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

doi:10.5246/jcps.2017.09.076

Journal of Chinese Pharmaceutical Sciences ›› 2017, Vol. 26 ›› Issue (9): 675-683.DOI: 10.5246/jcps.2017.09.076

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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).

Abstract

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

CLC Number:

R962.1

Supporting:

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.

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

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