ION CHANNELS, RECEPTORS AND TRANSPORTERS

The organic cation transporter 3 (OCT3) as molecular targetof psychotropic drugs: transport characteristics and acuteregulation of cloned murine OCT3

Vivian Massmann & Bayram Edemir & Eberhard Schlatter & Rouvier Al-Monajjed &

Saliha Harrach & Philipp Klassen & Svenja K. Holle & Aleksandra Sindic &

Marina Dobrivojevic & Hermann Pavenstädt & Giuliano Ciarimboli

Received: 4 July 2013 /Revised: 5 August 2013 /Accepted: 9 August 2013 /Published online: 28 August 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract The organic cation transporter 3 (OCT3) is a widelyexpressed transporter for endogenous and exogenous organiccations. Of particular interest is OCT3 expression and func-tion in the brain, where it plays a role in serotonin clearanceand influences mood and behavior. Protein kinase signalingmediates rapid modulation of cerebral processes, but little isknown about acute regulation of OCT3 by protein kinases.Therefore, we cloned mouse OCT3 (mOCT3) and generated ahuman embryonic kidney cell line stably expressing the trans-porter to study transport characteristics, acute regulation byprotein kinases, and interaction with psychotropic drugs. Up-take measurement was performed using the fluorescentcation 4-(4-(dimethylamino)styryl)-N -methylpyridiniumiodide (ASP+, 1 μM) as a substrate. The translationalvalue of these findings was determined by comparingresults obtained with cloned mouse and human OCT3.mOCT3-mediated transport is membrane potential de-pendent and pH independent. ASP+ uptake by mOCT3and human OCT3 (hOCT3) was efficiently inhibited by 1-methyl-4-phenylpyridinium, tetrapentylammonium (TPA+),corticosterone, serotonin, and histamine and by the drugs

ketamine, fluoxetine, and diazepam. The half maximal inhib-itory concentrations of mOCT3 and hOCT3 for TPA+, sero-tonin, diazepam, and ketamine are significantly different.Diazepam is a non-transported inhibitor. Furthermore, theactivities of mOCT3 and hOCT3 are acutely regulated bythe p56lck tyrosine kinase by decreasing their Vmax. Studieswith freshly isolated renal proximal tubules frommOCT1/2−/−

mice, in which mOCT3 is the only OCT present, confirmedthis regulation pathway. Only the activity of hOCT3 is regu-lated by calmodulin. These findings suggest that even thoughmany transport properties of mOCT3 and hOCT3 are similar,there are also species-specific aspects of OCT3 function.

Keywords Organic cation transport . Regulation . Serotoninclearance . Fluoxetine . Diazepam

Abbreviations

ASP+ 4-(4-(Dimethylamino)styryl)-N-methylpyridiniumiodide

DMEM Dulbecco's modified Eagle's medium5-HTT Serotonin reuptake transporterMEM Modified Eagle's mediumMPP+ 1-Methyl-4-phenylpyridiniumOCT Organic cation transporterTEA+ TetraethylammoniumTPA+ Tetrapentylammonium

Introduction

The organic cation transporter 3 (OCT3) is a widely expressedpolyspecific transporter for organic cations, which acceptssubstrates of endogenous (such as serotonin [28], histamine,

V. Massmann :B. Edemir : E. Schlatter (*) :R. Al-Monajjed :S. Harrach : P. Klassen : S. K. Holle :H. Pavenstädt :G. CiarimboliExperimental Nephrology, Department of Internal Medicine D,University of Münster, Albert-Schweitzer-Campus 1 (A14),48149 Münster, Germanye-mail: eberhard.schlatter@uni-muenster.de

A. SindicDepartment of Physiology, Croatian Institute for Brain Research,University of Zagreb, Zagreb, Croatia

M. DobrivojevicDepartment of Histology and Embryology, Croatian Institute forBrain Research, University of Zagreb, Zagreb, Croatia

Pflugers Arch - Eur J Physiol (2014) 466:517–527DOI 10.1007/s00424-013-1335-8

dopamine, and epinephrine [3]) and of exogenous (such as N-methylpyridinium [43] and metformin [35]) origin. Togetherwith its paralogs OCT1 and OCT2, this transporter belongs tothe solute carrier family 22 (SLC22). OCTs are characterizedby a specific organ and species-dependent expression andmediate the transport of organic cations in an electrogenicand Na+-independent manner. In opposite to the specific ex-pression of mouse OCT1 (mOCT1)/human OCT1 (hOCT1)and mOCT2/hOCT2 to the liver and kidney, respectively,OCT3 has a broad tissue distribution, and it is expressed inthe placenta, ovary, uterus, prostate, kidney, bronchial epithe-lium, liver, heart, adrenal gland, aorta, and brain (for a review,see [29]). In mice and rats, the cerebral OCT3 expression ishigh in the hippocampus and even denser in the pyramidal andgranule cell layer [3, 4, 40]. Other regions of OCT3 expressionin the brain are circumventricular organs, dorsomedial hypo-thalamus, frontal cortex, thalamic nuclei, and substantia nigra[16, 40, 41], where it shows both a neuronal [40, 41] as well asan extraneuronal expression [25, 41]. In the brain, OCT3seems to play an important role in the regulation of neuro-transmission by having an effect on the neurotransmitterclearance in the synaptic cleft, where it represents the so-called uptake 2 transport system [4, 13]. Indeed, experimentsin animals showed that its blockade robustly increases extra-cellular serotonin concentration in the medial hypothalamusand hippocampus [4].

