The Affinity of the Organic Cation Transporter rOCT1 Is Increased by Protein Kinase C-Dependent Phosphorylation

Cell Culture

HEK293-cells (CRL-1573 ; American Type Culture Collection, Manassas, VA) were stably transfected with rOCT1 as described previously (22). Cells (passages 16 to 50) were grown in 50-ml tissue culture flasks (Greiner, Frickenhausen, Germany) in Dulbecco's modified Eagle's medium containing 10% (vol/vol) fetal calf serum and 0.6 mg/ml geneticin (Life Technologies, Eggenstein, Germany). Cells were incubated at 37°C in an atmosphere of 95% air plus 5% CO₂. After 7 d, the confluent monolayers were trypsinized with Ca²⁺- and Mg²⁺-free phosphate-buffered saline (PBS) and 0.05% trypsin-ethylenediaminetetra-acetic acid (Biochrom, Berlin, Germany). For the experiments, cells were seeded on glass coverslips with areas of 2.25 cm². Cell confluence was reached after 3 to 5 d, and measurements were carried out after 4 to 9 d.

Fluorescence Measurements of ASP⁺ Uptake

Fluorescence microscopy experiments were performed as described previously, using an inverted microscope (Axiovert 135 ; Zeiss, Oberkochen, Germany) equipped with a ×100 oil immersion objective (19,20). The excitation light generated by a xenon-quartz lamp was passed through a 450- to 490-nm bandpass filter mounted in a filter wheel (U. Fröbe, Universität Freiburg, Germany) rotating at 10 Hz to generate a pulsating excitation light. The excitation light was reflected by a dichroic mirror (560 nm) to a perfusion chamber with the cell monolayers on cover slips forming the bottom of the chamber. The cells were superfused at a rate of 10 ml/min with a HCO₃-free Ringer's solution containing (in mM) : 145 NaCl ; 0.4 KH₂PO₄ ; 1.6 K₂HPO₄ ; 5 D-glucose ; 1 MgCl₂ ; 1.3 Ca-gluconate ; and pH adjusted to 7.4 at 37°C. Fluorescence emission was measured after passing through a 575- to 640-nm bandpass filter by a photon counting tube (Hamamatsu H3460-04 ; Herrsching, Germany). Experiments were controlled and data were analyzed with a computer-aided system and specific software (U. Fröbe). As a measurement of ASP⁺ uptake, the increase of cellular fluorescence was plotted versus time. The signals of 10 pulses every second were averaged, yielding a time resolution of 1 Hz. Because ASP⁺ fluorescence is bleached by light, the whole device was protected from light. ASP⁺ uptake was recorded for up to 10 min, and the maximal cellular fluorescence reached as well as the initial slope of the fluorescence increase (linearly fitted during the first 30 s) were analyzed as transport parameters. Background fluorescence of 20 to 50 photon counts per second measured for each monolayer in the absence of ASP⁺ was subtracted from every experiment. The fluorescence signal was obtained from approximately five cells using an adjustable iris diaphragm. A comparable number of control experiments and experiments with the test substances were always performed on the same day with cells of the same passage and age to reduce variability between the unpaired observations. For all series, data from at least two different days were used.

Patch-Clamp Experiments

Recordings of membrane voltages (V_m) of transfected and nontransfected HEK293 cells were obtained using the fast-whole-cell patch-clamp method. The experiments were performed at 37°C in a chamber superfused with the standard solution (see above) with a flow rate of 10 to 20 ml/min. Patch pipettes had an input resistance of approximately 5 mΩ and were filled with a solution containing (in mM) : 95 K-gluconate ; 30 KCl ; 1.2 NaH₂PO₄ ; 4.8 Na₂HPO₄ ; 5 D-glucose ; 0.73 Ca-gluconate ; 1 ethyleneglycol-bis(β-aminoethyl ether)-N,N'-tetra-acetic acid ; 1.03 MgCl₂ ; 1 ATP ; and pH adjusted to 7.2 V_m was recorded continuously with a patch-clamp amplifier (U. Fröbe) and plotted by a pen recorder (WeKaGraph WK-250R ; WKK, Kaltbrunn, Switzerland).

[³H]MPP Uptake Studies

Confluent HEK293 cells were washed with PBS, suspended by shaking, collected by 10 min centrifugation at 1000 × g, and suspended at 37°C in PBS. For uptake measurements, the cells were suspended for 1 s with PBS (37°C) containing 0.15 μM [³H]MPP without or with different concentrations of ASP⁺, or with 50 μM cyanine 863. The uptake reactions were stopped with ice-cold PBS containing 100 μM quinine (stop solution), and the cells were washed three times by 5 min centrifugation with the ice-cold stop solution. In the stop solution no significant cation efflux from the cells could be observed. The nonspecific uptake was determined by adding the ice-cold stop solution before the incubation buffers with [³H]MPP.

