发展基于活细胞的FlexStation3高通量钙荧光方法及筛选TRPV3通道抑制剂

doi:10.5246/jcps.2017.09.076

中国药学(英文版) ›› 2017, Vol. 26 ›› Issue (9): 675-683.DOI: 10.5246/jcps.2017.09.076

发展基于活细胞的FlexStation3高通量钙荧光方法及筛选TRPV3通道抑制剂

孙晓梅, 卫宁宁, 孙晓颖, 王克威^*

青岛大学药学院, 山东青岛 266071

收稿日期:2017-05-12 修回日期:2017-07-08 出版日期:2017-09-30 发布日期:2017-08-15
通讯作者: Tel.: +86-0532-82991070, E-mail: wangkw@qdu.edu.cn
基金资助:
The National Natural Sciences Foundation of China (Grant No. 81573410), the Ministry of Science and Technology of China (Grant No. 2014ZX09507003-006-004) and the Natural Sciences Foundation of Shandong Province (Grant No. ZR2015QL008).

Development of a cell-based HTS adaptable calcium fluorescent assay in FlexStation3 for the screening of TRPV3 inhibitors

Xiaomei Sun, Ningning Wei, Xiaoying Sun, Kewei Wang^*

Department of Pharmacology, Qingdao University School of Pharmacy, Qingdao 266021, China

Received:2017-05-12 Revised:2017-07-08 Online:2017-09-30 Published:2017-08-15
Contact: Tel.: +86-0532-82991070, E-mail: wangkw@qdu.edu.cn
Supported by:
The National Natural Sciences Foundation of China (Grant No. 81573410), the Ministry of Science and Technology of China (Grant No. 2014ZX09507003-006-004) and the Natural Sciences Foundation of Shandong Province (Grant No. ZR2015QL008).

摘要/Abstract

摘要：

为了建立高通量筛选TRPV3通道调节剂的方法, 本研究利用FlexStation3酶标仪, 针对瞬时转染TRPV3通道的HEK293T细胞的密度、Cal-520染料的浓度及染料孵育时间进行了条件优化。结果显示, 40 000个细胞/孔、5.0%的Cal-520染料浓度及1.5 h的染料孵育时间为最佳实验条件。应用该筛选方法, 筛选并发现来源于睡莲科植物的有效成分甲基莲心碱(neferine)能够抑制TRPV3通道, 该抑制作用经全细胞膜片钳实验证实并测得其IC₅₀为24.5±4.1 μM (n = 6)。对其它TRPV通道成员的选择性电生理学评价结果显示, 甲基莲心碱对TRPV1和TRPV4通道无抑制作用。总之, 实验数据显示, FlexStation3高通量钙荧光筛选可以用于发现TRPV3以及其他TRP通道的调节剂, 筛选发现的天然产物甲基莲心碱可以作为TRPV3通道的工具药研究通道的药理学功能。

关键词: TRPV3通道抑制剂, 甲基莲心碱, FlexStation3, 全细胞膜片钳, 钙离子

Abstract:

To develop a cell-based high-throughput calcium fluorescent assay for screening of TRPV3 channel modulators, we employed the FlexStation3 microplate reader and optimized conditions of the high-throughput screening (HTS) adaptable calcium fluorescent assay, such as cell density, incubation time and concentration of Cal-520 calcium fluorescent dye. The optimized FlexStation3 assay included the cell density of 40 000 cells per well, 5.0% of Cal-520 calcium fluorescent dye and 1.5 h of incubation time. Using the FlexStation3 assay in a 96-well format, we screened and identified a natural neferine from Nymphaeaceae plant that inhibited TRPV3 channel. To further confirm the inhibitory effect of neferine on TRPV3, we utilized the whole-cell patch clamp recordings and determined the dose-dependent inhibition of TRPV3 current by neferine with an IC₅₀ at 24.5±4.1 μM (n = 6) without inhibition of TRPV1 or TRPV4 channels. Taken together, our data showed that this validated HTS adaptable calcium fluorescent assay in FlexStation3 format could be used for screening and identification of modulators for either TRPV3 channel or other calcium permeable TRP channels. The identified natural neferine could be used as a tool for pharmacological investigations of TRPV3 channel function.

Key words: TRPV3 inhibitor, Neferine, FlexStation3, Whole-cell patch clamp, Calcium

中图分类号:

R962.1

Supporting:

孙晓梅, 卫宁宁, 孙晓颖, 王克威. 发展基于活细胞的FlexStation3高通量钙荧光方法及筛选TRPV3通道抑制剂[J]. 中国药学(英文版), 2017, 26(9): 675-683.

Xiaomei Sun, Ningning Wei, Xiaoying Sun, Kewei Wang. Development of a cell-based HTS adaptable calcium fluorescent assay in FlexStation3 for the screening of TRPV3 inhibitors[J]. Journal of Chinese Pharmaceutical Sciences, 2017, 26(9): 675-683.

参考文献

[1] Pedersen, S.F.; Owsianik, G.; Nilius, B. Cell. Calcium. 2005, 38, 233–252.

[2] Planells-Cases, R.; Garcia-Sanz, N.; Morenilla-Palao, C.; Ferrer-Montiel, A. Pflugers. Arch. 2005, 451, 151–159.

[3] Craig, M.; Lutz, B.; Flockerzi, V. Cell. 2002, 108, 595–598.

[4] Todaka, H.; Taniguchi, J.; Satoh, J.; Mizuno, A.; Suzuki, M. J. Biol. Chem. 2004, 279, 35133–35138.

[5] Vriens, J.; Watanabe, H.; Janssens, A.; Droogmans, G.; Voets, T.; Nilius, B. Proc. Natl. Acad. Sci. USA. 2004, 101, 396–401.

[6] Hu, H.Z.; Gu, Q.; Wang, C.; Colton, C.K.; Tang, J.; Kinoshita-Kawada, M.; Lee, L.Y.; Wood, J.D.; Zhu, M.X. J. Biol. Chem. 2004, 279, 35741–35748.

[7] Chung, M.K.; Lee, H.; Mizuno, A.; Suzuki, M.; Caterina, M.J. J. Neurosci. 2004, 24, 5177–5182.

[8] Nilius, B.; Vriens, J.; Prenen, J.; Droogmans, G.; Voets, T. Am. J. Physiol. Cell. Physiol. 2004, 286, 195–205.

[9] Bandell, M.; Story, G.M.; Hwang, S.W.; Viswanath, V.; Eid, S.R.; Petrus, M.J.; Earley, T.J.; Patapoutian, A. Neuron. 2004, 41, 849–857.

[10] Bang, S.; Yoo, S.; Yang, T.J.; Cho, H.; Hwang, S.W. J. Biol. Chem. 2010, 285, 19362–19371.

[11] Zhai, C.; Liu, Q.; Zhang, Y.; Wang, S.; Zhang, Y.; Li, S.; Qiao, Y. Molecules. 2014, 19, 18733–18746.

[12] Chen, J.; Lake, M.R.; Sabet, R.S.; Niforatos, W.; Pratt, S.D.; Cassar, S.C.; Xu, J.; Gopalakrishnan, S.; Pereda-Lopez, A.; Gopalakrishnan, M.; Holzman, T.F.; Moreland, R.B.; Walter, K.A.; Faltynek, C.R.; Warrior, U.; Scott, V.E. J. Biomol. Screen. 2007, 12, 61–69.

[13] Watanabe, H.; Davis, J.B.; Smart, D.; Jerman, J.C.; Smith, G.D.; Hayes, P.; Vriens, J.; Cairns, W.; Wissenbach, U.; Prenen, J.; Flockerzi, V.; Droogmans, G.; Benham, C.D.; Nilius, B. J. Biol. Chem. 2002, 277, 13569–13577.

[14] Moqrich, A.; Hwang, S.W.; Earley, T.J.; Petrus, M.J.; Murray, A.N.; Spencer, K.S.; Andahazy, M.; Story, G.M.; Patapoutian, A. Science. 2005, 307, 1468–1472.

[15] Wang, S.; Zhai, C.; Zhang, Y.; Yu, Y.; Zhang, Y.; Ma, L.; Li, S.; Qiao, Y. Molecules. 2016, 21, 1145–1155.
[16] Bang, S.; Kim, K.Y.; Yoo, S.; Lee, S.H.; Hwang, S.W. Neurosci Lett. 2007, 425, 120–125.

发展基于活细胞的FlexStation3高通量钙荧光方法及筛选TRPV3通道抑制剂

Development of a cell-based HTS adaptable calcium fluorescent assay in FlexStation3 for the screening of TRPV3 inhibitors

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