Studies in mice, where OCT3 was genetically deleted(mOCT3−/−), showed an important role of mOCT3 in deter-mining behavioral characteristics, even though the results arecontradictory. In one study, these mice displayed an increasedsensitivity to psychostimulants and an increased level of anx-iety [41]. On the other hand, there are also data showing thatantisense knockdown of OCT3 in mice led to a decreasedimmobility time in the forced swimming test [27] and thatmOCT3−/− mice were significantly more active and less anx-ious compared to wild-type animals [45]. The reason for thisdiscrepancy is not known. Recently, it was shown that ratOCT3-mediated uptake of the fluorescent organic cation4-(4-(dimethylamino)styryl)-N -methylpyridinium iodide(ASP+) could be inhibited by antidepressant drugs such asdesipramine, sertraline, paroxetine, amitriptyline, imipramine,and fluoxetine [49], suggesting that at least a part of theirantidepressant effects may be mediated by an interaction withOCT3. Indeed, translational studies in mice with complete(serotonin reuptake transporter (5-HTT)−/−) or partial (5-HTT+/−) genetic deletion of the 5-HTT, a gene whose poly-morphism in the promoter region is associated with an in-creased risk for psychiatric diseases like major depression,alcoholism, and anxiety [32], have shown that the OCT3mRNA and protein expression was upregulated in the hippo-campus compared to wild-type controls, supposedly as acompensation mechanism [4]. As the OCT blocker decynium22 exerts antidepressive-like effects in both 5-HTT+/− and 5-

HTT−/− mice [4], identification of further OCT3 inhibitorsmight be of interest for the future treatment of mood disorders.Therefore, we cloned mOCT3 from mouse kidney and stablyexpressed it in the human embryonic kidney 293 (HEK293)cells. The transport mediated by cloned mOCT3 was charac-terized with respect to its interaction with known OCT sub-strates and its acute regulation by several protein kinases.Protein kinases are important rapid modulators of brain func-tion, and changes in their functions have been associated withpathological conditions such as Alzheimer's disease [8]. Thephysiological importance of such an acute regulation wasconfirmed using freshly isolated kidney proximal tubulesfrom double OCT1 and OCT2 knockout mice, where OCT3is the only OCT subtype expressed. Moreover, the interactionof selective serotonin reuptake inhibitors (SSRI), anxiolyticdrugs, and other psychoactive substances like fluoxetine, di-azepam, and ketamine, withmOCT3was also investigated. Toexplore the translational meaning of these findings, theseexperiments were also performed in HEK293 cells stablytransfected with hOCT3.

Materials and methods

Cloning of mOCT3, stable transfection, and culture oftransfected cells The coding region of mOCT3 (acc. no.NM_011395, 381–2,036 bp) was amplified by PCR frommouse kidney cDNA using primers with extension for restric-tion sites at the 5′ ends. An EcoRI was introduced with theforward, and Xho I restriction site, with the reverse primer. Theamplicon and the vector pcDNA3.1 were restricted usingEcoRI and XhoI, and the DNA fragments were gel elutedand ligated overnight. The vector was transformed intoEscherichia coli Dh5-β-competent bacteria for plasmid am-plification. The plasmid preparation was performed using theInvitrogen Zyppy™ PlasmidMiniprep Kit (Invitrogen, Darm-stadt, Germany). After validation of the correct sequences, theplasmid was transfected into HEK293 cells (CRL-1573;American Type Culture Collection, Rockville, MD) usingLipofectamine (Invitrogen). Cells were cultured in DMEM(Biochrom, Berlin, Germany) containing 3.7 g/l NaHCO3,1.0 g/l D-glucose, 2.0 mML-glutamine, 100 U/l penicillin,100 mg/l streptomycin (Biochrom), and 10 % of fetal bovineserum at 37 °C in an atmosphere of 8 % CO2. For the selectionof transfected cells, 24–48 h after transfection, 0.8 mg/mlGeneticin (PAA Laboratories, Coelbe, Germany) wasadded to the media. As control, HEK293 cells weretransfected with an empty pcDNA3.1 vector. The cells werediluted, and single clones were picked to generate a stableHEK293–mOCT3 clone. The cells stably transfected withhOCT3 were a generous gift of Prof. Koepsell [31]. Fortransport measurements, cells from passages 15–80 wereseeded onto 96-well microplates (Nunclon 96 Flat Bottom;

518 Pflugers Arch - Eur J Physiol (2014) 466:517–527

Nunc, Wiesbaden, Germany) and grown until confluencewas achieved (after 2–3 days). Right before starting mea-surements, the cell culture medium was replaced byHCO3

−-free Ringer-like solution containing the following(in millimolar): NaCl, 145; K2HPO4, 1.6; KH2PO4, 0.4; D -glucose, 5; MgCl2, 1; and calcium gluconate 1.3, with pHadjusted to 7.4 at 37 °C. Each set of experiments wasperformed on the same day with cells of the same age andpassage.