Western Blot Analysis

For Western blotting, the total protein of approximately 10⁷ transfected rOCT1-HEK293 or nontransfected HEK293 cells with or without stimulation (sn-1,2-dioctanoyl glycerol [DOG] 1 μM for 15 min) was isolated and resolved in 10% sodium dodecyl sulfate (SDS). Protein concentration was measured by the method of Lowry. The probes were diluted with Laemmli buffer and afterward protein was fractionated by a continuous 10% SDS-polyacrylamide gel (20 μg/lane). The protein on the gel was transferred to a nitrocellulose membrane (0.25 μm) (Schleicher & Schuell, Dassel, Germany) in 50 mM sodium phosphate buffer (pH 7.4) for 3 h at 4°C. The membranes were blocked in 2% bovine serum albumin containing PBS for 1 h and afterward were incubated overnight with either the rabbit polyclonal antibody to phosphoserin (1 μg/ml) (Zymed Laboratory, San Francisco, CA) or an affinity-purified antibody against the large extracellular loop of rOCT1 (23). After removal of the antibodies, the membranes were washed 3 times and incubated with the secondary ¹²⁵I-labeled goat anti-rabbit IgG antibody (2 to 4 μCi/ml) for the rOCT1 antibody and with the ¹²⁵I-labeled protein A anti-IgG antibody (2 μCi/ml) for the phosphoserine antibody (both from Amersham, Braunschweig, Germany) for 3 h at room temperature. The membranes were washed again 3 times, dried, and analyzed by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Chemicals

ASP⁺ was obtained from Molecular Probes (Leiden, The Netherlands) ; cimetidine, tetraethylammonium (TEA⁺), tetrapenthylammonium (TPA⁺), quinine hydrochloride, cyanine 863, forskolin, and all standard chemicals were from Sigma (Deisenhofen, Germany) ; DOG, tamoxifen, KT5720, genestein, aminogenestein, and tyrphostin AG1295 were from Calbiochem (Bad Soden, Germany). All chemicals for SDS-polyacrylamide electrophoresis were purchased from Bio-Rad (Munich, Germany). [³H]MPP was obtained from E. I. Du Pont de Nemours & Co. (Dreieich, Germany)

Statistical Analyses

Data are presented as mean values ± SEM, with n referring to the number of monolayers (fluorescence measurements) or cells (patchclamp experiments). IC₅₀ values were obtained after biexponential curve fittings. Unpaired (fluorescence measurements) or paired (patch-clamp experiments) two-sided t test was used, and a P value <0.05 was considered statistically significant.

Fluorescence Measurements of ASP⁺ Uptake

To characterize the transport kinetics of HEK293 cells transfected with rOCT1 (rOCT1-HEK293), the concentration dependence of the ASP⁺ uptake was measured. ASP⁺ uptake by rOCT1-HEK293 cells followed a saturation curve with an apparent K_m value of approximately 1 μM and a maximal cellular fluorescence above 10 μM (Figure 1). The initial slope of the ASP⁺ uptake did not reach saturation at 5 mM, which was the highest concentration we were able to dissolve in an aqueous solution at pH 7.4. In uptake studies using [³H]MPP as substrate for the rOCT1, an IC₅₀ value for ASP⁺ of 1.4 ± 0.6 μM was determined (Figure 2). For all additional fluorescence experiments, an ASP⁺ concentration of 0.1 μM was used, which resulted in significant increases in cellular fluorescence. Figure 3 shows the time courses of the ASP⁺ (0.1 μM) uptake in nontransfected HEK293 and transfected rOCT1-HEK293 cells. In addition, the effects of the competitive cation transporter inhibitor cimetidine (1 mM) are shown for both cell types. The curves represent averages of 11 to 16 individual experiments during the observation period of 10 min. In the transfected cells, the initial uptake rate and the maximal fluorescence were markedly increased, and the uptake was inhibited by cimetidine. The increase in the cellular ASP⁺ fluorescence was also completely inhibited by the organic cations TEA⁺, TPA⁺, and quinine. Half-maximal inhibitions (IC₅₀) of the initial uptake rates were reached with 74 ± 15 nM TPA⁺, 1.5 ± 1.3 μM quinine, and 58 ± 16 μM TEA⁺ (see Figures 8 to 10Figures 8 to 10Figures 8 to 10). At a concentration of <10 μM TPA⁺, <100 μM quinine, or <1 mM TEA⁺, an almost complete inhibition of ASP⁺ transport was obtained. From tracer flux measurements after expression of rOCT1 in Xenopus laevis oocytes, IC₅₀ values for TPA⁺ of 0.43 μM and for quinine of 0.93 μM were determined (1).

