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A Reevaluation of Substrate Specificity of the Rat Cation Transporter rOCT1

1997; Elsevier BV; Volume: 272; Issue: 51 Linguagem: Inglês

10.1074/jbc.272.51.31953

1083-351X

Georg Nagel, Christopher Volk, Thomas Friedrich, Jochen C. Ulzheimer, Ernst Bamberg, Hermann Koepsell,

Pregnancy and Medication Impact

The substrate specificity of the previously cloned rat cation transporter rOCT1, which is expressed in kidney, liver, and small intestine, was reevaluated. rOCT1 is the first member of a new protein family comprising electrogenic and polyspecific cation transporters that transport hydrophilic cations like tetraethylammonium, choline, and monoamine neurotransmitters. Previous electrical measurements suggested that cations like quinine, quinidine, and cyanine 863, which have been classified as type 2 cations in the liver, are also transported by rOCT1, since they may induce inward currents in rOCT1 expressing Xenopus oocytes (Busch, A. E., Quester, S., Ulzheimer, J. C., Waldegger, S., Gorboulev, V., Arndt, P., Lang, F., and Koepsell, H. (1996) J. Biol. Chem. 271, 32599–32604). Tracer flux measurements with oocytes and with stably transfected human embryonic kidney cells showed that [3H]quinine and [3H]quinidine are not transported by rOCT1. The voltage dependence observed for the quinine- or quinidine-induced inward currents in rOCT1-expressing oocytes, and tracer efflux measurements indicate that the inward currents by type 2 cations are generated by the inhibition of electrogenic efflux of transported type 1 cations. Therefore, rOCT1 cannot contribute to transport of type 2 cations in the liver and the hepatic transporter for type 2 cations remains to be identified. The substrate specificity of the previously cloned rat cation transporter rOCT1, which is expressed in kidney, liver, and small intestine, was reevaluated. rOCT1 is the first member of a new protein family comprising electrogenic and polyspecific cation transporters that transport hydrophilic cations like tetraethylammonium, choline, and monoamine neurotransmitters. Previous electrical measurements suggested that cations like quinine, quinidine, and cyanine 863, which have been classified as type 2 cations in the liver, are also transported by rOCT1, since they may induce inward currents in rOCT1 expressing Xenopus oocytes (Busch, A. E., Quester, S., Ulzheimer, J. C., Waldegger, S., Gorboulev, V., Arndt, P., Lang, F., and Koepsell, H. (1996) J. Biol. Chem. 271, 32599–32604). Tracer flux measurements with oocytes and with stably transfected human embryonic kidney cells showed that [3H]quinine and [3H]quinidine are not transported by rOCT1. The voltage dependence observed for the quinine- or quinidine-induced inward currents in rOCT1-expressing oocytes, and tracer efflux measurements indicate that the inward currents by type 2 cations are generated by the inhibition of electrogenic efflux of transported type 1 cations. Therefore, rOCT1 cannot contribute to transport of type 2 cations in the liver and the hepatic transporter for type 2 cations remains to be identified. Recently a cation transporter has been sequenced from a cDNA library of rat kidney (rOCT1) that mediates electrogenic transport of organic cations with different molecular structures (1Gründemann D. Gorboulev V. Gambaryan S. Veyhl M. Koepsell H. Nature. 1994; 372: 549-552Crossref PubMed Scopus (608) Google Scholar, 2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 3Busch A.E. Quester S. Ulzheimer J.C. Gorboulev V. Akhoundova A. Waldegger S. Lang F. Koepsell H. FEBS Lett. 1996; 395: 153-156Crossref PubMed Scopus (90) Google Scholar). rOCT1 is the first member of a new, rapidly growing protein family (2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 4Zhang L. Dresser M.J. Chun J.K. Babbitt P.C. Giacomini K.M. J. Biol. Chem. 1997; 272: 16548-16554Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 5Schweifer N. Barlow D.P. Mamm. Genome. 1996; 7: 735-740Crossref PubMed Scopus (73) Google Scholar, 6Gorboulev V. Ulzheimer J.C. Akhoundova A. Ulzheimer-Teuber I. Karbach U. Quester S. Baumann C. Lang F. Busch A.E. Koepsell H. DNA Cell Biol. 1997; 16: 871-881Crossref PubMed Scopus (548) Google Scholar, 7Zhang L. Dresser M.J. Gray A.T. Yost S.C. Terashita S. Giacomini K.M. Mol. Pharmacol. 1997; 51: 913-921Crossref PubMed Scopus (350) Google Scholar, 8Gründemann D. Babin-Ebell J. Martel F. Örding N. Schmidt A. Schömig E. J. Biol. Chem. 1997; 272: 10408-10413Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 9Okuda M. Saito H. Urakami Y. Takano M. Inui K.-I. Biochem. Biophys. Res. Commun. 1996; 224: 500-507Crossref PubMed Scopus (326) Google Scholar, 10Sekine T. Watanabe N. Hosoyamada M. Kanai Y. Endou H. J. Biol. Chem. 1997; 272: 18526-18529Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar) that belongs to a major superfamily of transmembrane facilitators (12Marger M.D. Saier Jr., M.H. Trends Biochem. Sci. 1993; 18: 13-20Abstract Full Text PDF PubMed Scopus (753) Google Scholar). The OCT family includes a second subtype, OCT2 (2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 9Okuda M. Saito H. Urakami Y. Takano M. Inui K.-I. Biochem. Biophys. Res. Commun. 1996; 224: 500-507Crossref PubMed Scopus (326) Google Scholar), and the polyspecific renal anion transporter OAT1, which is known as the paraaminohippurate transporter (10Sekine T. Watanabe N. Hosoyamada M. Kanai Y. Endou H. J. Biol. Chem. 1997; 272: 18526-18529Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 11Lopez-Nieto C.E. You G. Bush K.T. Barros E.J.G. Beier D.R. Nigam S.K. J. Biol. Chem. 1997; 272: 6471-6478Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar,13Roch-Ramel F. Besseghir K. Murer H. Windhager E.E. Handbook of Physiology. Oxford University Press, New York1992: 2189-2262Google Scholar). rOCT1 probably mediates the first step in the excretion of many organic cations in the kidney, small intestine, and liver, since it was localized in the basolateral membrane of renal proximal tubules and intestinal enterocytes and in the sinusoidal membrane of hepatocytes (14Koepsell H. Annu. Rev. Physiol. 1998; 60: 243-266Crossref PubMed Scopus (287) Google Scholar). 1F. Meyer-Wentrup, U. Karbach, and H. Koepsell, unpublished data. The functional properties of rOCT1 have been characterized after expression in Xenopus oocytes and in human embryonic kidney (HEK 293) 2The abbreviations used are: HEK, human embryonic kidney; TEA, tetraethylammonium; MPP, 1-methyl-4-phenylpyridinium. cells (1Gründemann D. Gorboulev V. Gambaryan S. Veyhl M. Koepsell H. Nature. 1994; 372: 549-552Crossref PubMed Scopus (608) Google Scholar, 2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 3Busch A.E. Quester S. Ulzheimer J.C. Gorboulev V. Akhoundova A. Waldegger S. Lang F. Koepsell H. FEBS Lett. 1996; 395: 153-156Crossref PubMed Scopus (90) Google Scholar). By tracer flux measurements it was shown that rOCT1 mediates transport of structurally different cations, such as tetraethylammonium (TEA), N 1-methylnicotinamide, choline, 1-methyl-4-phenylpyridinium (MPP), and dopamine. In voltage-clamped oocytes rOCT1 was also characterized by electrical measurements (2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). When TEA, choline, MPP, or dopamine was added to the bath of rOCT1 expressing oocytes, inward currents were induced, and it was concluded that the uptake of these cations is electrogenic. Electrical measurements were also performed with the more hydrophobic cations, quinine, quinidine, d-tubocurarine, and cyanine 863 (2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). In transport measurements with isolated rat hepatocytes, these cations had been previously classified as type 2 cations, and it had been postulated that these cations were translocated by a separate transport system to the more hydrophilic type 1 cations like TEA and choline (15Groothuis G.M.M. Meijer D.K.F. J. Hepatol. 1996; 24: 3-28PubMed Google Scholar, 16Elferink R.P.J.O. Meijer D.K.F. Kuipers F. Jansen P.L.M. Groen A.K. Groothuis G.M.M. Biochim. Biophys. Acta. 1995; 1241: 215-268Crossref PubMed Scopus (384) Google Scholar). Since, in rOCT1 expressing oocytes, large inward currents were also induced by quinine, quinidine,d-tubocurarine, and cyanine 863, it was proposed that rOCT1 translocates type 1 and type 2 cations (2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) to a similar extent. The present paper disproves this hypothesis. Oocytes were injected with 10 ng of rOCT1 cRNA per oocyte as described before (1Gründemann D. Gorboulev V. Gambaryan S. Veyhl M. Koepsell H. Nature. 1994; 372: 549-552Crossref PubMed Scopus (608) Google Scholar, 2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) and were stored 2–4 days in 5 mm Hepes-Tris, pH 7.4, 96 mm NaCl, 2 mm KCl, 1.8 mmCaCl2, 1 mm MgCl2 (ORi) containing 50 mg/l gentamicin. Tracer uptake and efflux experiments were performed as described earlier (2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Initial efflux rates were estimated from monoexponential curves that were fitted to the data. For electrical measurements the oocytes were permanently superfused with fresh solution at room temperature (∼3 ml/min, 22–24 °C). For the determination of current-voltage relations, steady-state current was measured during the last 100 ms of 500-ms voltage pulses to different potentials in the absence and presence of the agonist. The averages of the currents before and after agonist application were subtracted from the currents during agonist application. HEK 293 cells were transfected and selected as described (3Busch A.E. Quester S. Ulzheimer J.C. Gorboulev V. Akhoundova A. Waldegger S. Lang F. Koepsell H. FEBS Lett. 1996; 395: 153-156Crossref PubMed Scopus (90) Google Scholar). A stably transfected single clone was isolated and grown in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum and 0.6 mg/ml Geneticin. Suspended cells were incubated 0, 1, 3, and 5 s at 37 °C in the presence of 137 mm NaCl, 2.7 mmKCl, 8 mm Na2HPO4, 1.6 mm KH2PO4, pH 7.4 (PBS) plus the radioactively labeled cations without or with 40 μmcyanine 863 (3Busch A.E. Quester S. Ulzheimer J.C. Gorboulev V. Akhoundova A. Waldegger S. Lang F. Koepsell H. FEBS Lett. 1996; 395: 153-156Crossref PubMed Scopus (90) Google Scholar). The short term incubations with [3H]MPP showed linearity for only 1 s. [3H]Quinine (0.56 TBq/mmol) and [3H]quinidine (0.54 TBq/mmol) were obtained from Biotrend (Köln, Germany) and 1-[3H]methyl-4-phenylpyridinium (3.1 TBq/mmol) from E. I. du Pont de Nemours & Co. (Dreieich, Germany). Geneticin G418 was from Life Technologies, Inc. (Eggenstein, Germany). The other chemicals were obtained as described earlier (2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 3Busch A.E. Quester S. Ulzheimer J.C. Gorboulev V. Akhoundova A. Waldegger S. Lang F. Koepsell H. FEBS Lett. 1996; 395: 153-156Crossref PubMed Scopus (90) Google Scholar). We determined the uptake of radioactively labeled quinine and quinidine after expression of rOCT1 in Xenopusoocytes and in HEK 293 cells. Fig. 1shows an experiment in which the time course of the cyanine 863-inhibitable uptake of [3H]MPP and [3H]quinine was measured in water-injected control oocytes and in rOCT1 cRNA-injected oocytes employing cation concentrations around their apparent K i values, as determined for inhibition of TEA uptake in rOCT1 cRNA-injected oocytes (1Gründemann D. Gorboulev V. Gambaryan S. Veyhl M. Koepsell H. Nature. 1994; 372: 549-552Crossref PubMed Scopus (608) Google Scholar, 2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). After 1-h incubation of the rOCT1-injected oocytes with 14 μm [3H]MPP, we measured a cyanine-inhibitable uptake of 73.5 ± 3.1 pmol/oocyte, which showed substrate saturation (2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) and was trans-stimulated about 2-fold when the oocytes had been preincubated with 1 mm choline (data not shown). After 1-h incubation of rOCT1-injected oocytes with 0.3 μm [3H]quinine in the absence and presence of 36 μm cyanine 863, 1.3 ± 0.1 and 0.6 ± 0.1 pmol/oocyte were found associated with the oocytes, respectively. This small amount of cyanine-inhibitable quinine uptake did not increase with larger concentrations, but (because of increased unspecific uptake) was not detected with quinine concentrations larger than 3 μm. Since the cyanine-inhibitable uptake of 0.3 μm quinine was not stimulated by preincubation of the oocytes with 1 mm choline and was partially removed by extensive (30 min at 19 °C) washing of the oocytes (data not shown), it probably represents quinine binding to rOCT1, which might appear increased if endocytosis occurred during the 1-h incubation with 0.3 μm [3H]quinine. The initial uptake rates of 14 μm [3H]MPP, 0.3 μm [3H]quinine, and 2 μm[3H]quinidine were also compared in stably transfected and in nontransfected HEK 293 cells. TableI shows that no cyanine-inhibitable uptake of 0.3 μm [3H]quinine or 2 μm [3H]quinidine was detected, whereas a significant cyanine inhibitable uptake of 14 μm[3H]MPP was measured in the transfected cells. Also with 3 μm [3H]quinine or 20 μm[3H]quinidine no significant cyanine-inhibitable uptake could be observed (data not shown). The data show that rOCT1 does not translocate quinine and quinidine at a detectable rate. Trying to understand why quinine induces large inward currents (2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) without being transported, we tested whether quinine and other type 2 cations may inhibit an rOCT1 mediated efflux of cations from the oocytes. Fig. 2 shows efflux experiments with [3H]MPP, which were performed with rOCT1-injected and water-injected oocytes (control). As reported earlier (2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), it was found that rOCT1-induced MPP efflux is observed when no organic cations were present in the bath and is trans-stimulated by extracellular MPP. Fig. 2 also shows that rOCT1-mediated efflux of [3H]MPP is trans-inhibited by quinine, d-tubocurarine, and cyanine 863. Inhibition was also observed for quinidine (data not shown). The stimulatory trans-effect of MPP on the initial efflux rate and the inhibitory trans-effects of the type 2 cations were significant (p < 0.05, see legend of Fig. 2). This suggests that the transporter loaded with type 2 cations exhibits a much slower (if at all) reorientation to its inward conformation than the unloaded transporter, supporting the notion that rOCT1 does not significantly translocate type 2 cations.Table IAttempt to measure transport of quinine and quinidine after expression of rOCT1 in an eukaryotic cell lineTransport measurementUptake raterOCT1-transfectedNontransfectedpmol × mg protein−1 × s−1MPP without cyanine2.1 ± 0.40.01 ± 0.01MPP with cyanine0.01 ± 0.010.01 ± 0.01Quinine without cyanine0.01 ± 0.010.03 ± 0.01Quinine with cyanine0.01 ± 0.010.01 ± 0.02Quinidine without cyanine0.3 ± 0.20.1 ± 0.1Quinidine with cyanine0.4 ± 0.20.2 ± 0.2 Open table in a new tab Fig. 3 a shows a typical current recording from an oocyte that had been preincubated for 18 h with 1 mm choline and was clamped at −50 mV. As reported earlier (2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) quinine (10 and then 100 μm) induced a large inward current in these oocytes, which might suggest that quinine is transported. Surprisingly, the following application of choline led to a drastically decreased signal which recovered slowly. Fig. 3 b shows a current recording from the same oocyte, which was maintained at a holding potential of −100 mV. Whereas the choline-induced signal increased slightly at this more negative potential, the quinine-induced signal disappeared virtually completely, although it is obvious from the following decreased choline-induced signal that quinine has interacted with rOCT1. Next we tested the response to quinine on rOCT1-injected oocytes that were not preincubated with choline. By the application of quinine at concentrations that inhibited the choline-induced currents almost completely, only small and transient inward currents were induced at a holding potential of −50 mV. Fig. 3c shows a typical experiment in which 3 μm quinine and/or 5 mm choline were added to the bath. From the onset observed for quinine inhibition of the choline induced current, and from the slow recovery of the choline current after quinine removal, we estimated a k off for the dissociation (∼0.015 s−1) and a k on for association of quinine (∼3 × 104m−1 × s−1). This leads to a K 0.5 of ∼0.5 μm for quinine-induced inhibition of the choline current, which is in good agreement with earlier flux and electrical measurements (2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Very similar results to quinine were obtained with the other type 2 cations quinidine, cyanine 863 and d-tubocurarine (data not shown). Fig. 4 shows the difference current voltage dependence obtained by current measurements before, during, and after superfusion of rOCT1-expressing oocytes with saturating concentrations of choline or quinine. When the rOCT1-expressing oocytes without choline preincubation in Fig. 4 a were superfused with 5 mm choline, inward difference currents were obtained that increased with increasingly negative membrane potential, i.e. the current voltage relationship showed a positive slope, as expected for electrogenic transport of choline. After superfusion of these oocytes with 100 μm quinine, a much smaller difference current with negative slope and a reversal potential at ∼−70 mV was observed, contrary to what would be expected for electrogenic transport of quinine. Preincubating, however, rOCT1-expressing oocytes with 1 mm choline resulted in different current voltage relationships (Fig. 4 b). Here the currents induced by superfusion with choline were independent of the membrane potential, whereas the quinine-induced current voltage relationship showed a steep negative slope, as expected for quinine-induced inhibition of electrogenic choline efflux. The electrical data confirm our conclusion from the flux measurements that the type 2 cation quinine is not significantly transported by rOCT1, but is a potent inhibitor of rOCT1-mediated electrogenic influx and efflux of the type 1 cation choline. Tracer influx and efflux measurements performed in Xenopus oocytes and stably transfected eukaryotic cells showed that the type 1 cations TEA, choline,N 1-methylnicotinamide, MPP, and dopamine are transported by rOCT1, rOCT2, and hOCT2. Electrical measurements with voltage-clamped oocytes indicated that the cation transport is potential-dependent and electrogenic ((2, 3, 6, 14). 3P. Arndt, C. Volk, V. Gorboulev, G. Nagel, T. Friedrich, S. Gambaryan, and H. Koepsell, unpublished data. Since rOCT1 is localized in the basolateral membranes of rat hepatocytes, we wondered whether rOCT1 may mediate the two previously distinguished cation transport activities in isolated hepatocytes,i.e. the transport of small relatively hydrophilic type 1 cations like choline, and the transport of larger, more hydrophobic type 2 cations like quinine (17Mol W.E.M. Fokkema G.N. Weert B. Meijer D.K.F. J. Pharmacol. Exp. Ther. 1988; 244: 268-275PubMed Google Scholar, 18Steen H. Oosting R. Meijer D.K.F. J. Pharmacol. Exp. Ther. 1991; 258: 537-543PubMed Google Scholar, 19Steen H. Merema M. Meijer D.K.F. Biochem. Pharmacol. 1992; 44: 2323-2331Crossref PubMed Scopus (45) Google Scholar). In an attempt to detect rOCT1-mediated transport of nonradioactively labeled type 2 cations, rOCT1 expressing Xenopus oocytes, preincubated with 1 mm choline and clamped at −50 mV, were superfused with type 2 cations and inward currents of similar magnitude to those obtained with TEA or choline were found (2Busch A.E. Quester S. Ulzheimer J.C. Waldegger S. Gorboulev V. Arndt P. Lang F. Koepsell H. J. Biol. Chem. 1996; 271: 32599-32604Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The present study shows that these results could only be obtained in a limited voltage range (see Fig. 4). We now demonstrate that the type 2 cations quinine, quinidine, cyanine 863, and d-tubocurarine are inhibitors of cation transport by rOCT1 and are not significantly translocated themselves. We suggest that the quinine-induced current is generated by the inhibition of electrogenic choline efflux through rOCT1. In hepatocytes rOCT1 is responsible for electrogenic uptake of type 1 cations over the basolateral membrane, whereas type 2 cations are taken up by a different nonidentified transport system. In renal proximal tubules type 1 cations may be translocated over the basolateral membrane by OCT2 and in the rat also by rOCT1, 4U. Karbach, F. Meyer-Wentrup, and H. Koepsell, unpublished data. whereas no functional evidence for a type 2 cation transporter has been presented (14Koepsell H. Annu. Rev. Physiol. 1998; 60: 243-266Crossref PubMed Scopus (287) Google Scholar). Our electrical measurements show that rOCT1 does not only mediate influx of type 1 cations into epithelial cells but may also mediate their electrogenic efflux. Further experiments with excised giant patches (20Hilgemann D.W. Pfluegers Arch. 1989; 415: 247-249Crossref PubMed Scopus (98) Google Scholar, 21Nagel G. Möckel B. Büldt G. Bamberg E. FEBS Lett. 1995; 377: 263-266Crossref PubMed Scopus (70) Google Scholar) from rOCT1-expressing oocytes should help to characterize the different steps in the transport cycle to understand how rOCT1 is operating in vivo. We thank W. Schwarz, R. Clarke, V. Gorboulev, and P. Arndt for their help and acknowledge the expert technical assistance of D. Stiegert and I. Schatz.