2007 JASN IMPACT FACTOR 7.111	HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP
advanced	CURRENT ISSUE	ARCHIVES	JASN Express	ONLINE SUBMISSION

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MEHRENS, T. Articles by SCHLATTER, E. Search for Related Content PubMed PubMed Citation Articles by MEHRENS, T. Articles by SCHLATTER, E.

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

THOMAS MEHRENS^*, SILKE LELLECK^*, IBRAHIM ÇETINKAYA^*, MARION KNOLLMANN^*, HELGE HOHAGE^*, VALENTIN GORBOULEV, PETER BOKNÍK, HERMANN KOEPSELL and EBERHARD SCHLATTER^*

^* Medizinische Poliklinik, Experimentelle Nephrologie, Westfälische Wilhelms-Universität Münster, Germany
Institut für Pharmakologie und Toxikologie, Westfälische Wilhelms-Universität Münster, Germany
Anatomisches Institut, Universität Würzburg, Germany.

Correspondence to Dr. Eberhard Schlatter, Medizinische Poliklinik, Experimentelle Nephrologie, Westfälische Wilhelms-Universität Münster, Domagkstraße 3a, D-48149 Münster, Germany. Phone : +49 2518 356991 ; Fax : +49 2518 356973 ; E-mail : schlate{at}uni-muenster.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract. Members of the organic cation transporter (OCT) family are mainly expressed in kidney, liver, intestine, and brain. The regulation of the OCT type 1 from rat (rOCT1) stably transfected in HEK293 cells was examined using a fluorimetric technique, 1-[³H]methyl-4-phenylpyridinium uptake studies, and fast-whole-cell patch-clamp recordings. For the fluorescence measurements, the cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP⁺) was used as substrate. Uptake of ASP⁺ via rOCT1 was electrogenic, and its inhibition by other organic cations was consistent with previously reported radioactive tracer flux measurements. The inhibitor quinine was not translocated by the organic cation transporter in contrast to tetraethylammonium. Stimulation of diacyl glycerol-dependent protein kinase C (PKC) by sn-1,2-dioctanoyl glycerol (1 µM) resulted in an increase in initial ASP⁺ uptake rate by 216 ± 28% (n = 29). The effect was completely antagonized by the PKC inhibitor tamoxifen (20 µM, n = 22). Forskolin (1 µM), which activates adenylate cyclase and thereby protein kinase A (PKA), stimulated the initial rate of ASP⁺ accumulation by 51 ± 6% (n = 19). This effect was inhibited by the specific PKA inhibitor KT5720 (1 µM, n = 12). Inhibition of tyrosine kinases by aminogenestein (10 µM) reduced ASP⁺ uptake by 63 ± 7% (n = 7), while genestein or tyrphostin AG1295 (each 10 µM) were without significant effects. Incubation of the cells with sn-1,2-dioctanoyl glycerol (1 µM) increased the affinities of the transporter to tetraethylammonium, tetrapenthylammonium, and quinine by a factor of 58, 14.5, and 2.4, respectively. Western blot analysis revealed that rOCT1 protein was phosphorylated at a serine residue upon stimulation of PKC. In conclusion, it has been demonstrated that the organic cation transport by rOCT1 is stimulated by PKC, PKA, and endogenous tyrosine kinase activation. The PKC phosphorylates rOCT1 and leads to a conformational change at the substrate binding site.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Many drugs such as antihistamines, antibiotics, cytostatics, antiarrhythmics, opiates, and endogenous metabolites are organic cations. Since the identification of a polyspecific organic cation transporter in rats (rOCT1), which translocates a variety of small organic cations and is inhibited by large cations, a better understanding for drug interactions on renal cation absorption and excretion has been achieved (1). Homology cloning techniques allowed the identification of organic cation transporter subtypes of rat, mouse, rabbit, pig, and humans (2,3,4,5,6,7,8,9,10). Immunohistochemistry and functional studies with rOCT1-transfected cells in comparison to in vivo experiments suggest that rOCT1 is the basolateral organic transporter in proximal tubules, small intestinal enterocytes, and sinusoidal membranes of hepatocytes (9,10). In these tissues, rOCT1 facilitates transport of organic cations from the blood compartment into the cells.