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Figure 1.

Concentration-response curves for the increase of maximal fluorescence (in counts) and initial slope of the fluorescence increase (in counts per second) in the organic cation transporter type 1 from rat (rOCT1)-expressing HEK293 cells after addition of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP⁺) to the bathing solution. Mean values ± SEM are presented. The numbers of monolayers are given in parentheses.

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Figure 2.

Inhibition of rOCT1-mediated 1-[³H]methyl-4-phenylpyridinium ([³H]MPP) uptake by ASP⁺. Initial uptake rates of 0.15 μM MPP were determined in the presence of the different ASP⁺ concentrations or of 100 μM cyanine 863. The uptake rates in the absence and presence of cyanine 863 were 0.129 ± 0.012 and 0.011 ± 0.003 fmol/mg protein per s, respectively. The relative cyanine-inhibitable MPP uptake in the presence of ASP⁺ is indicated. Mean ± SD values from pairs of three determinations are shown, and the Hill equation was fitted to the data.

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Figure 3.

Time course of the increase in cellular fluorescence of nontransfected HEK293 and transfected rOCT1-HEK293 cells with or without 1 mM cimetidine. At time 0, 0.1 μM ASP⁺ was added to the bathing solution. The curves represent the mean values ± SEM of several individual experiments. n indicates the number of monolayers used.

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Figure 8.

Concentration-response curves for the effects of TEA⁺ on uptake of 0.1 μM ASP⁺ by rOCT1-HEK293 cells under control conditions and after incubation with 1 μM DOG for 10 min. Data are presented as initial slope of the uptake in percentages of the control experiments performed on the same day with cells of the same passage and age. IC₅₀ values calculated from these curves are 58 ± 16 μM before (□) and 1.0 ± 1.5 μM after (▪) DOG stimulation. The number of monolayers used for each concentration is given in parentheses.

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Figure 9.

Concentration-response curves for the effects of tetrapenthylammonium (TPA⁺) on uptake of 0.1 μM ASP⁺ by rOCT1-HEK293 cells under control conditions and after incubation with 1 μM DOG for 10 min. Data are presented as initial slope of the uptake in percentages of the control experiments performed on the same day with cells of the same passage and age. IC₅₀ values calculated from these curves are 74 ± 20 nM before (□) and 5.1 ± 1.4 nM after (▪) DOG stimulation. The number of monolayers used for each concentration is given in parentheses.

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Figure 10.

Concentration-response curves for the effects of quinine on uptake of 0.1 μM ASP⁺ by rOCT1-HEK293 cells under control conditions and after incubation with 1 μM DOG for 10 min. Data are presented as initial slope of the uptake in percentages of the control experiments performed on the same day with cells of the same passage and age. IC₅₀ values calculated from these curves are 1.5 ± 1.3 μM before (□) and 0.62 ± 0.12 μM after (▪) DOG stimulation. The number of monolayers used for each concentration is given in parentheses.

Previous functional data from Xenopus laevis oocytes expressing rOCT1 showed that this organic cation transporter is electrogenic (11). Therefore, we investigated the influence of changes in the membrane potential (V_m) on ASP⁺ uptake by rOCT1-HEK293 cells. Cells were depolarized by raising the extracellular K⁺ concentration from 3.6 mM to 50 and 145 mM with equimolar reductions in Na⁺. These elevations in extracellular K⁺ reduced V_m significantly from -44 ± 2 mV to -17 ± 1 mV and +6 ± 1 mV (n = 6), respectively. ASP⁺ uptake was significantly reduced in 13 experiments with 50 mM K⁺ by 36 ± 8% (initial rate) and 50 ± 9% (maximal fluorescence), and in six experiments with 145 mM K⁺ by 73 ± 7% (initial rate) and 87 ± 6% (maximal fluorescence).