Animals Male OCT1/2-deficient (OCT1/2−/−) mice [26] wereprovided by A.H. Schinkel (The Netherlands Cancer Institute,Amsterdam, The Netherlands). Mice were kept under temper-ature, light, and humidity control and had free access to waterand standard mice chow (Sniff, Soest, Germany). Experi-ments were approved by a governmental committee on animalwelfare and were performed in accordance with national an-imal protection laws. Proximal tubules (S2 and S3 segments)from male mice were mechanically isolated as customary inour laboratory [21, 24] in MEM-Earle medium (Biochrom)and transferred to the HCO3

−-free Ringer-like control solu-tion. S2 and S3 segments were used, as these are easier toisolate mechanically in larger numbers with less mechanicaldamage during the isolation process. Mostly, two experi-menters isolated between 300 and 600 tubule segments fromone or two kidneys of a mouse within 2 h. Three segments ofproximal tubules each were transferred in a well of a 384-wellmicrotiter plate (Deep Well Small Volume; Greiner,Frickenhausen, Germany) with 10 μl of control solution. Thisallows obtaining 100 to 300 separate data points from oneanimal. Plates were mildly centrifuged to ensure settling ofsegments at the flat well bottom and to remove air bubbles.

Uptake measurements with cloned mOCT3, hOCT3, andisolatedmouse proximal tubules The fluorescent organic cationASP+was used in a 1-μMconcentration as substrate for OCTs ascustomary in our laboratory [9, 11, 42]. Microfluometric detec-tion of ASP+ uptake was accomplished using a fluorescenceplate reader (Infinity M200; Tecan, Crailsheim, Germany)equipped with a monochromator system (excitation at 450 nm,emission at 590 nm) as described for cells stably transfected withOCT [42] and for mouse proximal tubules [21, 24]. Emissionchanges between ASP+ in solution and after transport across thecell membrane allow monitoring of cellular ASP+ accumulation[42]. Figure 1 shows recordings of emission measured in a wellcontaining empty vector or mOCT3 stably transfected HEK293cells in the presence or not of the organic cation 1-methyl-4-phenylpyridinium (MPP+, 1 mM).

Dynamics of OC transport were measured at 37 °C as theinitial (~100 s) rate of fluorescence increase [42]. Emissionfrom the complete area of the well bottom was analyzed ninetimes (96 wells) or four times (384 wells) and averaged foreach well. Slopes of fluorescence changes were linearly fitted

and used as ASP+ uptake parameter. ASP+ was injected intothe well after three fluorescence measurements. The increasein fluorescence after the third value in Fig. 1 is due to ASP+

addition to the well. When investigating regulation pathways,a 10-min incubation step with the respective agonist or inhib-itor preceeded the addition of ASP+. In some experiments, theinfluence of membrane voltage on the ASP+ uptake bymOCT3 was investigated by substituting Na+ by K+ graduallyincreasing the extracellular K+ concentration right beforemeasurement to 18.6, 88.6, or 145 mM, which resulted in adepolarization of 10, 30, and 40 mV, respectively. Membranevoltages of HEK293 cells in the presence of increasing K+

concentrations were measured by slow whole-cell patch-clamp experiments as described before [19]. Briefly, cover-slips with HEK293 cells were mounted at the bottom of aperfusion chamber, which was placed on the stage of aninverted microscope (Axiovert 10; Zeiss, Goettingen, Germa-ny). HCO3

−-free Ringer-like solution was used for experi-ments with pH adjusted to 7.4 (see above). The filling solutionfor patch-clamp pipettes contained the following (in millimo-lar): potassium gluconate, 95; KCl, 30; Na2HPO4, 4.8;NaH2PO4, 1.2; D-glucose, 5; calcium gluconate, 0.73; EGTA,1; MgCl2, 1.03; and ATP 1, with pH adjusted to 7.2. To allowelectrical access to the cell cytosol, 160 μM nystatin wasadded to the pipette solution. The membrane potential wasmeasured with a patch-clamp amplifier (U. Fröbe;Physiologisches Institut, Universität Freiburg, Germany) andrecorded continuously by a pen chart recorder (WeKa graphWK-250R; WKK, Kaltbrunn, Switzerland). To investigatewhether the observed effects are Na+ dependent, in someexperiments Na+ was replaced by N -methyl-D-glucamine(NMDG).

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Fig. 1 ASP+ uptake by mOCT3-transfected HEK293 cells in the pres-ence (open circles) or absence (closed circles) of 1 mMMPP+ comparedto that observed in cells transfected with the empty vector (triangles).Increase of fluorescence intensity after the addition of 1 μM ASP+ withtime is shown as arbitrary units (a.u.). ASP+ was injected into the wellafter three fluorescence measurements. The step after the third value isdue to ASP+ addition to the solution

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Detection of cellular diazepam and serotonin accumulation Formeasurement of cellular diazepam uptake, cells were incubat-ed for 5 min with 100 μM diazepam in Ringer solution at37 °C in the presence or absence of 20μMMPP+ as an OCT3-specific competitive substrate.