Radioactive tracer flux measurements with transfected oocytes from Xenopus laevis expressing rOCT1 characterized the transport as electrogenic, but NaCl- and pH-independent (11). Type 1 organic cations such as tetraethylammonium, choline, N'-methylnicotinamide, and monoamines are competitive substrates that are translocated by this transporter, whereas type 2 organic cations such as quinine, quinidine, cyanine 863, and d-tubocurarine are nontransported inhibitors of the transporter (12). Regulation of substrate transporters has been under investigation only recently. We demonstrated that organic anion and cation transport in nonperfused rabbit renal proximal S2 tubules was stimulated by protein kinase C (PKC) (13,14). Furthermore, we investigated organic cation transport in cell lines (IHKE-1 and LLC-PK₁) with typical characteristics of proximal tubule cells. Organic cation transport in the human cell line IHKE-1 was stimulated by protein kinase A (PKA), PKC, and protein kinase G (PKG) activation, whereas in porcine LLC-PK₁ cells it was inhibited by PKC activation without effects of PKA or PKG stimulation (15).

In the present study, the effects of protein kinase activation on transport of the fluorescence organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP⁺) (16) in human embryonic epithelial cells (HEK293) stably transfected with rOCT1 (rOCT1-HEK293 cells) were investigated using dynamic fluorescence microscopy, electrophysiologic patchclamp recordings, and 1-[³H]methyl-4-phenylpyridinium (MPP) uptake measurements. This fluorimetric technique, which was established by Ullrich et al. (17,18) in vivo and recently modified by us for in vitro systems (19,20), allows the dynamic analysis of organic cation transport by measurements of cellular uptake of the fluorescent dye ASP⁺. ASP⁺ has been shown to be a substrate for luminal as well as basolateral organic cation transport systems of the rat renal proximal tubule in vivo (21).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 x100 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 m 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(ß-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 x 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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
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).

View larger version (23K): [in this window] [in a new window] [as a PowerPoint slide]   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.

View larger version (12K): [in this window] [in a new window] [as a PowerPoint slide]   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.

View larger version (38K): [in this window] [in a new window] [as a PowerPoint slide]   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.

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   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.

View larger version (17K): [in this window] [in a new window] [as a PowerPoint slide]   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.

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   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 V_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).

View larger version (14K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Effects of ASP+, quinine, and tetraethylammonium (TEA+) on membrane voltages (Vm) 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.

View larger version (37K): [in this window] [in a new window] [as a PowerPoint slide]   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.

View larger version (27K): [in this window] [in a new window] [as a PowerPoint slide]   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.

View larger version (31K): [in this window] [in a new window] [as a PowerPoint slide]   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.

View larger version (44K): [in this window] [in a new window] [as a PowerPoint slide]   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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study shows that the cationic fluorescent dye ASP⁺ is a substrate of the polyspecific cation transporter rOCT1 and can be used to continuously monitor cation transport activity with high temporal resolution (15,19,20). The measurements were performed with HEK293 cells stably transfected with rOCT1, which proved to be the first member of a large protein family that includes five different transporter subtypes (10). Transport activity by rOCT1 expressed in HEK293 cells could be analyzed by measuring the fluorescence change and in addition by measuring inhibition of [³H]MPP uptake after addition of ASP⁺ to the superfusion solution. Both methods revealed an affinity of the rOCT1 for ASP⁺ of approximately 1 µM. By this fluorescence method, comparable IC₅₀ values for transport inhibition of TEA⁺, TPA⁺, and quinine were obtained as reported previously (1). Furthermore, it could be demonstrated that the ASP⁺ uptake by rOCT1 is stimulated by PKA and PKC and that the PKC stimulation leads to the phosphorylation of rOCT1 at a serine residue. rOCT1 expressed in these HEK293 cells is apparently also endogenously stimulated by tyrosine kinases. In addition, we describe that the affinity of rOCT1 to TEA⁺, TPA⁺, and quinine was increased by variable amounts after stimulation by PKC.

