Anal. Chem. 2008, 80, 1493-1496
Noninvasive Monitoring of Transporter-Substrate Interaction at Cell Membrane Toshiya Sakata† and Yuji Miyahara*,‡
Center for NanoBio Integration, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, and Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan
We report noninvasive monitoring of the transportersubstrate interaction at the cell membrane using an oocyte-based field effect transistor (FET), which is based on detection of extracellular potential change induced as a result of the interaction between transporting peptide and substrate at the cell membrane. The interface potential change at the cell membrane/gate insulator interface can be monitored during the uptake of substrate mediated by transporter without any labeling materials and fracturing oocyte. Moreover, we can discriminate the transporting kinetics of the substrate mediated by the wild-type and the mutant-type transporters by use of the oocyte-based FETs. Our findings on the time course of the interface potential would provide important information to understand the molecular mechanism of the uptake kinetics for the OATP-C transporter. The liver plays a primary role in the excretion of drugs and drug metabolites. The clearance process involves membrane transport systems that mediate the hepatocellular uptake of bile acids, organic anions, and organic cations.1-3 In particular, the organic anion transport pathway has been shown to mediate the elimination of various drugs. Xenopus laevis oocytes efficiently express the membrane-bound transporters and can be used as a convenient model system in pharmaceutical lead discovery to predict drug disposition, drug clearance, and drug-drug interactions.4,5 Oocyte expression systems have been used extensively to study the function of membrane proteins such as transporters, ion channels, and pumps because of their low background and high expression levels. In case of the uptake measurement of substrates mediated by transporters, radioisotope (RI)-labeled compounds are usually used and fundamental characteristics such as substrate selectivity and uptake rates are investigated. In this method, oocytes need to be solved and fractured before detecting specific radioactivity. On the other hand, the patch clamp * Corresponding author. Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan); phone, +81-29-860-4506; fax, +81-29-860-4714. E-mail:
[email protected]. † The University of Tokyo. ‡ National Institute for Materials Science. (1) Moller, J. V.; Sheikh, M. I. Pharmacol. Rev. 1982, 34, 315-358. (2) Wolkoff, A. W. Semin. Liver Dis. 1996, 16, 121-127. (3) Muller, M.; Jansen, P. L. Am. J. Physiol. 1997, 272, G1258-G1303. (4) Abe, T.; Kakyo, M.; Tokui, T.; Nakagomi, R.; Nishio, T.; Nakai, D.; Nomura, H.; Unno, M.; Suzuki, M.; Naitoh, T.; Matsuno, S.; Yawo, H. J. Biol. Chem. 1999, 274, 17159-17163. (5) Cha, S. H.; Sekine, T.; Fukushima, J. I.; Kanai, Y.; Kobayashi, Y.; Goya, T.; Endou, H. Mol. Pharmacol. 2001, 59, 1277-1286. 10.1021/ac701977e CCC: $40.75 Published on Web 02/02/2008
© 2008 American Chemical Society
technique has been accepted as a label-free standard method for studies of ion channel proteins by use of oocyte expression systems. The patch clamp technique is basically an invasive method. A fine glass pipet tip has to be pressed against the cell membrane carefully and a tiny area of the cell membrane is sucked into it, although the chip-based planar patch clamp technique has been developed.6 In the whole cell patch clamp mode, the cell membrane is broken by suction to exchange cytoplasm. It is therefore not possible to obtain information on cell activity noninvasively. Moreover, high skill is required to achieve a good contact between the cell membrane and the pipet tip and to measure the ionic currents resulting from openings of ion channels without any leakage. It would be preferable to monitor the interaction between membrane proteins/transporters and ligands at the cell membrane noninvasively while the cells are cultured on the materials. We have been investigating electrostatic detection of biomolecular recognition events using a biologically coupled field effect transistor (bio-FET).7-10 The principle of bio-FET is based on potentiometric detection of charge density change which is induced at a gate insulator/solution interface by specific biomolecular recognition. The FET devices have also been used in combination with pulsative living cells such as cardiac myocytes or neurons to detect action potentials as extracellular potential changes.11-15 The response pattern obtained with FET devices was similar to those with the conventional voltage clamp technique using a fine glass electrode, although the amplitude of the response was much smaller. In this paper, we propose an oocyte-based field effect transistor (oocyte-based FET) for drug transport analysis, in which target transporters are expressed at the cell membrane of the oocyte. We describe noninvasive monitoring of the uptake kinetics of (6) Schmidt, C.; Mayer, M.; Vogel, H. Angew. Chem., Int. Ed. 2000, 39, 31373140. (7) Sakata, T.; Miyahara, Y. ChemBioChem 2005, 6, 703-710. (8) Sakata, T.; Kamahori, M.; Miyahara, Y. Jpn. J. Appl. Phys. 2005, 44 (No. 4B), 2854-2859. (9) Sakata, T.; Miyahara, Y. Angew. Chem., Int. Ed. 2006, 45, 2225-2228. (10) Sakata, T.; Miyahara, Y. Proceedings of the 23rd Sensor Symposium, Takamatsu, Kagawa, October 5-6, 2006; 23, pp 334-337. (11) Krause, M.; Ingebrandt, S.; Richter, D.; Denyer, M.; Scholl, M.; Spro¨ssler, C.; Offenha¨usser, A. Sens. Actuators, B 2000, 70, 101-107. (12) Offenha¨usser, A.; Knoll, V. Trends Biotechnol. 2001, 19, 62-66. (13) Zeck, G.; Fromherz, P. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1045710462. (14) Meyburg, S.; Goryll, M.; Moers, J.; Ingebrandt, S.; Meffert, S. B.; Lu ¨ th, H.; Offenha¨usser, A. Biosens. Bioelectron. 2006, 21, 1037-1044. (15) Peitz, I.; Voelker. M.; Fromherz. P. Angew. Chem.. Int. Ed. 2007, 46, 57875790.
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substrates mediated by membrane-bound transporters. We also report discrimination of transporting ability among genotypes of the transporters using the oocyte-based FET. EXPERIMENTAL SECTION Expression of Transporter in Xenopus laevis Oocyte and [3H]-Modified Substrate Transport Measurement. Defolliculated oocytes were injected with the capped human organic anion transporting peptide C (OATP-C) cRNA and incubated in Modified Barth’s Solution (MBS; 88.0 mM NaCl, 1.0 mM KCl, 0.33 mM Ca(NO3)2‚4H2O, 0.41 mM CaCl2‚2H2O, 0.82 mM MgSO4‚7H2O, 2.4 mM NaHCO3, and 10.0 mM HEPES, pH 7.4 with NaOH, Daiichi Pure Chemicals Co., Ltd., ADME/TOX Research Institute) containing 10 units/mL penicillin and 10 µg/mL streptomycin at 18 °C. The oocyte was incubated for 3 days so that the transporter expression at the cell membrane was completed. After 3 days of incubation, uptake experiments were performed at room temperature in Na+ buffer solution (100 mM NaCl, 2.0 mM KCl, 1.0 mM CaCl2‚2H2O, 1.0 mM MgCl2‚6H2O, and 10.0 mM HEPES, pH 7.4 with Tris, Daiichi Pure Chemicals Co., Ltd., ADME/TOX Research Institute). Radioisotopes (RI) of [3H] estrone-3-sulfate (E3S) and [3H] estradiol 17β-D-glucuronide (E217βG) were used for specific radioactivity measurements and were purchased from PerkinElmer Life Science Products. Each substrate was prepared at the concentration of 50 nM to ∼20 µM for uptake experiments. Uptake values were expressed as pmol/oocyte(/60 min) and presented as mean ( standard error. A minimum of eight oocytes were used in each experiment. Measurements of Interface Potentials of Oocyte-Based FETs. The structure of oocyte-based FET was shown in Figure 1. Four n-channel depletion mode FETs were integrated in a 5 mm × 5 mm chip (Figure 1b). The thicknesses of the Si3N4 layer and the SiO2 layer are 140 and 35 nm, respectively. The fabricated FET chip was mounted on a flexible polyimide film with patterned copper electrodes and wire-bonded. The FET chip was encapsulated with a polymeric cover made by laser stereolithography (SOLIFORM-250B, CMET Inc.) using a photosensitive resin (TSR821, CMET Inc.) except for the sensing areas. The polymer