Development And Optimization Of High-throughput Screening Adaptable Fluorescent Assay For The Screening Of TRPV3 Channel Inhibitors And Their Pharmacological Evaluations

TRPV3?Transient Receptor Potential Vanilliod 3?channel as a member of TRP channel family is a calcium-permeable,ligand-gated,non-selective cation channel.TRPV3 channel as a warm sensation sensor is activated by temperature?33?.TRPV3 is widely distributed in central nervous system and peripheral sensory system,such as dorsal root ganglion,trigeminal ganglion and skin keratinocytes.TRPV3 is not only associated with temperature sensory,but also involved in pain,itching and skin hair growth.Although TRPV3 is associated with pathophysiological condictions,the study of TRPV3pharmacology has been severely hindered due to the lack of specific modulators.Objective This study was to develop a high throughput screening assay and identify specific regulators for TRPV3 channel.Medthods We utilized the Flex Station 3 microplate reader and optimized the assay conditions including cell density,Cal-520 calcium fluorescent dye incubation time and concentration,the adding sequence of the activators?2-Aminoethoxydiphenyl borate,2-APB;Camphor?and inhibitor?Ruthenium Red,RR?.Ruthenium red was used to confirm the calcium fluorescent signal resulted from activation of TRPV3 channels by2-APB.Using the optimized assay,we screened TRPV3 channel regulators from a number of natural products.Whole-cell patch clamp recording was used to further confirm the effect of neferine on TRPV3,and the selectivity of neferine over other TRP channels was also evaluated.Techniques combined with positive and reverse thin-layer chromatography,HPD-100 Macroporous resin,ODS column,silica gel column and high performance liquid chromatograph?HPLC?were used to extract and isolate the active components of Gastrodia elata BL.The effects of the extracts of Gastrodia elata BL and gastrodin on TRP channels were tested by high-throughput screening adaptable calcium-fluorescent assay in Flex Station 3 format.Results The optimized FlexStation 3 assay includes the cell density of 40,000cells/well,5.0%of Cal-520 calcium fluorescent dye,the dye incubation time of 1.5 h,and the prior addition of test compound.In the FlexStation 3 assay ruthenium red inhibited TRPV3 channel with an IC₅₀ at 5.35±1.39mM.We found a new natural TRPV3 channel inhibitor:neferine.We determined its dose-dependent inhibition of TRPV3 current with an IC₅₀ at 24.5±4.1mM.We also found that neferine had no significant inhibitory effect on TRPV1 and TRPV4 channels.In addition,our preliminary screening shows that Gastrodia elata BL extracts had no effects on TRPV1-4,TRPA1 and TRPM8 channels.Conclusions We optimized the high-throughput screening adaptable assay using Flex Station 3 in 96-well format.Using this calcium fluorescent assay,we identified a natural TRPV3 channel inhibitor neferine.We confirmed the dose-dependent inhibition of TRPV3 by neferine using whole-cell patch clamp recordings and also determined the selectivity of neferine over TRPV1 or TRPV4.Neferine,as a new natural TRPV3 channel inhibitor,is of a great significance for pharmacological study of TRPV3 channel.