The initial slope of the cellular ASP⁺ fluorescence increase was used to characterize the transport kinetics of this transporter. We demonstrate that the uptake of the fluorescent dye ASP⁺ by rOCT1-HEK293 cells showed saturation, which was reached within minutes (Figures 2,5, and 6). The initial rate of ASP⁺ fluorescence increase directly reflects transport of ASP⁺ by the rOCT1. The maximal fluorescence obtained after several minutes, however, is the sum of transport of ASP⁺, leakage of ASP⁺ from the cells, and bleaching of the dye. The experiments presented in Figures 2 and 5 also indicate that specific accumulation of ASP⁺ in HEK293 cells is dependent on transfection with the rOCT1 protein. The small increase in cellular ASP⁺ accumulation in nontransfected cells could not be inhibited by the specific competitive substrate cimetidine. The presence of a so far undefined transport system for organic cations in these embryonic kidney cells is also evident from the small depolarization induced by ASP⁺. Another known characteristic of rOCT1 is its electrogeneity. This was confirmed in our study by the voltage dependence of the ASP⁺ uptake and the transport-specific depolarization of rOCT1-HEK293 cells induced by ASP⁺ and TEA⁺ (Figure 5). These depolarizations were even larger when the K⁺ conductances were blocked by Ba²⁺, which separates the effects of TEA⁺ as K⁺ channel inhibitor from those as cationic substrate of rOCT1. These enhanced depolarizations reflect the increased contribution of the rOCT1 to the membrane voltage when the otherwise dominating K⁺ conductances are blocked. The small hyperpolarization induced by quinine under these conditions is probably due to its effects on Cl^- and nonselective cation conductances (24) and confirms the previous observation that quinine is an inhibitor of organic cation transporters, which is not transported itself (12).

One intention of this study was to examine a possible regulation of the rOCT1 transporter by protein kinases. In previous studies, PKC stimulation of organic cation as well as anion transport was found in freshly isolated S2 segments of rabbit proximal tubules (13,14). Recently, we demonstrated an activation of organic cation transport across the apical membrane of human cultured proximal tubule cells (IHKE-1) by PKA, PKC, and PKG (15). Here, we present the first evidence for a protein kinase-mediated regulation of a molecularly identified organic cation transporter. Comparable observations have only been made for the multispecific organic anion transporter OAT1, in which an inhibition of transport activity was demonstrated after PKC activation (25). The accumulation of ASP⁺ by rOCT1-HEK293 cells was stimulated by an activation of PKC and, to a lesser extent, of PKA. An endogenous activation of this transporter is also evident from the decrease in transport rates seen after inhibition of the p56lck tyrosine kinases. The involvement of the protein kinases when DOG or forskolin was added is evident from the inhibition of these effects by the specific protein kinase inhibitors tamoxifen (PKC) and KT5720 (PKA). The stimulation of these two protein kinase pathways resulted in an increase of the initial slope of ASP⁺ uptake (Figures 5 through 7 Figures 5 through 7Figures 5 through 7). Furthermore, stimulation of ASP⁺ transport was also demonstrated by the slightly increased depolarizations of rOCT1-HEK293 cells induced by addition of ASP⁺ after stimulation of PKA or PKC (Table 1). The fact that the concentration-response curve for the inhibition of initial ASP⁺ uptake by TEA⁺ or TPA⁺ was shifted to the left by more than one order of magnitude after activation of PKC (Figures 8 and 9) indicates that the increase in transport is due to an increase in the affinity of the rOCT1 molecule. Furthermore, the shift in the IC₅₀ value for the inhibition of ASP⁺ transport by quinine as well indicates that the binding domain of the protein is the same for the transported substrates as well as for the nontransported inhibitors.

