The P2X 7 receptor (P2X7R) is an important member of the P2X family of ligand-gated ion channels that respond to ATP as the endogenous agonist. Studies suggest that P2X7R plays a potentially pivotal role in a variety ...The P2X 7 receptor (P2X7R) is an important member of the P2X family of ligand-gated ion channels that respond to ATP as the endogenous agonist. Studies suggest that P2X7R plays a potentially pivotal role in a variety of physiological functions, including peripheral and central neuronal transmission, smooth muscle contraction, and inflammation. Thus, P2X7R may be a potential target for drug development. Here, we used a FlexStation to examine the function of recombinant P2X7R stably expressed in human embryonic kidney 293 cells and to compare three high-throughput screening assays: a membrane potential assay, an ethidium bromide uptake assay, and a calcium influx assay. We found that all three assays were suitable for the analysis of P2X7R, but the calcium influx assay was the most robust and is the best choice as a first high-throughput screening assay when embarking on a P2X7R drug discovery project.展开更多
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 （...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 ICs0 at 24.5~4.1 p.M （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.展开更多
文摘The P2X 7 receptor (P2X7R) is an important member of the P2X family of ligand-gated ion channels that respond to ATP as the endogenous agonist. Studies suggest that P2X7R plays a potentially pivotal role in a variety of physiological functions, including peripheral and central neuronal transmission, smooth muscle contraction, and inflammation. Thus, P2X7R may be a potential target for drug development. Here, we used a FlexStation to examine the function of recombinant P2X7R stably expressed in human embryonic kidney 293 cells and to compare three high-throughput screening assays: a membrane potential assay, an ethidium bromide uptake assay, and a calcium influx assay. We found that all three assays were suitable for the analysis of P2X7R, but the calcium influx assay was the most robust and is the best choice as a first high-throughput screening assay when embarking on a P2X7R drug discovery project.
基金The National Natural Sciences Foundation of China(Grant No.81573410)the Ministry of Science and Technology of China(Grant No.2014ZX09507003-006-004)the Natural Sciences Foundation of Shandong Province(Grant No.ZR2015QL008)
文摘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 ICs0 at 24.5~4.1 p.M （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.
