| Assay Method Information | |
| | Monitoring the TRPM3 Ion Channel Driven Ca2+ Uptake |
| Description: | In order to monitor the inhibition of the mouse TRPM3α2 (mTRPM3) ion channel by the compounds of the invention, a cellular system making use of an mTRPM3alpha2 or hTRPM3 overexpressing cell line (flip-in HEK293) was used. The TRPM3 channel was stimulated/opened with Pregnenolone sulfate (PS) (50 μM) which results in Ca2+ influx. For mTRPM3, the intracellular Ca2+ was measured with a Calcium responsive dye. Fluor-4 AM ester (Invitrogen). Cells were cultured until a confluence of 80-90%, washed with Versene (Invitrogen) and detached from the surface by a short incubation with 0.05% Trypsin (Invitrogen). The trypsination process was stopped by the addition of complete cell culture medium (DMEM, glutamax, 10% FCS,NEAA,Pen-Strep). Cells were collected and resuspended in Krebs buffer without Calcium at RT. Prior the cell seeding (±2000 cells/well into a black, 384 well plate (Greiner)) the diluted compound was added in the assay plate, together with the PS dissolved in Krebs buffer containing Calcium. This resulted in a 2.4 mM Ca2+ assay solution. Directly after cell addition the plates were read on an Envision fluorescence reader (Perkin Elmer) by an Excitation of 485 nM and emission at 535 nM. Channel inhibition was calculated compared to a non-PS stimulated control versus a condition stimulated with PS (50 μM) with vehicle. The ability of the compounds of the invention to inhibit this activity was determined as: Percentage inhibition=[1-((RFU determined for sample with test compound present—RFU determined for sample with positive control inhibitor) divided by (RFU determined in the presence of vehicle—RFU determined for sample with positive control inhibitor))]*100. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |