Robust Electrophysiological Assays using Solid Supported Membranes: the Organic Cation Transporter OCT2
Olga Gaiko A B , Ingo Janausch A C , Sven Geibel D E , Henning Vollert D F , Petra Arndt D , Sigrid Gonski D and Klaus Fendler A GA MPI for Biophysics, Max-von-Laue Str.3, 60438 Frankfurt/Main, Germany.
B Present address: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Dr, Mail Code 0718, La Jolla, CA 92093-0718, USA.
C Present address: Universität Heidelberg, Im Neuenheimer Feld 325, 69120 Heidelberg, Germany.
D Sanovi-Aventis Germany GmbH, Industriepark Höchst, 65926 Frankfurt, Germany.
E Present address: Bayer BioScience NV, Technologiepark 38, 9052 Ghent, Belgium.
F Present address: BioActive Food GmbH, am Ihlsee 36a, 23975 Bad Segeberg, Germany.
G Corresponding author. Email: klaus.fendler@biophys.mpg.de
Australian Journal of Chemistry 64(1) 31-35 https://doi.org/10.1071/CH10322
Submitted: 31 August 2010 Accepted: 12 October 2010 Published: 14 January 2011
Abstract
An electrophysiological assay platform based on solid supported membranes (SSM) for the organic cation transporter (OCT) is presented. Stable Chinese hamster ovary (CHO) cell lines overexpressing the human (hOCT2) and rat transporters (rOCT2) were generated and validated. Membrane preparations from the cell lines were investigated using SSM-based electrophysiology. Baculovirus transfected insect cells (HighFive and Mimic Sf9) were also tested with the same assay but yielded less than optimal results. The assays were validated by the determination of substrate affinities and inhibition by standard inhibitors. The study demonstrates the suitability of the SSM-based electrophysiological OCT assay for rapid and automatic screening of drug candidates.
References
[1] C. Ganea, K. Fendler, Biochim. Biophys. Acta 2009, 1787, 706.| 1:CAS:528:DC%2BD1MXntVSitrY%3D&md5=50332994b95cb9249a15ee5187c117c8CAS | 19366604PubMed |
[2] P. Schulz, J. J. Garcia-Celma, K. Fendler, Methods 2008, 46, 97.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhtlyhsbvE&md5=a66ad314c95dce8935fb70fb1902c4f4CAS | 18675360PubMed |
[3] J. Pintschovius, K. Fendler, Biophys. J. 1999, 76, 814.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXnsVanuw%3D%3D&md5=a0d54d614ca4534c074a4d40c07dbc69CAS | 9929483PubMed |
[4] R. Krause, N. Watzke, B. Kelety, W. Dorner, K. Fendler, J. Neurosci. Methods 2009, 177, 131.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhsFCgsL%2FO&md5=62120fa5854083a975df98f7d13c986bCAS | 18996149PubMed |
[5] B. Kelety, K. Diekert, J. Tobien, N. Watzke, W. Dörner, P. Obrdlik, K. Fendler, Assay Drug Develop. Technol. 2006, 4, 575.
| 1:CAS:528:DC%2BD28Xht1WqurvF&md5=c43f53a728346d230544882d1dd69c3dCAS |
[6] D. Gründemann, V. Gorboulev, S. Gambaryan, M. Veyhl, H. Koepsell, Nature 1994, 372, 549.
| Crossref | GoogleScholarGoogle Scholar | 7990927PubMed |
[7] M. R. Koehler, B. Wissinger, V. Gorboulev, H. Koepsell, M. Schmid, Cytogenet. Cell Genet. 1997, 79, 198.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXjslGqsb4%3D&md5=638f8e66e4efd0f092255e933917fbe4CAS | 9605850PubMed |
[8] H. Koepsell, Annu. Rev. Physiol. 1998, 60, 243.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXitVOhs7c%3D&md5=023b8704745e940e25b4fee5beb77adeCAS | 9558463PubMed |
[9] J. J. Garcia-Celma, B. Dueck, M. Stein, M. Schlueter, K. Meyer-Lipp, G. Leblanc, K. Fendler, Langmuir 2008, 24, 8119.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXns1SqtL8%3D&md5=d2981ff1146fb7a1f979d59883272529CAS | 18572928PubMed |
[10] P. Arndt, C. Volk, V. Gorboulev, T. Budiman, C. Popp, I. Ulzheimer-Teuber, A. Akhoundova, S. Koppatz, E. Bamberg, G. Nagel, H. Koepsell, Am. J. Physiol. Renal Physiol. 2001, 281, F454.
| 1:CAS:528:DC%2BD3MXmvFWisrY%3D&md5=f4c320bbefa6438890adf0ec9e70cf3aCAS | 11502595PubMed |
[11] M. Hayer-Zillgen, M. Bruss, H. Bonisch, Br. J. Pharmacol. 2002, 136, 829.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XlvV2rurk%3D&md5=998e9a368c5d36fc7750bb5da563cc78CAS | 12110607PubMed |
[12] B. M. Schmitt, H. Koepsell, J. Biol. Chem. 2005, 280, 24481.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXlsFyrur8%3D&md5=0c687252d9680d332c776490b957fa2cCAS | 15878879PubMed |
[13] J. H. Zhang, T. D. Chung, K. R. Oldenburg, J. Biomol. Screen. 1999, 4, 67.
| Crossref | GoogleScholarGoogle Scholar | 10838414PubMed |
[14] K. S. Lips, C. Volk, B. M. Schmitt, U. Pfeil, P. Arndt, D. Miska, L. Ermert, W. Kummer, H. Koepsell, Am. J. Respir. Cell Mol. Biol. 2005, 33, 79.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXmsVSiu7s%3D&md5=dcf7bc33c50a0989effab7c065e35735CAS | 15817714PubMed |
[15] Y. Urakami, M. Okuda, S. Masuda, H. Saito, K. I. Inui, J. Pharmacol. Exp. Ther. 1998, 287, 800.
| 1:CAS:528:DyaK1cXnsVerurk%3D&md5=53f09875bce2d39588913e145a3fb210CAS | 9808712PubMed |
[16] M. Okuda, H. Saito, Y. Urakami, M. Takano, K. Inui, Biochem. Biophys. Res. Commun. 1996, 224, 500.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK28Xktl2qurg%3D&md5=edb8822f11d10f8a59fd72b9b4732deaCAS | 8702418PubMed |
[17] V. Gorboulev, J. C. Ulzheimer, A. Akhoundova, I. Ulzheimer-Teuber, U. Karbach, S. Quester, C. Baumann, F. Lang, A. E. Busch, H. Koepsell, DNA Cell Biol. 1997, 16, 871.
| Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXltFGlsbk%3D&md5=03d0df6df300a9de2d25caa8703010d1CAS | 9260930PubMed |
