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High-throughput Screening Assay of α1G T-type Ca2+ Channels and Comparison with Patch-clamp Studies

2008년 8월 11일 16시 46분 55초
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목 <발표Ⅰ>
저자 및
김윤지, 최기현, 이재열1, 임혜원2
한국과학기술연구원 생체과학연구본부, Korea
1경희대학교 화학과, Korea
2한국과학기술연구원 의과학연구센터, Korea
Although whole-cell patch-clamp techniques are powerful tools for studying biophysical and pharmacological properties of ion channels, recent validation of ion channels as novel drug targets require the development of rapid, reliable, and sensitive cell-based high-throughput screening (HTS) methods for ion channels. In the previous study [1], we had devised HEK293/α1G/Kir2.1 cell lines which stably expressed α1G and Kir2.1 subunits and found that α1G T-type channel-sensitive Ca2+ signals were detected by application of high concentration of KCl under fura-2-based single cell measurements of intracellular Ca2+ concentration ([Ca2+]i). In the present study, we applied HEK293/α1G/Kir2.1 cells into the FDSS6000 (Functional Drug Screening System) system to develop a fast and reliable cell-based HTS method for α1G channels. After we detected 70 mM KCl-induced [Ca2+]i increases using the FDSS6000 system, we verified this new α1G channel HTS system by examining T-type Ca2+ channel blockers, Ni2+ and mibefradil, and measuring the Z´-factor (Z´ factor = 0.66) in 96-well plates. Furthermore, we assayed the selected 3,4-dihydroquinazoline derivatives using this FDSS6000-based α1G channel HTS system at the level of IC50 values and compared the results with the ones obtained from whole-cell patch-clamp recordings. Taken together, these results suggest that the FDSS6000-based α1G channel HTS system is a fast and feasible assay method for α1G T-type Ca2+ channels and can be utilized to screen chemicals as a primary screening method and to develop HTS systems for other types of ion channels.