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Am J Physiol Cell Physiol 290: C1521-C1531, 2006. First published January 4, 2006; doi:10.1152/ajpcell.00622.2005
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Characterization of regulatory mechanisms and states of human organic cation transporter 2

Jürgen Biermann,1 Detlef Lang,1 Valentin Gorboulev,2 Hermann Koepsell,2 Aleksandra Sindic,1 Rita Schröter,1 Aurelija Zvirbliene,3 Hermann Pavenstädt,1 Eberhard Schlatter,1 and Giuliano Ciarimboli1

1Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Universitätsklinikum Münster, Münster, Germany; 2Institut für Anatomie und Zellbiologie, Universität Würzburg, Germany; and 3Institute of Biotechnology, Vilnius, Lithuania

Submitted 13 December 2005 ; accepted in final form 2 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37°C. After subtraction of unspecific uptake determined in WT at 37°C or in hOCT2 at 8°C saturable specific uptake of both substrates was measured. Km values of hOCT2-mediated uptake of 95 µM amiloride and 24 µM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC50 (in µM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca2+/CaM complex (–55 ± 5%, n = 10 and –63 ± 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (–54 ± 5%, n = 14, and –31 ± 6%, n = 26) and PKA (–16 ± 5%, n = 16, and –18 ± 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 ± 6%, n = 8, and +55 ± 17%, n = 16). Inhibition of Ca2+/CaM complex resulted in a significant decrease of Vmax (160–99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (–22 ± 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca2+/CaM complex causes changes in transport capacity via hOCT2 trafficking.

organic cation transport; fluorescence measurement; 4-[4-(dimethylamino)-styryl]-n-methylpyridinium; amiloride

THE HUMAN ORGANIC CATION TRANSPORTER 2 (hOCT2) belongs to the superfamily solute carrier family 22 (SLC22 and SLC22A2) (21). Transport of organic cations by OCTs (OCT1–OCT3) has been characterized as polyspecific, bidirectional, electrogenic (3, 11, 14), and Na+-, Cl–-, and proton-gradient independent (4, 14, 23). Common structural properties of OCTs are the presence of 12 putative {alpha}-helical transmembrane domains (TMDs) with a hydrophilic extracellular loop connecting TMD1 and TMD2 and of several potential intracellular protein kinase phosphorylation sites (8). However, OCTs show different affinities to particular substrates and differ in their tissue and membrane localization (16) as well as in their regulation (8).

hOCT2, first cloned in 1997 (14) and localized on chromosome 6q26 (20), consists of 555 amino acids and has been found in human kidney, placenta, spleen, intestine, and central neurons (3, 14, 23). hOCT2 has been detected mainly in the basolateral membranes of proximal tubules of human kidneys (29) and is thought to be the major transporter for the uptake of various organic cations from the bloodstream into renal epithelial cells. Endogenous substrates transported by hOCT2 are the monoamine neurotransmitter 5-hydroxytryptamine, noradrenaline, histamine, agmatine, dopamine, and choline (3), as well as compounds such as creatinine (41). Examples of drugs that are transported by hOCT2 include the histamine receptor antagonist cimetidine (1), the antidiabetic drugs metformin and phenformin (12, 19), the anti-Parkinson's disease drugs memantine and amantadine, the neurotoxin 1-methyl-4-phenylpyridinium (3), and the antineoplastic drug cisplatin (7). The transporter is critical in the detoxification and elimination of xenobiotics from the systemic circulation and thus is a major determinant of drug response and sensitivity. An attractive model to explain the polyspecificity of OCTs (i.e., ability to accommodate substrates with different structures) has been proposed recently on the basis of mutation-function studies with rOCT1 and rOCT2. The results of these studies suggest that OCTs have a large binding pocket containing partially overlapping binding domains for different substrates (13, 33). Transport mediated by hOCT2 can be regulated by several kinases (5), similarly to observations in freshly isolated human proximal tubules (31) but in a fashion different from that of its rat renal counterpart rOCT1 (27) and from the human isoforms hOCT3 (26) and hOCT1 (9). Modulation of various regulatory pathways can modify transport of OCTs by changing the apparent substrate affinities. In the case of rOCT1, in which PKC regulation is associated with transporter phosphorylation and increase in apparent affinity (27), mutations of the putative PKC phosphorylation sites of rOCT1 changed not only the regulation but also the selectivity as well as, to different extents, the inhibition of transport by various competitive substrates (6). These findings were obtained almost exclusively with 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) as substrate and only IC50 values for other organic cations were calculated indirectly by the interaction of these substances with ASP uptake. The question remained how and to what extent the regulation of OCTs interferes with the large substrate-binding pockets of the transporters and whether the affinity of all substrates, many substrates, or only individual substrates is altered. In previous experiments, we were not able to estimate Km and Vmax values for ASP uptake by OCTs because the fluorescence increase with ASP did not reach saturation (5, 6, 27).

Therefore, the aims of the present study were to demonstrate hOCT2-mediated and saturable uptake of the fluorescence substrate ASP to search for a second fluorescent substrate and to further characterize the regulation of organic cation transport by hOCT2 using two fluorescence substrates and measuring the effect of regulation on the amount of hOCT2 within the plasma membrane. The present study has increased our understanding of how hOCT2, the most relevant OCT in human proximal tubules, is regulated.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Expression of hOCT2 in human embryonic kidney cells. Experiments were performed with human embryonic kidney (HEK)-293 cells (CRL-1573; American Type Culture Collection, Manassas, VA) as wild-type (WT) and HEK-293 cells stably expressing hOCT2 (3). In some immunohistological experiments, HEK-293 cells stably transfected with rOCT1 (17), rOCT2 (17), or hOCT1 (9) were also used (see below). Cells were grown at 37°C in 50-ml cell culture flasks (Greiner Bio-One, Frickenhausen, Germany) in DMEM (Biochrom, Berlin, Germany) containing 3.7 g/l NaHCO3, 1.0 g/l D-glucose, and 2.0 mM L-glutamine (Biochrom), and gassed with 8% CO2. Penicillin (100 U/ml), 100 mg/l streptomycin (Biochrom), 10% FCS, and, only for OCT-transfected cells, 0.8 mg/ml geneticin (PAA Laboratories, Coelbe, Germany) were added to the medium. Experiments were performed with confluent cells grown on glass coverslips and aged 6–10 days from passages 25–70. Each set of experiments was performed on the same day with cells of the same age and passage. Culture and functional analyses of these cells were approved by the state government Landesumweltamt Nordrhein-Westfalen, Essen, Germany (no. 521.-M-1.14/00).

Dynamic fluorescence microscopy. Uptake of the fluorescent substances in WT and hOCT2 cells was measured using dynamic fluorescence microscopy and also, for some substances, with confocal microscopy. Dynamic fluorescence measurements were performed as is customary in our laboratory: in the dark with an inverted microscope (Axiovert 135; Zeiss, Oberkochen, Germany) equipped with an oil-immersion lens objective (x100 magnification). Cells were excited by a pulsating excitation light (5–20 pulses/s) generated by a xenon quartz lamp (XBO, 75 W; Zeiss) after reflection to the perfusion chamber using a dichroic mirror, with cell monolayers on coverslips forming the bottom of the chamber. Cells were superfused at a rate of 10 ml/min with the fluorescent organic cations solubilized in HCO3–-free Ringer-like solution containing (in mM) 145.0 NaCl, 1.6 K2HPO4, 0.4 KH2PO4, 5.0 D-glucose, 1.0 MgCl2, and 1.3 Ca2+-gluconate, with pH adjusted to 7.4 at 37°C or at 8°C, respectively. The temperature of the bath solution was continuously controlled and regulated using a thermostat. Fluorescence emission was measured using a photon-counting tube (Hamamatsu H3460-04; Herrsching, Germany). Different excitation and emission filters corresponding to the distinct fluorescence spectra of the tested substances were applied (Table 1). Experiments were controlled and data were analyzed using a computer-aided system and software (provided by U. Fröbe, Universität Freiburg, Freiburg, Germany). We evaluated the initial slope measured during the first 10–30 s as the transport parameter. Although the initial slope directly represented the uptake of organic cations across the plasma membrane, the maximal cellular fluorescence was the sum of the substrate uptake into the cell, exit from the cell, intracellular compartmentalization with changes in the emission spectra, and bleaching of the dye (27, 31, 37, 38).


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  		Table 1. Excitation and emission spectra of investigated fluorescent organic cations

 
Because ASP and amiloride were identified as the most suitable fluorescence substrates for hOCT2 tested, their transport properties were investigated extensively. In some experiments, amiloride and ASP could be used simultaneously because of their different fluorescence spectra. Excitation light of 360 or 450–490 nm, dichroic mirrors reflecting up to 390 or 560 nm, and emission filters of 395–440 or 575–640 nm for amiloride or ASP, respectively, were applied. For the study of the regulation of hOCT2, cells were incubated with the potential regulator for 10 min at 37°C before the fluorescence substrate was added in the continued presence of the respective regulatory substance.

Confocal microscopy. For evaluation of specific hOCT2-mediated transport, confocal microscopy of WT and hOCT2 cells after incubation with ASP or quinacrine was performed. For confocal microscopy, we used a Zeiss LSM 510 Meta System with an Axiovert 200M inverted microscope (Carl Zeiss; kindly made available by Prof. Gerke, Center for Molecular Biology of Inflammation, Münster, Germany) using a x63 magnification lens objective. A laser generating 488 nm of light was used as the excitation source, fitted with the excitatory fluorescence spectra of ASP and quinacrine.

The hOCT2 and WT cells were prepared as follows: after being washed in HCO3–-free Ringer-like solution at 37°C, cells were incubated for 60 s at 37°C with ASP (1 µM) or quinacrine (1 µM), respectively. After incubation, cells were washed immediately three times with ice-cold HCO3–-free Ringer-like solution and then fixed with 4% paraformaldehyde (PFA) solution. After 30 min, cells were washed with PBS (Biochrom). For final fixation, MOWIOL 4-88, the semipermanent mounting medium used for fluorescence microscopy, was prepared and used as recommended by the manufacturer (Calbiochem Merck Biosciences, Schwalbach, Germany). Immediately before use, small amounts of the antifade agent 1,4-diazabicyclo[2.2.2]octane (Molecular Probes/Invitrogen, Karlsruhe, Germany) were added to reduce signal fading under intense light.

Patch-clamp experiments. Membrane voltage (Vm) recordings of transfected hOCT2 and nontransfected WT cells were obtained using the slow whole cell patch-clamp technique as performed previously by our group (5). Cells were superfused in a chamber with HCO3–-free Ringer-like solution (see above) at 37°C with a flow rate of 10–20 ml/min. Patch pipettes had an input resistance of ~10 M{Omega} and were filled with a solution containing (in mM) 95.0 K+-gluconate, 30.0 KCl, 4.8 Na2HPO4, 1.2 NaH2PO4, 5.0 D-glucose, 0.73 Ca2+-gluconate, 1.0 EGTA, 1.03 MgCl2, and 1.0 ATP, with pH adjusted to 7.2. Before use, nystatin (162 µM) was added to permeabilize the membrane patch under the pipette. Vm was measured in the current-clamp mode of a patch-clamp amplifier.

Preparation of MAb against extracellular loop of hOCT2 and its characterization using Western blot analysis and immunohistochemistry. The hOCT2 DNA fragment coding for the large extracellular loop of hOCT2 (amino acids 44–158) was cloned in the plasmid pET22b(+) into the NdeI and XhoI restriction enzyme sites. The His-tagged hOCT2 fragment expressed in Escherichia coli was purified using Ni2+-iminodiacetic acid-Sepharose CL-6B and DEAE-Sepharose chromatography dialyzed against 10 mM Tris·HCl, pH 8.1, and 150 mM NaCl and subjected to immunization. Mice BALB/c were immunized subcutaneously with 50 µg of recombinant hOCT2 fragment emulsified in complete Freund's adjuvant. Mice were boosted twice with the same dose of hOCT2 fragment in PBS on days 30 and 60 after primary immunization. Sera of the immunized mice were tested for the presence of specific antibodies using indirect ELISA. Spleen cells of the best responder animal were fused with mouse myeloma Sp2/0 cells using PEG 1500 as a fusion agent (HybriMax PEG/DMSO solution; Sigma-Aldrich, Munich, Germany). Hybrid cells were selected in growth medium supplemented with hypoxantine, aminopterin, and thymidine (50x HAT medium supplement; Sigma). Viable clones were screened by indirect ELISA using 96-well microtiter plates coated with recombinant hOCT2 fragment (5 µg/ml in 0.05 M Na+-carbonate buffer, pH 9.5) and peroxidase-labeled secondary antibody against mouse IgG raised in sheep (NXA 931; Amersham Biosciences Europe, Freiburg, Germany). Positive clones were stabilized by limiting dilution cloning on macrophage feeder layer using growth medium supplemented with recombinant human IL-6. Hybridoma cells were maintained in complete DMEM containing 15% FCS (Biochrom) and antibiotics.

For Western blot analysis, human renal cortex was homogenized in 50 mM Tris·HCl, pH 7.4, 250 mM sucrose, 1 mM EDTA, protease inhibitors (18), and Chinese hamster ovary (CHO) cells stably transfected with vector pcDNA3.1 or pcDNA3.1 containing hOCT1, hOCT2, or hOCT3 (25) were solubilized by incubation with 50 mM Tris·HCl, pH 7.4, 250 mM sucrose, 1 mM EDTA containing protease inhibitors, and 1% (wt/vol) Triton X-100. The homogenate of the renal cortex and the solubilized CHO cells were diluted 10-fold with sample buffer for SDS-PAGE. SDS-PAGE and Western blot analysis were performed as described previously (18). For Western blot analysis staining, we used the MAb against hOCT2 with hybridoma supernatant diluted 1:10. Bound peroxidase-conjugated secondary antibody (sheep anti-mouse IgG) was visualized using ECL (Amersham Biosciences Europe, Freiburg, Germany). MAb against hOCT2 was also tested using immunohistochemistry. Briefly, cells (hOCT1-, hOCT2-, rOCT1-, rOCT2-, or WT-HEK-293 cells) on coverslips were fixed in 4% PFA for 10 min at room temperature. After fixation, the cells were washed three times with PBS and incubated with 0.1% Triton X-100 for 3 min. After being washed extensively with PBS, nonspecific binding sites were blocked by overnight incubation at 4°C with 1% (vol/vol) gelatin (cold fish skin; Sigma). The cells were then incubated for 60 min at room temperature with hybridoma supernatant diluted 1:10. After being subjected to three washing steps in PBS, the secondary antibody (1:1,000 dilution, Alexa Fluor 594 goat anti-mouse; Invitrogen, Karlsruhe, Germany) was incubated for 60 min, followed by five more washing steps in PBS. Finally, the cells were covered with Crystal Mount (Sigma). Fluorescence photomicrographs were taken with an Axiocam camera mounted onto an Axiovert 100 microscope (Zeiss) using Axiovision software.

FACScan flow cytometry. The same number of cells (WT or hOCT2) of the same passage and age were incubated for 10 min with or without 5 µM calmidazolium at 37°C. After incubation, cells were fixed for 20 min with 4% PFA at 4°C and pH 7.4. After being washed three times with ice-cold HCO3–-free Ringer-like solution, the unspecific binding sites were blocked by 30-min incubation at 37°C with HCO3–-free Ringer-like solution containing 0.5% gelatin (Amersham Biosciences Europe). Finally, cells were incubated overnight at 4°C with hybridoma supernatant of mouse MAb (1:100 dilution) against the extracellular region of the transporter. After being washed, cells were incubated for 45 min in the dark with the FITC F(ab')2 fragment of rabbit anti-mouse IgG (1:10 dilution; DakoCytomation, Hamburg, Germany). Cell-associated fluorescence was measured using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). In some experiments, cells were incubated with or without calmidazolium, fixed, and then permeabilized with 0.1% saponin for 20 min at 4°C to evaluate the intracellular transporter pool.

Biochemicals. Acridine orange, acriflavine (synonym, euflavine), amiloride, daunomycin (synonym daunorubicin), doxorubicin, proflavine, quinacrine, quinine, rhodamine-123, and rhodamine B as potential fluorescence substrates were purchased from Sigma-Aldrich (Munich, Germany). ASP was bought from Molecular Probes/Invitrogen. Forskolin, 1,2-dioctanoyl-sn-glycerol (DOG), wortmannin, calmidazolium, and calphostin C were obtained from Calbiochem/Merck Biosciences. Compounds were dissolved in HCO3–-free Ringer-like solution and, if necessary, with methanol, ethanol, or DMSO as a solvent. The final concentration of these solvents did not affect the results of the experiments (data not shown). All other substances and standard chemicals were obtained from Sigma-Aldrich or Merck Biosciences.

Statistical analysis. Data are presented as means ± SE, with n referring to the number of cell monolayers. Km, Vmax, and IC50 values were obtained using sigmoid dose-response curve fitting (constant Hill slope) with Prism version 4.0 software (GraphPad, San Diego, CA). The same software was used to compare the fitted midpoints (log IC50) of two curves statistically. An unpaired, two-sided Student's t-test was used to prove the statistical significance of the effects. ANOVA with Tukey's test was applied as indicated. P < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Transport specificity of fluorescent substrates. To find new, suitable fluorescence substrates for microfluorometric analysis of OCT-mediated transport, we compared the uptake of doxorubicin (10 µM), proflavine (10 µM), daunomycin (10 µM), acridine orange (1 µM), quinine (100 µM), quinacrine (1 µM), rhodamine-123 (0.1 µM), rhodamine B (10 µM), acriflavine (1 µM), and amiloride (1 µM) in HEK-293 cells (WT) with the uptake by HEK-293 cells stably transfected with hOCT2. The indicated concentrations were the lowest concentrations inducing discrete, measurable fluorescence emission. Although all of these fluorescent cations showed significant cellular accumulation, only acriflavine, amiloride, and ASP had significantly higher uptake rates in hOCT2 cells compared with WT cells (Fig. 1). The specific uptake of these concentrations was calculated by subtracting the unspecific uptake in WT cells from the total uptake in hOCT2 cells. Practically, the total uptake of ASP in hOCT2 cells at 37°C was set to 100%, and the ASP uptake of the same number of WT cells was assumed to be unspecific uptake. The difference between total and unspecific represents the specific uptake and is expressed as a percentage of the total (Fig. 1).


Figure 1
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  		Fig. 1. Determination of specific uptake of various fluorescent organic cations by human polyspecific organic cation transporter 2 (hOCT2) cells. Specific uptake of fluorescent organic cations via hOCT2 was determined by subtracting the unspecific uptake in wild-type (WT) cells from the total uptake in hOCT2 cells at 37°C. Specific uptake rates are presented in %total uptake. Values are means ± SE of initial fluorescence increase with n = 3–8; gray columns show statistically significant results (P < 0.05).

 
For acriflavine, amiloride, and ASP, the specific uptake amounted to 39 ± 6% (n = 6), 91 ± 2% (n = 6), and 93 ± 1% (n = 6) of total uptake in hOCT2 cells, respectively. Because at these concentrations unspecific uptake rates for amiloride and ASP or uptake rates via transporters other than hOCT2 was <10% of the total uptake, these substrates were used for further investigation.

Direct determination of Km values of amiloride and ASP uptake in hOCT2 cells. Total initial uptake rates by hOCT2 for amiloride and ASP did not reach substrate saturation; only time-dependent saturation for maximal cellular fluorescence was observed (Fig. 2). Concentrations >10 mM for amiloride or 1 mM for ASP could not be dissolved in the buffer systems. To test the contribution of passive diffusion and/or endogenous transport, we compared the concentration-dependent uptake in hOCT2-expressing cells with that in WT cells (Fig. 3A) or with that in hOCT2 cells at 8°C (Fig. 3C). At 8°C, cellular fluorescence increased in a concentration-dependent manner without reaching a plateau in WT and in hOCT2 cells. For the uptake rate of 500 µM ASP in WT cells, no temperature dependence was observed, suggesting passive diffusion rather than transporter-mediated uptake (Fig. 3B). The curves obtained when either ASP uptake in WT cells or ASP uptake in hOCT2 cells measured at 8°C were subtracted from uptake in hOCT2 cells measured at 37°C reached saturation (Fig. 3, A and C). As specific uptake rates for ASP reached saturation, we were able to determine Km values directly (Fig. 3A, Km = 42 µM; Fig. 3C, Km = 24 µM). Km values of ASP obtained with the two described approaches were in agreement; the small difference can be attributed to residual transport activity of hOCT2 at 8°C. Similarly to these experiments with ASP, we compared uptake rates of amiloride in hOCT2 at 37°C with those at 8°C (Fig. 4). Again, the difference representing specific uptake of amiloride by hOCT2 reached saturation. The corresponding Km value was calculated directly as 95 µM. Vmax values cannot be compared directly, because they are expressed in arbitrary units and the emitted fluorescence intensity of the two substrates is different.


Figure 2
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  		Fig. 2. Concentration-dependent uptake of N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) in hOCT2 cells. Initial and maximal fluorescence increase with amiloride (A) and ASP (B) in hOCT2 cells at 37°C. In contrast to the maximal fluorescence (interrupted curves), the initial uptake rates (continuous curves) were not saturable with both substances up to concentrations of 10 mM amiloride or 1 mM ASP. Values are means ± SE of initial fluorescence increase, with n referring to the number of cell monolayers.

 

Figure 3
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  		Fig. 3. Specific and unspecific uptake rates of ASP. Determination of specific ASP initial uptake rates (interrupted line curves) and of corresponding Km values by subtracting the unspecific uptake (gray-lined curves) evaluated as uptake in WT cells (A; Km = 42 µM) or as uptake in hOCT2 cells at 8°C (C; Km = 24 µM) from the total ASP uptake at 37°C (continuous solid-lined curves). B: initial ASP uptake (500 µM) in WT cells was investigated at 37°C and 8°C. Values are means ± SE of initial fluorescence increase in photons/s (A and C) or %value at 37°C (B). n refers to number of cell monolayers.

 

Figure 4
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  		Fig. 4. Specific and unspecific uptake rates of amiloride. Comparison of total initial amiloride uptake in hOCT2 cells at the physiological temperature of 37°C (continuous solid-lined curve) with unspecific uptake at 8°C (gray-lined curve). Interrupted line curve is derived difference representing specific hOCT2-mediated transport of amiloride (Km = 95 µM). Values are means ± SE of initial amiloride uptake (photons/s), with n referring to number of cell monolayers.

 
Inhibition of hOCT2-mediated transport. Other known OCT substrates [tetraethylammonium (TEA), cimetidine, and tetrapentylammonium (TPA)] inhibited the uptake of amiloride and ASP in a concentration-dependent manner (both 1 µM) (Fig. 5, A–C) with corresponding IC50 values summarized in Table 2. Experiments with amiloride and ASP showed significantly different IC50 values for TEA and TPA, whereas IC50 values for cimetidine were not significantly different. TPA had the lowest IC50 value and was therefore the strongest inhibitor, followed by cimetidine and TEA as the weakest inhibitor. The hOCT2-mediated uptake of the fluorescent organic cation acriflavine (specific uptake, 39 ± 6% of total uptake; n = 6) was also inhibited completely by TEA (10 mM) (data not shown). Furthermore, mutual inhibition experiments with amiloride and ASP were performed (Fig. 5D). ASP was used as the fluorescence substrate and amiloride was used as the competitor or vice versa.


Figure 5
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  		Fig. 5. Interaction of other substrates with transport of amiloride and ASP. A–C: inhibition of initial amiloride (continuous curves) and ASP (interrupted line curves) uptake in hOCT2 cells by tetraethylammonium (TEA; n = 6–46), cimetidine (n = 5–24), and tetrapentylammonium (TPA; n = 6–16). D: mutual effects of amiloride on ASP uptake and vice versa were also measured (n = 7–12). Data were analyzed using sigmoid concentration-response curve fitting (constant Hill slope), and values are means ± SE of initial fluorescence increase. n refers to number of cell monolayers. IC50 values are summarized in Table 2.

 

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  		Table 2. IC50 values for inhibition of amiloride and ASP transport via hOCT2

 
Contribution of hOCT2 to quinacrine uptake. Quinacrine had been shown to inhibit rOCT2 mediated TEA transport significantly (39) and has been proposed as a suitable fluorescence probe for organic cation transport in choroid plexus cells (28). However, in contrast to amiloride and ASP (both 1 µM), quinacrine (1 µM) accumulated in hOCT2 cells similarly to WT cells. In relation to total uptake, only 14 ± 19% (n = 6) of quinacrine was transported specifically by hOCT2 (Fig. 1). Furthermore, quinacrine uptake in hOCT2 cells was not inhibitable by TEA (0.1 µM–10 mM) and was not influenced by calmidazolium incubation (2.5 and 5 µM, 10 min, 37°C), and an increase in extracellular K+ concentration from 3.6 to 145 mM to depolarize the cells had no significant effect on quinacrine uptake in hOCT2 cells (data not shown). In addition to these experiments performed with the photon-counting tube, we examined differences between specific hOCT2-mediated and unspecific transport of organic cations with fluorescence confocal microscopy (Fig. 6). ASP and quinacrine (both 1 µM) were used as examples of specific hOCT2-mediated transport or unspecific uptake, respectively. In comparing the confocal photomicrographs obtained using the same exposure time and the same numerical aperture, quinacrine was detected in WT and also at a slightly higher level in hOCT2 cells (Fig. 6, C and D), whereas ASP was accumulated only in hOCT2 cells but not in WT cells (Fig. 6, A and B). Fluorescent dyes were evident in fixed cells, probably because of cross-linking effects of fixation as demonstrated in previous studies with biogenic amines (30, 36) or because the dyes were trapped in subcellular compartments. Furthermore, the dissipation of these dyes from the cellular compartment when the dye was washed from the extracellular compartment required several minutes and was stopped by fixation.


Figure 6
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  		Fig. 6. Uptake of ASP and quinacrine compared with fluorescence confocal microscopy. Comparison of ASP (1 µM) or quinacrine (1 µM) uptake in hOCT2 cells (A and C) with uptake in WT cells (B and D) after incubation for 60 s at 37°C. All images were obtained using the same exposure time and the same numerical aperture.

 
To further demonstrate the unspecific transport of quinacrine not mediated by electrogenic hOCT2, electrophysiological experiments using the slow whole cell patch-clamp technique were performed. Superfusion of hOCT2 cells with quinacrine (100 µM) resulted in depolarization of Vm by 8.3 ± 2.5 mV (n = 4). In the presence of Ba2+ (3.0 mM), an inhibitor of K+ channels used to increase the relative contribution of hOCT2 to Vm and to avoid possible effects of quinacrine on K+ channels, Vm was depolarized by quinacrine to the same extent in hOCT2 cells ({Delta}Vm = 4.5 ± 1.2 mV; n = 4) as in WT cells ({Delta}Vm = 4.1 ± 1.1 mV; n = 7) (Fig. 7). The data support the interpretation that the observed cellular quinacrine accumulation was independent of hOCT2. ASP already has been shown to induce specific depolarization of the membrane potential in HEK-293 cells stably transfected with rOCT1 (37) or hOCT2 (5).


Figure 7
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  		Fig. 7. Depolarization of hOCT2 cells by quinacrine with or without Ba2+ ({Delta}Vm = 4.5 ± 1.2 mV, n = 4, or {Delta}Vm = 8.3 ± 2.5 mV, n = 4, respectively) and of WT cells by quinacrine with Ba2+ ({Delta}Vm = 4.1 ± 1.1 mV, n = 7) investigated using slow whole cell patch-clamp technique. Values are means ± SE of {Delta}Vm (in mV), with n referring to number of cells. All depicted effects are statistically significant (P < 0.05), but there was no difference between effects of quinacrine on hOCT2 or WT cells in presence of Ba2+.

 
Regulatory effects of PKA, Ca2+/CaM, PKC, or PI3-kinase. To date, regulation of transport mediated by hOCT2 has been demonstrated for ASP as the only substrate. We compared the effects of four known regulatory pathways of hOCT2 on the uptake of ASP with those of amiloride to examine whether the substrate may influence the regulation of transport activity (Fig. 8A). Stimulation of cAMP-dependent PKA with the adenylate cyclase activator forskolin (1 µM) significantly inhibited amiloride and ASP transport to the same extent (–16 ± 5%, n = 16, and –18 ± 4%, n = 40, respectively). DOG (1 µM), a membrane-permeable analog of diacylglycerol and a PKC activator, led to inhibition of uptake of both substances (–54 ± 5%, n = 14, with amiloride; –31 ± 6%, n = 26, with ASP). Because in a previous study the DOG-induced inhibition of ASP uptake did not reach significance (5), the specificity of the inhibitory effect in this study also was investigated using calphostin C (0.1 µM), a specific PKC inhibitor (Fig. 8B). Coincubation with calphostin C suppressed the inhibitory effect of DOG (–1 ± 10%; n = 10) and incubation only with calphostin C showed no significant effect on the transport rate (–7 ± 5%; n = 9), underlining that PKC is involved in the regulation of hOCT2. Inhibition of phosphatidylinositol 3-kinase (PI3-kinase) by 0.1 µM wortmannin increased the transport rate of both substrates similarly (+28 ± 6%, n = 8, and +55 ± 17%, n = 16, with amiloride or ASP, respectively). Calmidazolium (5 µM), known as a Ca2+/CaM complex inhibitor, decreased the transport of amiloride and ASP similarly (–55 ± 5%, n = 10, and –63 ± 2%, n = 15, respectively). Figure 9 summarizes the regulatory pathways of hOCT2.


Figure 8
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  		Fig. 8. Regulation of hOCT2. A: regulatory pathways for hOCT2-mediated transport by investigating uptake of amiloride or ASP (both 1 µM) in hOCT2 cells incubated at 37°C for 10 min with forskolin, 1,2-dioctanoyl-sn-glycerol (DOG), wortmannin, or calmidazolium. Initial uptake rates of amiloride or ASP after incubation with these different effectors are presented as %uptake without effector. Values are means ± SE of initial fluorescence increase, with n referring to number of cell monolayers. All depicted effects were statistically significant (P < 0.05). B: influences of calphostin C (PKC inhibitor) and/or DOG (PKC activator) on ASP (1 µM) uptake in hOCT2 cells. Values are means ± SE of initial fluorescence increase in %control with n referring to number of cell monolayers.

 

Figure 9
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  		Fig. 9. Proposed scheme of hOCT2 regulation with the investigated pathways for hOCT2-mediated transport, including PKA, Ca2+/CaM complex, phosphatidylinositol 3-kinase (PI3-kinase), and PKC. Pathways were proofed with amiloride and ASP as fluorescence tracers (see Fig. 8). TMD, transmembrane domain.

 
Regulation of ASP uptake by Ca2+/CaM inhibition influenced Vmax. To determine whether regulation of ASP uptake by Ca2+/CaM inhibition is linked to Km or Vmax changes, we performed a series of experiments in which concentration-dependent ASP uptake rates in hOCT2 cells of the same age and passage after incubation with or without calmidazolium (5 µM, 10 min, 37°C) were compared (Fig. 10). The unspecific uptake was determined in parallel experiments by measuring ASP uptake in hOCT2 cells of the same age and passage at 8°C. This unspecific uptake was not influenced by calmidazolium incubation (data not shown) and was subtracted from the total uptake with or without calmidazolium incubation at 37°C. With these curves, we could determine the corresponding Km and Vmax values directly. Km was 29 µM without and 26 µM with calmidazolium. Vmax decreased significantly from 160 photons/s without calmidazolium incubation to 99 photons/s with calmidazolium incubation (Fig. 10).


Figure 10
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  		Fig. 10. Direct determination of Km and Vmax under the regulatory influence of calmidazolium. A: initial uptake of increasing ASP concentrations in hOCT2 cells was measured with (interrupted line curve) or without (continuous line curve) calmidazolium (5 µM) incubation at 37°C and compared with uptake at 8°C (gray-lined curve). B: specific uptakes with (interrupted line curve) or without calmidazolium (continuous line curve) were determined by subtracting uptake at 8°C from uptake at 37°C. n refers to number of cell monolayers, and values are means ± SE of initial ASP uptake (photons/s).