To support the finding that the regulatory effect of PKC stimulation was a direct phosphorylation of the protein rather than an increased membrane trafficking and thereby an increase in the number of active transporters, we analyzed the phosphorylation of rOCT1 protein by Western blotting. By structural analysis of the rOCT1 protein, it was shown that the intracellular loops of the protein contain six putative PKC phosphorylation sites and that one of these was conserved between all members of the OCT family (1). Therefore, we used a specific antibody that detects phosphorylated phosphoserine residues. We demonstrate that the rOCT1 protein has a minor basal phosphorylation of phosphoserine sites, which increased markedly after stimulation of PKC (DOG). We conclude that direct phosphorylation of the rOCT1 protein regulates the transport capacity probably due to an increased affinity to the substrates. This modulation of the substrate affinity of rOCT1 may explain the differences in the substrate affinities of organic cation transporters determined in vivo or after expression of cloned transporters in different cellular systems. PKC regulation of substrate transport has been observed in various other systems. As mentioned above in isolated segments of nonperfused rabbit proximal tubules, organic anion and cation transport was stimulated (15), whereas para-aminohippuric acid transport in opossum kidney cells (26) and also the activity of the organic anion transporter OAT1 expressed in Xenopus laevis oocytes was inhibited by PKC activation (25). Activation of PKC also effects Na⁺/H⁺ exchange (27,28,29), the Na⁺/phosphate transporter, and the Na⁺/HCO₃^- exchanger of the proximal tubule (30). Regulation of organic substrate transport by PKA has only been reported for organic cation transport across the luminal membrane of a human proximal tubule cell line by us (15) and for the Na⁺/glucose cotransporter of rabbit and humans (31). PKA-mediated regulation of ion transporters in renal epithelia has also been shown for a variety of other transport mechanisms such as Na⁺/H⁺ exchange (32, 33), Na⁺/HCO₃^- exchange, and Na⁺/K⁺-ATPase (32).

In this study, we demonstrate that the rOCT1 protein is stimulated by PKA, PKC, and tyrosine kinases and that PKC-mediated phosphorylation of the protein leads to changes in its substrate affinity. Such a regulation of organic cation transport is certainly important for in vivo drug absorption and drug excretion.