Electrophysiologic Measurements

The effects of ASP⁺, quinine, and TEA⁺ on V_m of transfected and nontransfected HEK293 cells were measured to demonstrate the electrogeneity of the transport. Superfusion of the rOCT1-HEK293 cells with 10 or 100 μM ASP⁺ depolarized the cells significantly from -43 ± 2 mV by 5 ± 1 (n = 11) or 15 ± 2 mV (n = 25), respectively. In nontransfected HEK293 cells with a resting V_m of -49 ± 2 mV, ASP⁺ did not change V_m ΔV_m = 1 ± 1 mV, n = 4) at 10 μM and depolarized V_m significantly only by 7 ± 1 mV (n = 10) at 100 μM. In rOCT1-HEK293 cells, the membrane potential was also significantly depolarized by quinine (100 μM, n = 6) and TEA⁺ (1 mM, n = 6) (see open columns in Figure 4). Because quinine and TEA⁺ are also inhibitors of K⁺ channels, we tested their effects on rOCT1 after inhibition of the K⁺ channels by 1 mM Ba²⁺. In the presence of Ba²⁺, the membrane depolarizations by ASP⁺ and TEA⁺ were increased, whereas with quinine a small hyperpolarization was observed (Figure 4). The data are consistent with electrogenic transport of ASP⁺ and TEA⁺ by rOCT1 and with the previous observation that quinine is a nontransported inhibitor (12).

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Figure 4.

Effects of ASP⁺, quinine, and tetraethylammonium (TEA⁺) on membrane voltages (V_m) of rOCT1-HEK293 cells in the presence and absence of Ba²⁺ (1 mM). Results are given as mean values ± SEM of paired experiments with the number of experiments given in brackets. Differences between effects with or without Ba²⁺ are always significant (P < 0.05).

Regulation of ASP⁺ Uptake by Protein Kinases

When 1 μM of the adenylate cyclase activator forskolin was added to the control solution of rOCT1-HEK293 cells 10 min before transport measurements were started, both the initial rate of ASP⁺ uptake and the maximal fluorescence were increased (Figure 5). In contrast, no significant effect on ASP⁺ uptake could be observed in nontransfected HEK293 cells. The forskolin stimulation of ASP⁺ uptake by rOCT1-HEK293 cells was completely antagonized by the specific PKA inhibitor KT5720 (1 μM). In Figure 6, data on the effects of PKC stimulation on the ASP⁺ uptake by rOCT1 are shown. When 1 μM of the membrane-permeable diacylglycerol analog DOG was added to the control solution 10 min before the transport measurements were started, the initial slope and the maximal fluorescence were increased compared with control experiments without addition of DOG. When 20 μM of tamoxifen, a specific inhibitor of PKC, was added together with 1 μM DOG, the effect of DOG was nearly abolished. Next, we tested some concentrations of forskolin and DOG for their effects on ASP⁺ uptake. Forskolin and DOG were added 10 min before the uptake of ASP⁺ was recorded from the cellular fluorescence change. In Figure 7, both the initial rate of the cellular fluorescence change and the maximal cellular fluorescence are presented. The data show that at least 1 μM forskolin or DOG is required to obtain maximal effects. They suggest that the maximal fluorescence reflects a steady-state value that is dependent on the activity of rOCT1. To verify that the effects of forskolin and DOG on the ASP⁺-induced fluorescence reflect activity changes of rOCT1, we determined whether the effects of ASP⁺ on the membrane potential in rOCT1-HEK293 cells are changed by forskolin or DOG. The data from paired patchclamp experiments summarized in Table 1 show that the depolarizations induced by ASP⁺ (100 μM) were significantly increased with forskolin (1 μM) by 22% as well as with DOG (1 μM) by 20%. Forskolin did not significantly alter V_m, and DOG depolarized V_m only by 3 mV (Table 1) excluding significant changes in the driving force for ASP⁺ transport. To examine whether the increase in ASP⁺ uptake rates induced by DOG is due to a change in the affinity of the transporter, we measured the concentration dependence for the inhibition of 0.1 μM ASP⁺ uptake by TEA⁺ or TPA⁺ after stimulation of the cells with DOG (1 μM) for 10 min. Figure 8 shows that the affinity of rOCT1 for TEA⁺ is drastically changed after stimulation of PKC by DOG. The IC₅₀ value for TEA⁺ was 58 ± 16 μM in the absence and 1.0 ± 1.5 μM in the presence of DOG. As depicted in Figure 9, the IC₅₀ value for TPA⁺ was also increased from 74 ± 20 nM to 5.1 ± 1.4 nM after stimulation with DOG. Finally, we also examined whether the affinity of the rOCT1 to the nontransported organic cation quinine is also modulated by DOG. As can be seen from Figure 10, there was again a shift in the concentration-response curves for the quinine-induced inhibition of ASP⁺ uptake after DOG stimulation. The IC₅₀ values were 1.5 ± 1.3 μM before and 0.62 ± 0.12 nM after DOG incubation.