cover with four holes was used as a guide to set the oocyte on the gate surface correctly. After 3 days incubation in MBS, oocytes were ready for the measurements using the oocyte-based FETs and one oocyte was placed in a hole on the gate area of the FET (Figure 1a). The threshold voltage shift ∆VT was determined after introduction of the substrates into the oocyte-based FETs using a semiconductor parameter analyzer (4155C, Agilent). The time course of the surface potential at the gate surface was monitored during uptake of substrates using a circuit.8 In the present study, the gate voltage and the drain current were set to be 1 V and 700 µA, respectively. The concentration of introduced substrates was prepared as 20 to ∼100 µM. RESULTS AND DISCUSSION The schematic illustration of the oocyte-based FET is shown in Figure 1a. A Xenopus laevis oocyte is placed on the surface of the gate insulator of the FET. The oocyte-based FET is immersed in a measurement solution together with an Ag/AgCl reference electrode with a saturated KCl solution. The potential of a measurement solution is controlled and fixed by the gate voltage (VG) through the reference electrode. The extracellular potential 1494 Analytical Chemistry, Vol. 80, No. 5, March 1, 2008
Figure 1. Structure of oocyte-based field effect transistor (FET). (a) Schematic illustration for measurement of the surface potential of the oocyte-based FET. A shift of the threshold voltage VT can be determined from the gate voltage (VG)-drain current (ID) characteristics in a phosphate buffer solution (0.025 M Na2HPO4 and 0.025 M KH2PO4, pH 6.86). (b) Photograph of the fabricated FET chip. Four FETs are integrated in a 5 mm × 5 mm chip. The FET chip was encapsulated using a polymeric cover with holes except for the gate areas. An oocyte is placed in the hole on the gate surface.
changes are induced at the interface between the gate insulator and cell membrane by the uptake of substrates. The change in the electrical characteristics caused by interaction between transporters and substrates at the cell membrane is measured and monitored using the oocyte-based FET system. The FET chip was mounted on a polyimide printed circuit board and encapsulated with a polymer cover with holes in which cells are cultured (Figure 1b). The details of the FET device and the fabrication process have been reported previously.8 We have prepared two types of oocyte-based FETs; one is the FET with oocyte in which transporters are expressed, and the other is the FET with oocyte in which transporters are not expressed. With the use of these oocyte-based FETs, differential measurements were performed in order to eliminate the common background noises such as temperature change, change in ion concentration, and so on. The oocyte-based FET chip was treated as a single use tool for monitoring transporting function at cell membrane for the present. The reference FET without the oocyte itself was used in order to take the effect of oocyte placed at the gate surface into account. The changes in the surface potentials at the gate surface of the oocyte-based FETs were monitored after adding a substrate (Figures 2a-c). The estrone-3-sulfate (E3S) was used as a substrate in the uptake measurement for a human organic anion transporting peptide C (OATP-C). When the E3S was introduced into two kinds of oocyte-based FETs and the reference FET, the surface potential of the oocyte FET with OATP-C increased drastically during the uptake of E3S, while the oocyte FET without
Figure 2. Noninvasive monitoring of uptake of a substrate mediated by human organic anion transporting peptide C (OATP-C). (a) Surface potential change of the oocyte FET with the transporter based on the uptake of estrone 3-sulfate (E3S). (b) Surface potential change of the control oocyte FET based on the uptake of E3S. (c) Surface potential change of the reference FET based on the introduction of E3S. In parts a-c, 100 µM E3S was introduced at 10 min at room temperature. (d) Time-dependent uptake of [3H]-labeled E3S. Data were expressed as mean ( S.E. (n ) 8).
OATP-C expression and the reference FET showed little surface potential changes. This result was similar to those obtained with RI measurement (Figure 2d), in which [3H]-labeled E3S was used as a substrate. Monotonous increase of the E3S uptake during incubation time of 2 h was also reported.16 However, the time course of the uptake signal obtained with the oocyte-based FET was different from those of the conventional RI measurements. The surface potential of the oocyte-based FET reached steadystate in about 30 min after introduction of E3S, while the intensity of radioactivity increased over 2 h in the RI measurement. This means that the observed phenomena based on the oocyte-based FET would be disparate from the uptake amount of [3H]-labeled E3S. Although the molecular mechanism during the uptake of the estrone-3-sulfate through organic anion transporting peptide C (OATP-C) has not yet been elucidated, the uptakes of substrates mediated by some transporters are known to be associated with a substrate-dependent current under voltage-clamped conditions.17 In the case of OAT1, one of the family of organic anion transporters, entry of organic anion is reported to be accompanied by efflux of dicarboxylate by a 1:1 ratio.18 This exchange of singly charged organic anion with doubly charged dicarboxylates leads to a net loss of one negative charge per transporter cycle and (16) http://www.bdj.co.jp/gentest/1f3pro00000sgirm-att/tpc_oatp2_lc-ms.pdf. (17) Dresser, M. J.; Gray, A. T.; Giacomini, K. M. Pharmacology 2000, 292, 1146-1152. (18) Aslamkhan, A.; Han, Y.-H.; Walden, R.; Sweet, D. H.; Pritchard, J. B. Am. J. Physiol. Renal Physiol. 2003, 285, F775-F783.
change of the membrane potential in the positive direction. Since the positive change of the surface potential was obtained for the oocyte-based FET as shown in Figure 2, similar exchange of charged species is considered to take place during the uptake of E3S in the case of OATP-C. The constant flux of charged species continues during the uptake of E3S, which results in steady-state of the surface potential of the oocyte-based FET. Further investigation is underway to elucidate the response mechanism of the oocyte-based FET. OATP-C is a liver-specific transporter involved in the hepatocellular uptake of a variety of endogenous chemicals, such as taurocholate,19 estrone sulfate,20 estradiol 17β-D-glucuronide,4 leukotriene C4,4 prostaglandin E2,4 and thyroid hormone.4 Genetic polymorphisms in OATP-C have been investigated because of pharmacologic, toxicologic, and pathologic significances.21-23 We attempted to detect the difference of the transporting ability between the wild-type and mutant-type transporters using the (19) Hsiang, B.; Zhu, Y.; Wang, Z.; Wu, Y.; Sasseville, V.; Yang, W.-P.; Kirchgessner, T. G. J. Biol. Chem. 1999, 274, 37161-37168. (20) Tamai, I.; Nezu, J.; Uchino, H.; Sai, Y.; Oku, A.; Shimane, M.; Tsuji, A. Biochem. Biophys. Res. Commun. 2000, 273, 251-260. (21) Tamai, I.; Nozawa, T.; Koshida, M.; Nezu, J.; Sai, Y.; Tsuji, A. Pharm. Res. 2001, 18, 1262-1269. (22) Tirona, R. G.; Leake, B. F.; Merino, G.; Kim, R. B. J. Biol. Chem. 2001, 276, 35669-35675. (23) Nishizato, Y.; Ieiri, I.; Suzuki, H.; Kimura, M.; Kawabata, K.; Hirota, T.; Takane, H.; Irie, S.; Kusuhara, H.; Urasaki, Y.; Urae, A.; Higuchi, S.; Otsubo, K.; Sugiyama, Y. Int. J. Clin. Pharmacol. Ther. 2003, 73, 554-565.