After incubation, cells were washed with ice-cold Ringersolution, and hypo-osmotic lysis was induced with 0.1 %formic acid. For diazepam quantification, a high-pressureliquid chromatography (HPLC) method was used. The mobilephase consisted of acetonitrile (A) and 0.1 % formic acid (B)and was delivered at 0.3 ml/min in a gradient program. Alinear gradient from 20 % A and 80 % B to 80 % A and 20 %B was applied within 8 min. Thereafter, elution with 100 % Awas applied for 2 min, and then, the column was re-equilibrated with 20 % A with 80 % B for at least 3 min.The chromatographic system consisted of the Accela 600 witha UV detector (Thermo Fisher, Dreieich, Germany) set to254 nm for detection. Separation was performed on theAccucore C18 (100 mm×2.1 mm, 2.6 μm) equipped with aC18 guard column (10 mm×2.1 mm, 5 μm, Drop-In GuardCartridges) at room temperature. Instruments were piloted,and data were analyzed using the ChromQuest 5.0 software(all Thermo Fisher).

Cellular accumulation of serotonin was measured byexploiting serotonin intrinsic fluorescence [7]. Briefly,OCT3- and mock-transfected cells were incubated for10 min with 100 μM serotonin in Ringer-like solution at37 °C in the presence or absence of 1 to 100 μM ASP+ orcorticosterone as an OCT3-specific competitive substrate.After incubation, cells were washed with ice-cold Ringer-likesolution, and hypo-osmotic lysis was induced with water.Serotonin fluorescence emission was measured at 329 nmafter excitation at 280 nm.

Chemicals ASP+ was purchased from Molecular Probes(Invitrogen). Tetraethylammonium (TEA+), tetrapentylammonium(TPA+), MPP+, cimetidine, corticosterone, serotonin, fluoxetine,histamine, creatinine, diazepam, and forskolin were obtained fromSigma (Munich, Germany). Calmidazolium, aminogenistein, andwortmannin were purchased from Calbiochem (Bad Soden,Germany). Ketamine hydrochloride was purchased from CEVA(Düsseldorf, Germany).

All other substances and standard chemicals wereobtained from Sigma or Merck (Darmstadt, Germany).Compounds were dissolved in HCO3

−-free Ringer-likesolution and, if necessary, with ethanol or dimethyl sulf-oxide as a solvent. The final concentration of thesesolvents did not affect the results of the experiments(data not shown).

Statistical analysis Data are presented as mean values ± stan-dard error of the mean (SEM), with n referring to the numberof wells.Km, Vmax, and half maximal inhibitory concentration

(IC50) values were obtained by sigmoidal concentration–re-sponse curve fitting, and the best fit values of Km and IC50

were compared using the GraphPad Prism, version 5.3(GraphPad Software, Inc., San Diego, USA). Unpaired t testand ANOVA analysis with Tukey–Kramer multiple compar-isons test were used as appropriate to prove a statisticalsignificance of the effects. A P value of <0.05 was consideredstatistically significant.

Results

Characterization of ASP+ uptake by cloned mOCT3and hOCT3

The mouse kidney OCT3 cDNA is 3,391 bp long with anopen reading frame of 1,656 bp (including the terminationcodon), encoding a protein of 551 amino acids.

As shown in Fig. 1, mOCT3-transfected HEK cells wereable to take up ASP+, in contrast to empty vector-transfectedcells. This uptake was completely inhibited in the presence of1mMMPP+.ThesamewasobservedwithhOCT3(notshown).The uptake of ASP+ by mOCT3 or hOCT3 increased in aconcentration-dependent manner (Fig. 2). When subtractingthe unspecific ASP+ transport determined in the presence of1 mM TPA+ from the total ASP+ uptake, a specific saturationcurvewas obtained, andKm values could be determined (Km=1.8 and 1.1 μM for mOCT3 and hOCT3, respectively). TheVmax read from these curves cannot bedirectly compared, sincethey are given in arbitrary units, which depend on severalfactors such as transfection efficiency, fluorescent lamp perfor-mance, and signal amplification of the instrument.

As shown in Fig. 3a, the transport of ASP+ by mOCT3 isnot pH dependent, since the cells did not show a difference inthe ASP+ uptake after changing the pH in the medium to 6.4 or8.4. Depolarization of the cells by increasing the K+ concen-tration in the medium led to a concentration-dependent inhi-bition of transporter function, as shown by the decreaseduptake of ASP+ (Fig. 3b). Substitution of Na+ by NMDGhad no effect on the ASP+ transport by mOCT3, suggestingthat it is not Na+ dependent. These data show that mOCT3 is apotential-sensitive organic cation transporter and not an or-ganic cation H+ antiporter, as suggested in one report formOCT1 [18].

Comparison of transport inhibition of mOCT3 and hOCT3by typical organic cations

Substrate specificities of mOCT3 and hOCT3 transport inHEK293 cells were studied by inhibiting the ASP+ uptake byTEA+, TPA+,MPP+, and cimetidine, organic cations known tointeract with other OCT (for review, see [28]). TPA+, MPP+,

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and cimetidine inhibited ASP+ uptake with IC50 values of 73,110, and 140 μM for mOCT3 (Fig. 4a) and 28, 145, and193 μM for hOCT3, respectively (Fig. 4b). The IC50 forTPA+ was significantly different between the human and mu-rine transporter. Both mOCT3 and hOCT3 showed a lowapparent affinity for TEA+, making an exact determination ofIC50 difficult (IC50≈8 and 4 mM, respectively, Fig. 4a, b). Theendogenous organic cation creatinine showed no interactionwith ASP+ transport mediated by OCT3 (not shown).