	Acknowledgments

 
This work was supported by the German Federal Government, the Department of Education and Research (Grants 01 EC 9401 and 01 EC 9801), and the Deutsche Forschungsgemeinschaft (SFB 174 Grant A22). We thank Sabine Haxelmans, Ingrid Kleta, and Joachim Windau for their expert technical assistance.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Gründemann D, Gorboulev V, Gambaryan S, Veyhl M, Koepsell H : Drug excretion mediated by a new prototype of polyspecific transporter. Nature372 : 549-552,1994[Medline]
2. Lopez-Nieto CE, You G, Bush KT, Baros EJ, Beier DR, Nigam SK : Molecular cloning and characterization of NKT, a gene product related to the organic cation transporter family that is almost exclusively expressed in the kidney. J Biol Chem 272 :6471 -6478, 1997[Abstract/Free Full Text]
3. Gründemann D, Babin-Ebell J, Martel F, Örding N, Schmidt A, Schömig E : Primary structure and functional expression of the apical organic cation transporter from kidney epithelial LLC-PK₁ cells. J Biol Chem272 : 10408-10413,1997[Abstract/Free Full Text]
4. Zhang L, Dresser MJ, Gray AT, Yost SC, Terashita S, Giacomini KM : Cloning and functional expression of a human liver organic cation transporter. Mol Pharmacol 51 :913 -921, 1997[Abstract/Free Full Text]
5. Zhang L, Dresser MJ, Chun JK, Babbitt PC, Giacomini KM : Cloning and functional characterization of a rat renal organic cation transport isoform (rOCT1A). J Biol Chem272 : 16548-16554,1997[Abstract/Free Full Text]
6. Gorboulev V, Ulzheimer JC, Akhoundova A, Ulzheimer-Teuber I, Karbach U : Cloning and characterization of two human polyspecific organic cation transporters. DNA Cell Biol16 : 871-881,1997[Medline]
7. Terashita S, Dresser A, Zhang L, Gray AT, Yost SC, Giacomelli K : Molecular cloning and functional expression of a rabbit renal organic cation transporter. Biochim Biophys Acta 1369 : 1-6,1998
8. Zhang L, Brett CM, Giacomini KM : Role of organic cation transporters in drug absorption and elimination. Ann Rev Pharmacol Toxicol 38 :431 -460, 1998[Medline]
9. Koepsell H : Organic cation transporters in intestine, kidney, liver, and brain. Annu Rev Physiol60 : 243-266,1998[Medline]
10. Koepsell H, Gorboulev V, Arndt P : Molecular pharmacology of organic cation transporters in kidney. J Membr Biol167 : 103-117,1999[Medline]
11. Busch AE, Quester S, Ulzheimer JC, Waldegger S, Gorboulev V, Arndt P, Lang F, Koepsell H : Electrogenic properties and substrate specificity of the polyspecific rat cation transporter rOCT1. J Biol Chem 271 : 1-5,1996[Free Full Text]
12. Nagel G, Volk C, Friedrich T, Ulzheimer JC, Bamberg E, Koepsell H : A reevaluation of substrate specificity of the rat cation transporter rOCT1. J Biol Chem 272 :31953 -31956, 1997[Abstract/Free Full Text]
13. Hohage H, Löhr M, Querl U, Greven J : The renal basolateral transport system for organic anions : Properties of the regulation mechanism. J Pharmacol Exp Ther269 : 659-664,1994[Abstract/Free Full Text]
14. Hohage H, Mörth DM, Querl IU, Greven J : Regulation by protein kinase C of the contraluminal transport system for organic cations in rabbit kidney S2 proximal tubules. J Pharmacol Exp Ther 268 :897 -901, 1994[Abstract/Free Full Text]
15. Hohage H, Stachon A, Feidt C, Hirsch JR, Schlatter E : Regulation of organic cation transport in IHKE-1 and LLC-PK₁ cells : Fluorimetric studies with 4-(4-dimethylaminostyryl)-N-methylpyridinium. J Pharmacol Exp Ther 286 :305 -310, 1998[Abstract/Free Full Text]
16. Dobretsov GE, Morozova GI, Barenboim GM : Free energy of the accumulation of a fluorescent cation probe within lymphocyte mitochondria. Biofizika 30 :833 -836, 1985[Medline]
17. Pietruck F, Ullrich KJ : Transport interactions of different organic cations during their excretion by the intact rat kidney. Kidney Int 47 :1647 -1657, 1995[Medline]
18. Rohlicek V, Ullrich KJ : Simple device for continuous measurement of fluorescent anions and cations in the rat kidney in situ. Renal Physiol Biochem 17 : 57 -61, 1994
19. Stachon A, Hohage H, Feidt C, Schlatter E : Characterization of organic cation transport across the apical membrane of proximal tubular cells with the fluorescent dye 4-Di-1-ASP. Cell Physiol Biochem 7 :264 -274, 1997
20. Stachon A, Schlatter E, Hohage H : Dynamic monitoring of organic cation transport processes by fluorescence measurements in LLC-PK₁ cells. Cell Physiol Biochem 6 :72 -81, 1996
21. Ullrich KJ, Rumrich G : Luminal transport system for choline⁺ in relation to the other organic cation transport systems in the rat proximal tubule—Kinetics, specificity : Alkyl/arylamines, alkylamines with OH, O, SH, NH₂, ROCO, RSCO and H₂PO₄-groups, methylaminostyryl, rhodamine, acridine, phenanthrene and cyanine compounds. Pflügers Arch432 : 471-485,1996[Medline]
22. Busch AE, Quester S, Ulzheimer JC, Gorboulev V, Akhoundova A, Waldegger S, Lang F, Koepsell H : Monoamine neurotransmitter transport mediated by the polyspecific cation transporter rOCT1. FEBS Lett 395 :153 -156, 1996[Medline]
23. Meyer-Wentrup F, Karbach U, Gorboulev V, Arndt P, Koepsell H : Membrane localization of the electrogenic cation transporter rOCT1 in rat liver. Biochem Biophys Res Commun248 : 673-678,1998[Medline]
24. Gögelein H, Capek K : Quinine inhibits chloride and nonselective cation channels in isolated rat distal colon cells. Biochim Biophys Acta1027 : 191-198,1990[Medline]
25. Uwai Y, Okuda M, Takami K, Hashimoto Y, Inui KI : Functional characterization of the rat multispecific organic anion transporter OAT1 mediating basolateral uptake of anionic drugs in the kidney. FEBS Lett 438 :321 -324, 1998[Medline]
26. Takano M, Nagai J, Yasuhara M, Inui KI : Regulation of p-aminohippurate transport by protein kinase C in OK kidney epithelial cells. Am J Physiol271 : F469-F475,1996[Abstract/Free Full Text]
27. Wang T, Chan YL : Time- and dose-dependent effects of protein kinase C on proximal bicarbonate transport. J Membr Biol 117 :131 -139, 1995
28. Reboucas NA, Malnic G : Regulation of rat proximal tubule Na/H exchange by protein kinase C. Renal Physiol Biochem19 : 87-93,1996
29. Azuma KK, Balkovetz DF, Magyar CE, Lescale-Matys L, Zhang YB, Chambrey R, Warnock DG, McDonough AA : Renal Na⁺/H⁺ exchanger isoforms and their regulation by thyroid hormone. Am J Physiol 270 :C585 -C592, 1996[Abstract/Free Full Text]
30. Yamada H, Seki G, Taniguchi S, Uwatoko S, Nosaka K, Suzuki K, Kurokawa K : Roles of Ca²⁺ and PKC in regulation of acid/base transport in isolated proximal tubules. Am J Physiol271 : F1068-F1076,1996[Abstract/Free Full Text]
31. Hirsch JR, Loo DDF, Wright EM : Regulation of Na⁺/glucose cotransporter expression by protein kinases in Xenopus laevis oocytes. J Biol Chem271 : 14740-14746,1996[Abstract/Free Full Text]
32. Ruiz OS, Qiu YY, Wang LJ, Arruda JAL : Regulation of the renal Na-HCO₃ cotransporter. V. Mechanism of the inhibitory effect of parathyroid hormone. Kidney Int49 : 396-402,1996[Medline]
33. Schlatter E, Haxelmans S, Ankorina I, Kleta R : Regulation of Na⁺/H⁺ exchange by diadenosine polyphosphates, angiotensin II and vasopresin in rat cortical collecting duct. J Am Soc Nephrol 6 :1223 -1229, 1995[Abstract]

