and neuropsychiatric dis - have an important influence on pharmacokinetics of its eases. 266 T Sakata et al

controlWT T44M A116S T400I A439V G475S

hOCT3

Na,K-ATPase

merged

Fig. 2. Surface expression of hOCT3 wild-type and its variants at the plasma membrane of COS cells. Immunodetection with a specific antibody raised against the C terminus of OCT3 showed that the wild-type (WT) as well as the five mutant proteins (T44M, A116S, T400I, A439V, and G475S) are expressed at the plasma membrane detected by anti-Na,K-ATPase monoclonal antibody, in addition to the cytoplasm.

Acknowledgments 7 Martel F, Azevedo I. An update on the extraneuronal monoamine transporter (EMT): characteristics, distribution and regulation. The authors thank Akie Toki and Rie Kofuji for technical assis- Curr Drug Metab. 2003;4:313–318. tance. This work was supported in part by grants from the Japan Soci- 8 Hasannejad H, Takeda M, Narikawa S, Huang XL, Enomoto A, ety for the Promotion of Science (JSPS KAKENHI 21390073 and Taki K, et al. Human organic cation transporter 3 mediates the 21659216 to N.A. and 21790819 to T.K.), Research on Health Sci- transport of antiarrhythmic drugs. Eur J Pharmacol. 2004;19: ences focusing on Drug Innovation from the Japan Health Sciences 45–51. Foundation, Mutual Aid Corporation for Private Schools of Japan, the 9 Ishii T, Lemas MV, Takeyasu K. Na+ -, ouabain-, Ca2+ -, and Nakatomi Foundation, the Salt Science Research Foundation (No. thapsigargin-sensitive ATPase activity expressed in chimeras be- 0721), the Japan Foundation of Applied Enzymology, Astellas Foun- tween the calcium and the sodium pump alpha subunits. Proc dation for Research on Metabolic Disorders, and Gout Research Natl Acad Sci U S A. 1994;91:6103–6107. Foundation of Japan. 10 Zwart R, Verhaagh S, Buitelaar M, Popp-Snijders C, Barlow DP. Impaired activity of the extraneuronal monoamine transporter References system known as uptake-2 in Orct3/Slc22a3-deficient mice. Mol Cell Biol. 2001;21:4188–4196. 1 Ishikawa T, Tsuji A, Inui K, Sai Y, Anzai N, Wada M, et al. The 11 Aoyama N, Takahashi N, Kitaichi K, Ishihara R, Saito S, Maeno genetic polymorphism of drug transporters: functional analysis N, et al. Association between gene polymorphisms of SLC22A3 approaches. Pharmacogenomics. 2004;5:67–99. and methamphetamine use disorder. Alcohol Clin Exp Res. 2006; 2 Burckhardt G, Wolff NA. Structure of renal organic anion and 30:1644–1649. cation transporters. Am J Physiol Renal Physiol. 2000;278: 12 Kitaichi K, Fukuda M, Nakayama H, Aoyama N, Ito Y, Fujimoto F853–F866. Y, et al. Behavioral changes following antisense oligonucleotide- 3 Kerb R, Brinkmann U, Chatskaia N, Gorbunov D, Gorboulev V, induced reduction of organic cation transporter-3 in mice. Neuro- Mornhinweg E, et al. Identification of genetic variations of the sci Lett. 2005;382:195–200. human organic cation transporter hOCT1 and their functional 13 Ogasawara M, Yamauchi K, Satoh Y, Yamaji R, Inui K, Jonker consequences. Pharmacogenetics. 2002;12:591–595. JW, et al. Recent advances in molecular pharmacology of the 4 Sakata T, Anzai N, Shin HJ, Noshiro R, Hirata T, Yokoyama H, histamine systems: organic cation transporters as a histamine et al. Novel single nucleotide polymorphisms of organic cation transporter and histamine metabolism. J Pharmacol Sci. 2006; transporter 1 (SLC22A1) affecting transport functions. Biochem 101:24–30. Biophys Res Commun. 2004;313:789–793. 14 Schneider E, Machavoine F, Pléau JM, Bertron AF, Thurmond 5 Song IS, Shin HJ, Shin JG. Genetic variants of organic cation RL, Ohtsu H, et al. Organic cation transporter 3 modulates mu- transporter 2 (OCT2) significantly reduce metformin uptake in rine basophil functions by controlling intracellular histamine oocytes. Xenobiotica. 2008;38:1252–1262. levels. J Exp Med. 2005;202:387–393. 6 Lazar A, Gründemann D, Berkels R, Taubert D, Zimmermann T, 15 Vialou V, Amphoux A, Zwart R, Giros B, Gautron S. Organic Schömig E. Genetic variability of the extraneuronal monoamine cation transporter 3 (Slc22a3) is implicated in salt-intake regula- transporter EMT (SLC22A3). J Hum Genet. 2003;48:226–230. tion. J Neurosci. 2004;24:2846–2851.