 
Effect of inhibition of Ca2+/CaM complex by calmidazolium on trafficking of hOCT2. MAb against the extracellular loop of hOCT2 recognized specifically hOCT2 as demonstrated by performing Western blot and immunohistochemical analyses (Fig. 11). No immunofluorescence was detectable in immunohistochemical analysis of WT (Fig. 11, right) or of rOCT1, rOCT2, and hOCT1 HEK-293 cells (data not shown). Therefore, this antibody could be used to label the extracellular loop of hOCT2 in FACScan measurements. Figure 12 shows the effect of incubation with 5 µM calmidazolium on the cell-associated fluorescence in hOCT2 cells. Inhibition of Ca2+/CaM complex by calmidazolium caused a significant decrease of membrane-associated fluorescence in hOCT2 cells (–22 ± 6%; n = 9) compared with controls (0 ± 6%; n = 9). Exposition of the intracellular transporter pool by cell permeabilization resulted in a significant increase of fluorescence both in cells incubated with (+125 ± 4%; n = 5) or without (+108 ± 6%; n = 3) 5 µM calmidazolium (Fig. 12).


Figure 11
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  		Fig. 11. Characterization of MAb against extracellular loop of hOCT2. A: Western blot analysis (WB) of lysates from Chinese hamster ovary (CHO) cells stably transfected with hOCT1, hOCT2, or hOCT3, or only with vector from human kidney. B: immunohistochemical analysis (IHC) of human embryonic kidney (HEK)-293 cells stably transfected with hOCT2 compared with HEK-293 cells.

 

Figure 12
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  		Fig. 12. Effects of 10-min incubation with 5 µM calmidazolium (Calm.) on cell-associated fluorescence of hOCT2 cells. Transporters were labeled using a MAb against extracellular region of hOCT2. Effects of permeabilization with saponin are also shown. Data regarding change in cell-associated fluorescence as %control measured using FACScan. Asterisks indicate statistically significant differences from control measurements on the basis of ANOVA and Tukey's test.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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hOCT2 is the main OCT in the basolateral membrane of the human proximal tubule (14, 29). It plays a crucial role in absorption and excretion of drugs, xenobiotics, and endogenous compounds. The efficacy as well as the toxicity of cationic drugs could be improved by supplementary medication with substrates or inhibitors of hOCT2. In addition, endogenous or exogenous regulation of hOCT2 in the human kidney could modify the renal excretion of cationic drugs or toxins. Single-nucleotide polymorphisms have been identified in hOCT1 (35) and hOCT2 (24), with some of them affecting transport function when tested in heterologous expression systems.

Because of the manifold clinical effects and pathological implications of hOCT2, the regulation of hOCT2 was investigated further. Specifically, we addressed the question whether the regulation of hOCT2 affects transport of structurally distinct substrates differently. First, we searched for a fluorescent organic cation in addition to ASP that has been used in our previous studies (5, 34, 37, 38). Second, because in our earlier experiments substrate saturation in cells expressing hOCT2 or other OCTs was not demonstrated (5, 27), we subtracted unspecific uptake and were able to demonstrate saturable transport of amiloride and ASP mediated by hOCT2. This allowed us to estimate Km and Vmax directly. Third, we demonstrated that regulation of amiloride and ASP transport by hOCT2 was similar in most but not all aspects. Fourth, we showed that the downregulation of Vmax of ASP uptake observed after inhibition of the Ca2+/CaM complex included the downregulation of the amount of hOCT2 in the plasma membrane.

Specificity of transport via hOCT2 and determination of transport kinetics. Of the 11 tested organic cations with fluorescent properties, only amiloride, ASP, and, to some extent, acriflavine demonstrated uptake, which was significantly larger in hOCT2 than in WT cells, suggesting specific transport via hOCT2. Amiloride showed concentration-dependent but nonsaturable initial uptake rates in hOCT2 cells as previously reported for ASP and as confirmed in another study (5). ASP had a higher affinity for hOCT2 (Km = 24 µM) than it did for amiloride (Km = 95 µM).

Amiloride transport by hOCT2 could be inhibited by known competitors such as TEA, TPA, or cimetidine (2, 15, 22, 43, 44). Inhibition of amiloride uptake by these competitors revealed a similar sequence of IC50 values as we reported previously for ASP (5). Interference of amiloride with renal excretion of ASP in renal proximal tubule cells was indeed reported previously (32). However, even though ASP showed a higher affinity for hOCT2 than for amiloride, the IC50 values for TEA and TPA were significantly lower for ASP than for amiloride. This apparent discrepancy can be explained by presuming that amiloride and ASP bind at different but partially overlapping sites in the binding pockets of hOCT2 and that TEA and TPA bind closer to the ASP site than to the amiloride site. Because cimetidine has similar IC50 values for inhibition of amiloride and ASP uptake, its binding site may overlap more extensively with the ASP site, because ASP has a higher affinity for hOCT2 than for amiloride. The existence of overlapping interaction domains for different substrates already has been proposed for rOCT1 and rOCT2 (6, 13, 33).