This article has been cited by other articles:

				M. Mertl, H. Daniel, and G. Kottra Substrate-induced changes in the density of peptide transporter PEPT1 expressed in Xenopus oocytes Am J Physiol Cell Physiol, November 1, 2008; 295(5): C1332 - C1343. [Abstract] [Full Text] [PDF]

				A. Sturm, V. Gorboulev, D. Gorbunov, T. Keller, C. Volk, B. M. Schmitt, P. Schlachtbauer, G. Ciarimboli, and H. Koepsell Identification of cysteines in rat organic cation transporters rOCT1 (C322, C451) and rOCT2 (C451) critical for transport activity and substrate affinity Am J Physiol Renal Physiol, September 1, 2007; 293(3): F767 - F779. [Abstract] [Full Text] [PDF]

				R. D. Blakely and L. J. DeFelice All Aglow about Presynaptic Receptor Regulation of Neurotransmitter Transporters Mol. Pharmacol., May 1, 2007; 71(5): 1206 - 1208. [Abstract] [Full Text] [PDF]

				J. Biermann, D. Lang, V. Gorboulev, H. Koepsell, A. Sindic, R. Schroter, A. Zvirbliene, H. Pavenstadt, E. Schlatter, and G. Ciarimboli Characterization of regulatory mechanisms and states of human organic cation transporter 2 Am J Physiol Cell Physiol, June 1, 2006; 290(6): C1521 - C1531. [Abstract] [Full Text] [PDF]

				G. Ciarimboli, T. Ludwig, D. Lang, H. Pavenstadt, H. Koepsell, H.-J. Piechota, J. Haier, U. Jaehde, J. Zisowsky, and E. Schlatter Cisplatin Nephrotoxicity Is Critically Mediated via the Human Organic Cation Transporter 2 Am. J. Pathol., December 1, 2005; 167(6): 1477 - 1484. [Abstract] [Full Text] [PDF]

				H. Tahara, H. Kusuhara, H. Endou, H. Koepsell, T. Imaoka, E. Fuse, and Y. Sugiyama A Species Difference in the Transport Activities of H2 Receptor Antagonists by Rat and Human Renal Organic Anion and Cation Transporters J. Pharmacol. Exp. Ther., October 1, 2005; 315(1): 337 - 345. [Abstract] [Full Text] [PDF]