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Figure 3. Discrimination of transporting kinetics between wild-type and mutant-type human organic anion transporting peptide C (OATP-C). (a) Uptake of [3H]-labeled estradiol 17β-D-glucuronide (E217βG) mediated by the wild-type OATP-C*1a. (b) Uptake of [3H]-labeled E217βG mediated by the mutant-type OATP-C*15a. In parts a-b, uptake was expressed as pmol/oocyte/60 min. Each experiment was performed at room temperature, and the concentration of the substrate was 20 µM. (c) Surface potential changes of the oocyte-based FETs based on uptake of E217βG. An amount of 20 µM E217βG was introduced at 0 min at room temperature.
oocyte-based FETs. The estradiol 17β-D-glucuronide (E217βG) was used as a substrate mediated by the wild-type OATP-C*1a (the same transporter as OATP-C) and the mutant-type OATP-C*15. The uptake of the RI-labeled substrate was first measured for both the wild type and the mutant type transporters as a control experiment. The [3H]-labeled E217βG was detected in both OATPC*1a- and OATP-C*15- expressed oocytes (Figure 3a,b). The uptake amount of [3H]-labeled E217βG for the wild-type transporter was 2 times larger than that of the mutant-type transporter. The difference of the uptake amount among the genotypes in the OATP-C transporter has been reported in the previous work.23 The oocyte with the wild type OATP-C*1a or the mutant-type OATP-C*15 transporter was placed on the gate surface of the oocyte-based FET. Figure 3c shows the potential behaviors of the oocyte-based FETs during the uptake of E217βG without the RI label. The surface potentials of both oocyte-based FETs increased after the introduction of E217βG while the control oocyte FET showed a little change in the surface potential. The amount of the surface potential change of the oocyte-based FET with the wild-type transporter was approximately twice as large as that of the oocyte-based FET with the mutant-type transporter. The difference of the surface potential change between the wild type and the mutant type transporters was in good agreement with that obtained in the RI measurement (Figure 3a,b). Thus, the transporting kinetics of the substrate mediated by the wild-type and the mutant-type transporters were distinguished by use of the oocyte-based FETs. CONCLUSIONS In this paper, we have shown the noninvasive monitoring of the uptake kinetics of the substrate mediated by the OATP-C 1496 Analytical Chemistry, Vol. 80, No. 5, March 1, 2008
transporter using oocyte-based FETs. The surface potential of the oocyte-based FET reached steady-state in about 30 min after introduction of the substrate, suggesting a constant flux of charged species accompanied by the uptake of the substrate. Our findings on the time course of the surface potential would provide important information to understand the molecular mechanism of the uptake kinetics for the OATP-C transporter. It is possible to integrate multiple oocyte-based FETs and signal processing circuits in a single chip using advanced semiconductor technology. Simultaneous analyses of different transporters and membrane proteins can therefore be realized based on the integrated oocyte-based FETs. Because of the output of the oocytebased FET is an electrical signal, it would be easy to quantify the transporting ability of various transporters in the future. The platform based on the oocyte-based FETs is suitable for a simple, accurate, and inexpensive system for high-throughput screening in pharmaceutical lead discovery. ACKNOWLEDGMENT The authors wish to thank Drs. Y. Horiike, T. Tateishi, and S. Kita of National Institute for Materials Science in Japan, Profs. K. Kataoka, K. Ishihara, and M. Washizu of the University of Tokyo, Dr. M. Kamahori of Hitachi Ltd. in Japan, and Prof. P. Fortina of Thomas Jefferson University in the U.S.A. for their help and useful discussion.
Received for review September 20, 2007. Accepted December 18, 2007. AC701977E
