A biosensing system based on extracellular potential recording of ligand-gated ion channel function overexpressed in insect cells.
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 natural 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 potentiometric 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.
Haruyama
Tetsuya
T
Department of Biological Functions and Engineering, Kyushu Institute of Technology, Kitakyushu Science and Research Park, 2-4 Hibikino, Wakamatsu-ku, Fukuoka 808-0196, Japan. haruyama@life.kyutech.ac.jp
Bongsebandhu-Phubhakdi
Saknan
S
Nakamura
Ibuki
I
Mottershead
David
D
Keinänen
Kari
K
Kobatake
Eiry
E
Aizawa
Masuo
M
D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002460:Cell Line; D018690:Excitatory Amino Acid Agonists / Q000032:analysis*; D018698:Glutamic Acid / Q000032:analysis*; D007313:Insecta; D015640:Ion Channel Gating; D007473:Ion Channels; D008024:Ligands; D008839:Microelectrodes; D017470:Receptors, Glutamate / Q000235:genetics; D014162:Transfection
D018690:Excitatory Amino Acid Agonists; D007473:Ion Channels; D008024:Ligands; D017470:Receptors, Glutamate; D018698:Glutamic Acid