Regulation of hOCT2-mediated transport. We recently demonstrated that OCTs are regulated by various protein kinases and intracellular messengers (5, 8). Our previous results demonstrated activation of rOCT1 by PKC (27) but no effect on hOCT1 after PKC stimulation (9). Transport of ASP across the basolateral membrane of isolated human proximal tubules was inhibited by PKC (31). The possibility of determining exactly the kinetics of hOCT2-mediated transport with two different organic cations as fluorescence substrates allowed further functional examination of mechanisms of regulation of hOCT2. The binding pocket of OCTs apparently contains partially overlapping interaction domains for different substrates (13, 33) as also was demonstrated by our mutation-function studies in rOCT1 (6). In the latter study, we reported selective changes in IC50 values for the inhibition of ASP transport by various other organic cations by PKC activation or mutation of the putative PKC sites of rOCT1. Therefore, we have readdressed the question of how kinase-mediated regulation of hOCT2 influences the transport of different substrates.

Our previous findings based on IC50 values for ASP inhibition by other organic cations from rOCT1, hOCT1, or hOCT2 suggested that PKC or Ca2+/CaM activation selectively modifies apparent affinities for various substrates (5, 9, 27). These initial data did not allow for determination of whether these effects reflect changes in Km and/or Vmax or whether these effects are specific for ASP as substrate only. The qualitatively similar effects of regulation of hOCT2-mediated transport of amiloride and ASP via PKA, PKC, Ca2+/CaM, or PI3-kinase reported herein suggest that this regulation modifies more generally transport kinetics independent of the substrate transported. Interestingly, for both substrates, we demonstrated a small but significant decrease in transport activity with PKC activation in line with our findings in isolated human proximal tubules, in which basolateral OCT is mediated by hOCT2 (31). The absence of such a significant effect in our previous study (5) with ASP was probably due to the small sample size. The data regarding transport regulation obtained by measuring total uptake of amiloride or ASP in hOCT2 cells, however, did not allow us to identify the mechanisms of regulation, i.e., whether it changes substrate affinities by direct phosphorylation of the transporter as demonstrated for rOCT1 and PKC activation (6, 27). Because the largest effect on hOCT2-mediated transport was obtained by inhibition of the endogenously active Ca2+/CaM complex by calmidazolium, we used this pathway to address the question whether regulation of hOCT2 affects Km and/or Vmax. Vmax decreased significantly under calmidazolium incubation, but Km was not significantly influenced by inhibition of the Ca2+/CaM complex. Therefore, the reduced transport rate was linked to a decrease in Vmax, suggesting that the Ca2+/CaM complex affects the number of transporters expressed on the cell membrane. This assumption matches the decreased cell-associated fluorescence of hOCT2 cells after calmidazolium incubation, implying that inhibition of Ca2+/CaM complex affects hOCT2 trafficking. To explain the quantitative differences in Vmax (–38%) and trafficking (–22%) effects, other possible mechanisms, such as changes in the turnover rate or in the number of active transporters, also could be considered. Similar to this effect of calmidazolium on hOCT2, experiments with hOAT1 expressed in HEK-293 cells revealed that PKC-induced hOAT1 downregulation is achieved through carrier retrieval from the cell membrane and does not involve phosphorylation of the predicted classic hOAT1 PKC consensus sites (42). Previously, we reported that calmidazolium significantly increased the IC50 value of TEA for the inhibition of hOCT2-mediated ASP uptake (5). This apparent discrepancy underlines that IC50 values cannot be viewed as affinity values. We cannot exclude the possibility that regulation of hOCT2 involves changes in the binding pocket of hOCT2, thus influencing TEA and ASP transport differently.

Contribution of OCTs to quinacrine transport. Surprisingly, quinacrine, known as an antimalarial drug and described as interfering with basolateral transport of organic cations in rat proximal tubules in situ (40), did not show specific interaction with hOCT2 in this study. Microfluorimetry and confocal microscopy clearly demonstrated quinacrine accumulation in WT cells similar to that in hOCT2 cells. Furthermore, quinacrine accumulation in hOCT2 was neither temperature dependent nor electrogenic as determined using patch-clamp analysis, suggesting that quinacrine uptake is not hOCT2 mediated. In choroid plexus cells, quinacrine transport was inhibitable by TEA and TPA but not by PAH, a substrate of organic anion transport (28). Although hOCT2 is also expressed in neurons of the human central nervous system (21), a member of the SLC22 family other than hOCT2 could be responsible for OCT in choroid plexus cells. In line with these findings, in mouse brain endothelial cells (MBEC4), P-glycoprotein inhibitors increased the apicobasolateral quinacrine transport and uptake was reduced by TEA and cimetidine but was not affected by amiloride (10). These authors suggested the involvement of P-glycoproteins in quinacrine transport out of the cells and OCTN1, a member of the SLC22 family, mediating quinacrine uptake in MBEC4 cells.

In summary, amiloride and ASP are suitable fluorescence substrates for microfluorometric analysis of hOCT2 transport. Both were transported specifically, ASP with a higher affinity than amiloride, and have partially overlapping binding sites in the binding pockets of hOCT2. Both underlie the same qualitative regulation, and the inhibition of the Ca2+/CaM complex causes a change in Vmax via hOCT2 trafficking.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by Deutsche Forschungsgemeinschaft Grant DFG Schl277/8-3 (to E. Schlatter) and Innovative Medizinische Forschung Grant CI 120437 (to G. Ciarimboli).


   ACKNOWLEDGMENTS
 
We thank C. Ludwig (Center for Molecular Biology of Inflammation, Münster, Germany) for supervising confocal microscopy experiments and K. Beul, A. Dirks, U. Kleffner, R. Thanos, and T. V. Le for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Ciarimboli, Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Universitätsklinikum Münster, Domagkstrasse 3a, D-48149 Münster, Germany (e-mail: gciari{at}uni-muenster.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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