A Biosensing System Based on Extracellular Potential Recording of Ligand-Gated Ion Channel Function Overexpressed in Insect Cells Tetsuya Haruyama,* ,² Saknan Bongsebandhu-Phubhakdi, ‡ Ibuki Nakamura, ‡ David Mottershead, § Kari Keina 1 nen, § Eiry Kobatake, ‡ and Masuo Aizawa ‡ Department of Biological Functions and Engineering, Kyushu Institute of Technology, Kitakyushu Science and Research Park, 2-4 Hibikino, Wakamatsu-ku, Fukuoka 808-0196, Japan, Department of Biological Information, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan, and Department of Biosciences, Division of Biochemistry, Viikki Biocenter, 00014, University of Helsinki, P.O. Box 56, Helsinki, Finland We have used outer cell potential measurement to record agonist-dependent cellular responses in cells engineered to express ligand-gated ion channels and grown on a microelectrode surface. Application of glutamate, a natu- ral agonist, induced a complex and robust potentiometric response in cells expressing homomeric GluR-D glutamate receptor, but not in nonexpressing control cells. The response consisted of an initial decrease in outer potential followed by a transient increase and was not obtained for other amino acids devoid of agonist activity at glutamate receptors. Furthermore, the pharmacological agonist of the GluR-D receptor, kainate, also produced the poten- tiometric response whereas 6 -cyano-7 -nitroquinoxaline- 2 ,3 -dione, a competitive antagonist, was not active in itself but attenuated the responses to glutamate. The time course of the measured changes was slow, which may be partially due to the ligand being applied by free diffusion but may also reflect a contribution by secondary changes in the behavior of the cells. This novel approach should be applicable to other ligand-gated ion channels and holds promise as a cell-based biosensor for high-throughput drug screening and other applications. The highly specific ligand recognition and intrinsic signal amplification by ligand-gated ion channels make them attractive molecules for biosensor development. Both naturally occurring 1 and engineered 2 ligand-gated channels have been employed as the signal-generating component in both molecular (nonliving)- and cell-based biosensors. One obvious application of biosensors that use natural receptors as the element responsible for chemical specificity would be in high-throughput drug screening, as this class of molecules includes a number of important drug targets. For ligand-gated ion channels, a large-scale functional assay may be achieved by coupling the agonist-triggered ion flux in cultured cells to an optical readout, via use of fluorescent indicators sensitive to changes in the concentration of the permeant ions or to changes in the membrane potential. 3 Although electrophysi- ological assays of ion channel function have an exquisite sensitivity and resolution, the level of technical expertise involved and the time-consuming nature of the experiments largely exclude the use of glass electrode implade in cells from drug screening. Nonin- vasive extracellular measurement of the electrical activity in cells grown on electrode grids would provide a potentially more robust method that may lend itself to automation. Numerous studies have demonstrated measurement of spontaneous and pharmacologically manipulated electric activity (action potentials) in networks of cultured neurons grown on electrode arrays. The measured signal consists of complex spikes that reflect temporal and spatial patterns of activity of voltage-gated ion channels. These patterns can be modified by changes in the external conditions, e.g., by the presence of pharmacological agents, and therefore this kind of system may also hold promise for biosensor applications. 4 Although a successful measurement of maxi-K channel function by a field effect transistor was recently reported, 5 direct nonin- vasive electronic measurement of ligand-gated ion channel func- tion has not been reported. Indirectly, microphysiometry that senses pH changes in the cellular environment, due to a metabolic response to ion channel activation, however, has been used to monitor the activity of ligand-gated channels. 1 Ionotropic glutamate receptors (iGluR) are ligand-gated cation channels that mediate the majority of fast excitatory neurotrans- mission in the brain and comprise three subclasses known as AMPA, kainate, and NMDA receptors, which differ in their molecular composition, biophysical properties, and relative affini- ties to pharmacological compounds. 6 AMPA receptors are hetereo- and homomeric assemblies of four homologous subunits, GluR- * Corresponding author. E-mail: haruyama@ life.kyutech.ac.jp. † Kyushu Institute of Technology. ‡ Tokyo Institute of Technology. § University of Helsinki. (1) Hahnenberger, K. M.; Krystal, M.; Esposito, K.; Tang, W.; Kurtz, S. Nat. Biotechnol. 1996 , 14, 880-883. (2) Cornell, B. A.; Braach-Maksvytis, V. L.; King, L. G.; Osman, P. D.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997 , 387, 580-583. (3) Frostig, R. D.; Lieke, E.; Ts’o, D. Y.; et al. Proc. Natl. Acad. Sci. U.S.A. 1990 , 87, 6082-6086. (4) Ziegler, C. J. Anal. Chem. 2000 , 366, 552-559. (5) Zeck, G.; Fromherz, P. Proc. Natl. Acad. Sci. U.S.A. 2001 , 98, 10457- 10462. (6) Dingledine, R.; Borges, K.; Bowie, D.; Traynelis, S. F. Pharmacol. Rev. 1999 , 51,7-61. Anal. Chem. 2003, 75, 918-921 918 Analytical Chemistry, Vol. 75, No. 4, February 15, 2003 10.1021/ac025670x CCC: $25.00 © 2003 American Chemical Society Published on Web 01/21/2003