OCT3-mediated ASP+ transport is inhibitedby neurotransmitters and substances known to interactwith neurotransmitter signaling pathways

ASP+ uptake by mOCT3 and hOCT3 was inhibited with lowapparent affinities by the neurotransmitters serotonin and his-tamine with IC50 values of 1.8 and 1.4 mM for mOCT3(Fig. 5a) and 4.2 and 1.1 mM for hOCT3, respectively(Fig. 5b). Since in an animal stress model, a correlationbetween serotonin and corticosterone concentrations in thebrain has been observed [15, 16], the interaction of cortico-sterone with OCT3 has also been studied. Corticosteroneshowed a high apparent affinity for both mOCT3 and hOCT3(IC50 values 3.9 and 3.3 μM, respectively, Fig. 5a, b). Drugswith different effects on synaptic processes such as fluoxetine,diazepam, and ketamine also showed a significant interactionwith the ASP+ uptake mediated by mOCT3 or hOCT3. Diaz-epam had a high apparent affinity for both mOCT3 andhOCT3 (IC50 values of 40 μM for mOCT3 and 2 μM forhOCT3, Fig. 6), while ketamine and fluoxetine had an inter-mediate affinity (IC50 values of 200 and 474 μM for mOCT3and 440 and 274 μM for hOCT3, respectively, Fig. 6). TheIC50 values for serotonin, diazepam, and ketamine were

significantly different between the human and murine trans-porters. Even though diazepam was able to inhibit ASP+

transport by mOCT3 and hOCT3 with high apparent affinity,it was not transported into the cell by the transporter as shownin experiments, where the uptake of diazepam in emptyvector-, mOCT3-, and hOCT3-transfected cells was quanti-fied by HPLC determination (Fig. 7). Diazepam accumulation

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Fig. 2 Specific ASP+ uptake of mOCT3-transfected (closed circles) andhOCT3-transfected (open circles) HEK293 cells was defined by addingrising concentrations of ASP+ to the cells and detecting the fluorescenceintensity. The specific uptake was detected by subtracting the total uptakefrom the unspecific uptake determined in the presence of 1 mM TPA+

(n =at least 8 for all data points)

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Fig. 3 a ASP+ uptake by mOCT3-transfected HEK293 cells in depen-dence on extracellular pH as percentage of what was observed at pH 7.4(100±2 %, n =55). The medium was changed to a more acidic (6.4) orbasic (8.4) pH during the uptake measurements (ASP+ uptake, 95±2 %,n =55, and 94±3 %, n =55, in percentage of control, respectively). Thiswas done by titrating the solution with HCL or NaOH. b Membranepotential dependence of ASP+ uptake by mOCT3 is shown in the per-centage of the uptake in Ringer-like buffer with a K+ concentration of3.6 mM (100±2 %, n=72; mean membrane potential of −63±3 mV, n =21). Different extents ofmembrane depolarization (10±3mV, n =21; 30±6 mV, n =7; and 40±3 mV, n =7, in parentheses on the x-axis) wasachieved by increasing K+ concentration to 18.8, 88.8, and 145 mM,respectively, causing changes in ASP+ uptake to 85±4 %, n =40; 63±4 %, n=32; and 62±1 %, n =32, in percentage of control, respectively.The black column shows the effect on ASP+ uptake observed when Na+

was replaced by NMDG (95±2 %, n =32) and no changes in the K+

concentration, which hyperpolarized cells by 8±3 mV, n =7 mV (−8).Above the columns, the number of experiments is indicated. Asteriskshows a significant difference compared to controls (ANOVA)

Pflugers Arch - Eur J Physiol (2014) 466:517–527 521

in cells was neither changed by low temperature nor by addingthe specific OCT inhibitor TPA+ (0.1 mM) (not shown).Conversely, compared to mock cells, OCT3-transfected cellsshowed a significant cellular accumulation of serotonin(Fig. 7), which could be inhibited by ASP+ or corticosterone(not shown).

Table 1 shows a summary of the Km (only for ASP+) andIC50 values determined for the ASP+ uptake by mOCT3 andhOCT3.

Acute regulation of OCT3

Acute regulation of OC transport by various protein kinaseshas been demonstrated for isolated human, rat, and mouseproximal tubules [21, 24, 37] and for various rat and humanOCT orthologs and paralogs [10]. Here, we studied for thefirst time the acute regulation of mOCT3 and hOCT3 by

several kinases. Activation of protein kinase A (PKA) byforskolin (1 μM) and of PKC by DOG (1 μM) had no effecton ASP+ uptake mediated by mOCT3 (+3±4 %, n =32, and +5±2 %, n =24, respectively) and hOCT3 (−2±1 %, n =43, and−7±1 %, n =67, respectively) (Fig. 8). Inhibition of thephosphatidylinositol-3-kinase (PI3 kinase) by wortmannin(0.1 μM) had also no effect on mOCT3 and hOCT3 activity(+0±2%, n =32, and +1±2%, n =35, respectively). Converse-ly, inhibition of Ca2+/calmodulin by calmidazolium (5 μM)had no effect on ASP+ uptake by mOCT3, but significantlydownregulated the transport mediated by hOCT3 (−4±2 %,n =32, and −23±1 %, n =48, respectively) (Fig. 8). Inhibitionof p56lck tyrosine kinase by aminogenistein (5 μM) signifi-cantly reduced ASP+ uptake by mOCT3 (−22±2 %, n =32)and also by hOCT3 (−25±1 %, n =56), suggesting endoge-nous activity of this kinase.