				R. D. Blakely, L. J. DeFelice, and A. Galli Biogenic Amine Neurotransmitter Transporters: Just When You Thought You Knew Them Physiology, August 1, 2005; 20(4): 225 - 231. [Abstract] [Full Text] [PDF]

				S. Gu, C. J. Villegas, and J. X. Jiang Differential Regulation of Amino Acid Transporter SNAT3 by Insulin in Hepatocytes J. Biol. Chem., July 15, 2005; 280(28): 26055 - 26062. [Abstract] [Full Text] [PDF]

				P. Chandra, P. Zhang, and K. L. R. Brouwer Short-term regulation of multidrug resistance-associated protein 3 in rat and human hepatocytes Am J Physiol Gastrointest Liver Physiol, June 1, 2005; 288(6): G1252 - G1258. [Abstract] [Full Text] [PDF]

				G. Ciarimboli, H. Koepsell, M. Iordanova, V. Gorboulev, B. Durner, D. Lang, B. Edemir, R. Schroter, T. Van Le, and E. Schlatter Individual PKC-Phosphorylation Sites in Organic Cation Transporter 1 Determine Substrate Selectivity and Transport Regulation J. Am. Soc. Nephrol., June 1, 2005; 16(6): 1562 - 1570. [Abstract] [Full Text] [PDF]

				C. Popp, V. Gorboulev, T. D. Muller, D. Gorbunov, N. Shatskaya, and H. Koepsell Amino Acids Critical for Substrate Affinity of Rat Organic Cation Transporter 1 Line the Substrate Binding Region in a Model Derived from the Tertiary Structure of Lactose Permease Mol. Pharmacol., May 1, 2005; 67(5): 1600 - 1611. [Abstract] [Full Text] [PDF]

				S. H. Wright and W. H. Dantzler Molecular and Cellular Physiology of Renal Organic Cation and Anion Transport Physiol Rev, July 1, 2004; 84(3): 987 - 1049. [Abstract] [Full Text] [PDF]

				B. Grover, D. Buckley, A. R. Buckley, and W. Cacini Reduced Expression of Organic Cation Transporters rOCT1 and rOCT2 in Experimental Diabetes J. Pharmacol. Exp. Ther., March 1, 2004; 308(3): 949 - 956. [Abstract] [Full Text] [PDF]

				Y. Hagos, B. C. Burckhardt, A. Larsen, C. Mathys, T. Gronow, A. Bahn, N. A. Wolff, G. Burckhardt, and J. Steffgen Regulation of sodium-dicarboxylate cotransporter-3 from winter flounder kidney by protein kinase C Am J Physiol Renal Physiol, January 1, 2004; 286(1): F86 - F93. [Abstract] [Full Text] [PDF]

				O. Dohan, A. De la Vieja, V. Paroder, C. Riedel, M. Artani, M. Reed, C. S. Ginter, and N. Carrasco The Sodium/Iodide Symporter (NIS): Characterization, Regulation, and Medical Significance Endocr. Rev., February 1, 2003; 24(1): 48 - 77. [Abstract] [Full Text] [PDF]

				I. Cetinkaya, G. Ciarimboli, G. Yalcinkaya, T. Mehrens, A. Velic, J. R. Hirsch, V. Gorboulev, H. Koepsell, and E. Schlatter Regulation of human organic cation transporter hOCT2 by PKA, PI3K, and calmodulin-dependent kinases Am J Physiol Renal Physiol, February 1, 2003; 284(2): F293 - F302. [Abstract] [Full Text] [PDF]

				K. B. Goralski, G. Lou, M. T. Prowse, V. Gorboulev, C. Volk, H. Koepsell, and D. S. Sitar The Cation Transporters rOCT1 and rOCT2 Interact with Bicarbonate but Play Only a Minor Role for Amantadine Uptake into Rat Renal Proximal Tubules J. Pharmacol. Exp. Ther., December 1, 2002; 303(3): 959 - 968. [Abstract] [Full Text] [PDF]

X. Zhang, K. K. Evans, and S. H. Wright Molecular c