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Figure 5.

Effects of forskolin (1 μM) in the absence and presence of the protein kinase A (PKA)-specific inhibitor KT5720 (1 μM) on the uptake of 0.1 μM ASP⁺ by rOCT1-HEK293 cells. Monolayers were incubated with these substances for 10 min before 0.1 μM ASP⁺ was added at time 0. The two lower curves show that forskolin (1 μM) does not change the fluorescence increase in nontransfected HEK293 cells. The curves represent the mean values ± SEM of several separate experiments. n indicates the number of monolayers used.

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Figure 6.

Effects of 1 μM sn-1,2-dioctanoyl glycerol (DOG) in the absence and presence of the protein kinase C (PKC)-specific inhibitor tamoxifen (20 μM) on the uptake of 0.1 μM ASP⁺ by rOCT1-HEK293 cells. Monolayers were incubated with these substances for 10 min before ASP⁺ was added at time 0. The curves represent mean values ± SEM of several separate experiments. n indicates the numbers of monolayers used.

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Figure 7.

Effects of different concentrations of forskolin or DOG on the uptake of 0.1 μM ASP⁺. The columns on the left indicate initial cellular fluorescence changes after addition of ASP⁺ to the bathing solution. The columns on the right show the maximal fluorescence change that was obtained. Data are given in percentages above control measurements, which were performed with the same batch of rOCT1-HEK293 cells without addition of forskolin or DOG. Mean values ± SEM from the indicated number of monolayers (n) are presented.

To examine whether rOCT1-mediated ASP⁺ uptake is also regulated by endogenously activated tyrosine kinases, we examined the effects of three tyrosine kinase inhibitors : the broad-range tyrosine kinase inhibitor genestein, which is most specific for the endothelial growth factor receptor kinase at the used concentration of 10 μM ; the specific inhibitor of the platelet-derived growth factor receptor kinase tyrphostin AG1295 (10 μM) ; and the p561ck tyrosine kinase inhibitor aminogenestein (10 μM). Although genestein and tyrphostin AG1295 were without significant effects on basal ASP⁺ uptake (-14.4 ± 6.4%, n = 17 and 0.7 ± 10.6%, n = 12, respectively), aminogenestein reduced the basal ASP⁺ uptake by 63.4 ± 6.9% (n = 7).

Phosphorylation of rOCT1

Because rOCT1 contains several potential PKC-dependent phosphorylation sites (1) and one of these sites at serine 286 is conserved throughout the whole OCT family consisting of more than 20 different proteins (10), we tested whether a serine in rOCT1 is phosphorylated after PKC stimulation. Control HEK293 cells and rOCT1-HEK293 cells grown in parallel were incubated for 15 min without or with 1 μM DOG, harvested, and solubilized with 10% SDS. The total cellular protein was separated by SDS gel electrophoresis, transferred to nitrocellulose, and stained with a previously described polyclonal antibody against the extracellular loop of rOCT1 (23) or with a commercial antibody against phosphoserine. Figure 11 shows that DOG stimulates the phosphorylation of a serine residue in rOCT1-HEK293 cells. The polyclonal antibody against rOCT1 detects a band at about 60 kD in these cells (left panel). Also, in nontransfected HEK293 cells, a weak antibody staining was observed at this position that may be due to a reaction with an endogenous OCT type cation transporter, which had been suggested also by tracer uptake studies (our unpublished observations). There is an additional antibody staining at a lower molecular weight band that was not observed in Xenopus laevis oocytes or insect cells (23). The right panel of Figure 11 shows that an antibody against phosphoserine reacts in rOCT1-expressing cells at the same position where expressed rOCT1 protein was detected with the loop antibody. rOCT1 only shows a minor basal phosphorylation ; however, the phosphorylation was markedly increased when the cells were incubated with 1 μM DOG.

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Figure 11.

Demonstration that the phosphorylation of rOCT1 protein expressed in HEK293 cells is stimulated by DOG. Stably transfected rOCT-1HEK293 cells (rOCT1-HEK) and nontransfected HEK293 cells (HEK) were incubated in the absence (CTR) and presence (DOG) of 1 μM DOG, solubilized in sodium dodecyl sulfate (SDS), and then 20 μg of protein per lane was aplied to SDS-polyacrylamide gels. Western blots are shown that were developed either with an antibody against rOCT1 (left panel) or with an antibody against phosphoserine (right panel). The position of the expressed rOCT1 protein is indicated by an arrow.