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Fig. 4 Concentration–response curves for the inhibition of initial ASP+

uptake mediated by mOCT3 (a) and hOCT3 (b) by the organic cationsTPA+, MPP+, cimetidine, and TEA+. Values are means ± SEM expressedas percentage of ASP+ uptake in the absence of the inhibitor

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522 Pflugers Arch - Eur J Physiol (2014) 466:517–527

Ex vivo regulation of mOCT3

In order to confirm the importance of mOCT3 regulationin a more physiological system, we have investigatedthese pathways in freshly isolated proximal tubules fromOCT1/2−/− mice, which express only the mOCT3 on thebasolateral membrane of the tubules. The results of theseexperiments confirmed what was found in the in vitrosystem; the ASP+ uptake was significantly decreased byinhibition of p56lck tyrosine kinase with aminogenistein(−29±6 %, n =38, Fig. 8). The activation of PKA byforskolin, the inhibition of the PI3 kinase by wortmannin,and of Ca2+/calmodulin by calmidazolium had no signif-icant effect on ASP+ uptake mediated by mOCT3 in thefreshly isolated tubules (+11±7 %, n =39, for forskolin;

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uptakemediated bymOCT3 (a) and hOCT3 (b) by diazepam, fluoxetine,and ketamine. Values are means ± SEM expressed as percentage of ASP+

uptake in the absence of the inhibitor

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Fig. 7 Uptake of diazepam in HEK293 cells stably transfected with theempty vector (EV, white , n=8), mOCT3 (light gray, n =8), or hOCT3(dark gray, n=8) after 5 min of incubation with 100 μM diazepam. Theconcentration of diazepam per gram protein in cell lysates is shown (mean± SEM). The mOCT3 cell line was also incubated with diazepam solu-tions containing the OCT inhibitor TEA+ (0.1 mM) (not shown). Nosignificant differences could be detected. On the right side of the graph ,the uptake of serotonin into empty vector-transfected (n =4) and mOCT3-transfected (n =10) cells is shown

Table 1 Km±SEM (only for ASP+) and IC50 (both given in micromolar)values determined for the ASP+ uptake by mOCT3 and hOCT3

Substances Km ± SEM or IC50 (log IC50±SEM) in μM andnumber of observations (n)

mOCT3 hOCT3

ASP+ 1.8±0.3 1.1±0.1

n =7–8 n =17–61

TEA+ ≈8,000 (−2.11±0.08) ≈4,000 (−2.38±0.00)

n =8–40 n =6–28

TPA+ 73 (−4.14±0.04) 28* (−4.56±0.05)

n =8–16 n =6–30

MPP+ 110 (−3.97±0.04) 145 (−3.84±0.05)

n =8–50 n =6–30

Cimetidine 140 (−3.87±0.07) 193 (−3.71±0.04)

n =15–76 n =6–44

Serotonin 1,800 (−2.76±0.06) 4,200* (−2.38±0.03)

n =8–39 n =8–16

Histamine 1,400 (−2.84±0.05) 1,100 (−2.95±0.02)

n =16–24 n =8–48

Corticosterone 3.9 (−5.41±0.04) 3.3 (−5.48±0.04)

n =8–32 n =6–44

Diazepam 40 (−4.41±0.12) 2* (−5.59±0.03)

n =8–67 n =8–24

Fluoxetine 50 (−3.32±0.28) 27 (−3.56±0.18)

n =8–32 n =8–72

Ketamine 200 (−3.69±0.08) 440* (−3.36±0.04)

n =8–48 n =8–32

* Statistically significant difference from mOCT3

Pflugers Arch - Eur J Physiol (2014) 466:517–527 523

−3±6 %, n =46, for wortmannin; and −2±7 %, n =42, forcalmidazolium, Fig. 8). Similar regulation experimentswith wortmannin and aminogenistein in the isolated tubulepreparation were already performed in the study of Holle et al.[24]. In this work, we have increased the number of observa-tions. For this reason, the downregulation already observedunder incubation with wortmannin in the literature [24] be-came significant here.

Mechanisms of mOCT3 and hOCT3 regulation by p56lck

tyrosine kinase and calmodulin

The regulation of mOCT3 by p56lck tyrosine kinase wasassociated with a significant Vmax decrease, while the Km

remained unchanged (Fig. 9a) (Vmax=22.8±0.8 arbitrary units(a.u.) andKm=3.8±0.4μM, n =6–11, for control experiments;Vmax=15.4±0.7 a.u. and Km=2.6±0.4 μM, n =6–11, for ex-periments under p56lck inhibition, Table 2). Both the regula-tions of hOCT3 by calmodulin and by p56lck tyrosine kinasewere associated with a significant Vmax decrease of the trans-porter, while the Km remained unchanged only under calmod-ulin inhibition (Vmax=21.4±0.6 a.u. and Km=4.4±0.4 μM,n =57–65, for control experiments; Vmax=15.6±0.6 a.u. andKm=4.2±0.4 μM, n =8–32, for experiments with calmodulininhibition; and Vmax=18.5±0.7 a.u. and Km=6.3±0.7 μM,n =18–24, for experiments under p56lck inhibition, Fig. 9b,Table 2). These results suggest a possible involvement oftrafficking processes in the regulation pathways in mOCT3as well as in hOCT3. The p56lck tyrosine kinase seems toinfluence also the affinity of hOCT3 for ASP+.

Discussion

Transport mediated by the cloned mOCT3 is membrane po-tential dependent and pH and Na+ independent, as alreadydescribed for other OCT subtypes [17, 20]. Both mOCT3 andhOCT3 transport ASP+ with similar affinities and also interactwith some organic cations with remarkable similarities. How-ever, the apparent affinities of these transporters for TPA+,serotonin, diazepam, and ketamine are significantly different,underlining that the results obtained in the mouse cannotbe completely translated to humans. Interestingly, TEA+, aknown substrate for other OCT subtypes, is here con-firmed to have a low apparent affinity both for mOCT3and hOCT3.

The regulation of mOCT3 observed in the in vitro systemappears to be the same in the ex vivo setup. It is known thatorganic cation transporters are acutely regulated via differentkinases [10, 33]. This is also the case for OCT-mediatedtransport in the mouse proximal tubule [21, 24]. Both mOCT3and hOCT3 are stimulated by the p56lck tyrosine kinase. Forboth transporters, these effects are mediated by Vmax changesof ASP+ transport, suggesting a modulation of transportertrafficking to/from the plasma membrane. The physiologicalrelevance of these findings was confirmed by studies withfreshly isolated proximal tubules of OCT1/2−/− mice, wheremOCT3 is the only OCT expressed on the basolateral mem-brane. Here, the p56lck tyrosine kinase regulates ASP+ trans-port in the same fashion as in in vitro experiments. This resultis of importance, considering that this enzyme is expressed inthe kidney [47] and also considering the role played by OCT3in the brain. Studies by Omri et al. [36] showed that p56lck

tyrosine kinase activity is present in the neurons of mousebrain, where it is especially expressed in the granule cells ofthe dentate gyrus and in the pyramidal layer of the Ammon'shorn in the hippocampus. These are exactly those regionswhere OCT3 is expressed at high density [3, 4, 38, 40, 44],leading to the assumption that the regulation of OCT3 by thetyrosine kinase activity of p56lck may be involved in modu-lating the functions of OCT3 in the brain.

Besides p56lck, also calmodulin regulates the transportactivity of the human OCT3, but not in mOCT3, which is,again, probably linked to a modulation of transporter traffick-ing to/from the plasma membrane as already demonstrated forhOCT2 [5], but in contrast to what has been observed forrOCT2 [42], where this regulation pathway was associatedwith Km changes. The reasons for this difference are notknown; since calmodulin and OCT do not seem to directlyinteract, the different regulations may be attributed to specificcomponents of the calmodulin signaling pathway [42]. Qual-itative differences in the regulation pattern between differentOCT paralogs of a given species or between orthologs ofdifferent species have been reported before [10]. When com-pared to OCT1 and OCT2 of different species, acute kinase-

0

20

40

60

80

100

120

Aminogenistein CalmidazoliumWortmannin Forskolin

* * **

AS

P+ -u

pta

ke r

ate

(% o

f co

ntr

ol)

Fig. 8 Acute regulation (after 10min of incubation) of ASP+ transport bycloned mOCT3 (white), cloned hOCT3 (black), and in freshly isolatedproximal tubules of OCT1/2−/− mice (gray). The regulation was investi-gated by inhibiting the PI3 kinase with wortmannin (0.1 μM), the p56lck

tyrosine kinase with aminogenistein (10 μM for tubules and 5 μM forHEK293 cells), and calmodulin with calmidazolium (5 μM). Activationof the PKAwas tested with forskolin (1 μM). Initial uptake rates of ASP+

after incubation with these different effectors are presented in percentageof controls. Similar regulation experiments with wortmannin andaminogenistein in the isolated tubule preparation were already performedin the study of Holle et al. [24]. In this work, we have increased thenumber of observations. Values are means ± SEM with n =32–166

524 Pflugers Arch - Eur J Physiol (2014) 466:517–527

mediated regulation of mOCT3 or hOCT3 seems to be re-stricted to only a few pathways.

Human or rat OCT3 is known to be capable of transportingneurotransmitters including dopamine, norepinephrine, sero-tonin, histamine, and epinephrine [2]. In our experiments,histamine and serotonin appear to be a low apparent affinityinhibitor of the ASP+ uptake in mOCT3- and hOCT3-transfected cells. Nevertheless, OCT3 may play an importantrole in modulating neurotransmission or in inflammatory

reaction, since, locally, a high concentration of these sub-stances is achieved. For example, the serotonin concentrationin synapsis of the substantia nigra reticulata has been calcu-lated to be as high as 6 mM [6]. These findings can explain theobservation that in mice and rats in vivo, the blockade ofOCT3 robustly increases the extracellular serotonin concen-tration in the medial hypothalamus and in the hippocampus[15].

In this way, OCT3 may be a possible target of antidepres-sant therapy. Indeed, in this study, we show that the SSRIfluoxetine and the anxiolytic benzodiazepine diazepam inter-act with mOCT3- and hOCT3-mediated transport, suggestingthat a part of their antidepressive and anxiolytic actions,respectively, may be mediated by OCT3. In this context,ketamine is also an interesting psychoactive substance, andit is not only used in analgesia and anesthesia in emergencymedicine, for induction and maintenance of general anesthe-sia, but also in the treatment of bronchospasm, but it has beenshown to act as an antidepressant, particularly in patients withbipolar disorder [12, 14]. Ketamine is described as anoncompetitive NMDA receptor antagonist [22], but addi-tionally, a wide range of potential sites of action are described(e.g. on opioid receptors, muscarinergic receptors, and sigmareceptors [23, 34]). Descending monoaminergic pain path-ways are inhibited, as ketamine inhibits noradrenergic andserotonergic uptake. There is current work to test its useful-ness in pain therapy, depression, and in the treatment ofalcoholism [12, 30, 48]. In the present work, ketamine wasfound to inhibit both mOCT3 and hOCT3, confirming whathas already been demonstrated for rOCT3 and hOCT3 [3] andsuggesting that some effects are at least, in part, mediated byits interaction with OCT3. The inhibition of mOCT3 byketamine also plays an important role when mice are anesthe-tized with this drug, and metabolic studies including organiccations are performed.

It is known that ketamine and diazepam, according to ourobservation of a potent inhibitor of OCT3, may decrease the

0 5 10 15 200

5

10

15

20

+ aminogenistein

control

+ calmidazolium

ASP+[µM]

AS

P+ u

pta

ke r

ate

(a.u

.)

0 5 10 15 200

5

10

15

20a

b

+ aminogenistein

control

ASP+[µM]

AS

P+ u

pta

ke r

ate

(a.u

.)

Fig. 9 The specific ASP+ uptake of mOCT3-transfected (a) and hOCT3-transfected (b) HEK293 cells was defined by subtracting the unspecificfrom the total ASP+ uptake. For the control uptake, we calculated Km

values of 3.8 and 4.4 μM and Vmax values of 22.8 and 21.4 a.u. formOCT3 (n=6–11) and hOCT3 (n =57–65), respectively. Under inhibi-tion of p56lck with 5 μM aminogenistein, Km values of 2.6 and 6.3 μMand Vmax values of 15.4 and 18.5 a.u. for mOCT3 (n =6–11) and hOCT3(n =18–24), respectively, were calculated. Under the inhibition of cal-modulin with calmidazolium, the specific uptake of ASP+ by hOCT3showed a Km value of 4.2 μM and a Vmax value of 15.6 a.u. (n=8–32)

Table 2 Km and Vmax values determined for the ASP+ uptake bymOCT3 and hOCT3 under control conditions and under inhibition ofp56lck (mOCT3 and hOCT3) and calmodulin (only hOCT3) by 5 μMaminogenistein and 5 μM calmidazolium, respectively

Km (μM) Vmax (arbitrary units) n

Transporter mOCT3

Control 3.8±0.4 22.8±0.8 6–11

p56lck inhibition 2.6±0.4 15.4±0.7* 6–11

Transporter hOCT3

Control 4.4±0.4 21.4±0.6 57–65

p56lck inhibition 6.3±0.7* 18.5±0.7* 18–24

Calmodulin inhibition 4.2±0.4 15.6±0.6* 8–32

* Significant difference compared to controls, calculated by ANOVA

Pflugers Arch - Eur J Physiol (2014) 466:517–527 525

severity of ischemic cerebral injury [1, 39, 46]. Interestingly,transient focal ischemia in OCT3−/− mice led to a reducedinfarct volume compared to WT mice [50]. This suggests thatOCT3 may be the responsible molecule for clearance ofischemia-induced histamine in the brain, and targeted disrup-tion of OCT3 ameliorates ischemic brain damage through anincrease in regulatory T cells, as their concentration increasesin relation to histamine.

In conclusion, in this work, we have compared the trans-port characteristics of mouse and human OCT3 in vitro andex vivo to obtain information on its capability to transportpsychogenic drugs and to establish the translational relevanceof studies conducted in mice. Moreover, we showed that thetransporters can be acutely regulated in a similar but notidentical fashion. Such a regulation seems to be of physiolog-ical relevance, since it is also present in freshly isolated renaltubules from mice expressing only mOCT3. Our findingssuggest that OCT3 is an important component in the homeo-static regulation of serotonergic neurotransmission, indicatingthe OCT3 to become a novel molecular target to treat disor-ders related to monoaminergic neural systems.

Acknowledgments We thank Astrid Dirks for the excellent tech-nical assistance. This work was supported by the DeutscheForschungsgemeinschaft (DFG CI107/4-2 to G.C. and E.S.).

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