Development of TRPC Assays on Automated Electrophysiology Platforms

TRPC3, TRPC6 and TRPC7 are Ca2+ permeable non-selective cation channels that have been implicated in cancer, cardiovascular, respiratory and kidney diseases. In this study, CHO-K1 cells stably expressing human M3 muscarinic acetylcholine receptors were infected with human TRPC3, TRPC6 or TRPC7 BacMam viruses and measured on QPatch and IonWorks Quattro automated electrophysiology platforms. In the QPatch HT mode (single cell recording), TRPC currents were rapidly activated by the muscarinic agonist carbachol and then decayed irreversibly with a rank order of TRPC7>TRPC3>TRPC6. In both HT and HTX (10 cells per well) mode there was large variation in current levels between recordings. The lack of a good well-to-well comparison means that QPatch has low suitability for TRPC screening assays. The Quattro in PPC mode (64 cells per well), sufficiently normalised well-to-well variation in carbachol activated TRPC currents to enable assays to be developed for TRPC3 and TRPC6. The time delay on the Quattro between carbachol addition and the first current measurement resulted in TRPC7 currents having almost completely decayed so remaining currents were too small. The TRPC3 and TRPC6 assays were validated with two recently described TRPC inhibitors. Compound 8 [1] inhibited TRPC3 and TRPC6 currents with respective IC50 values of 1.1 ± 0.2 μM and 24 ± 7 nM (Mean ± SD, n=4-5). 2-(amino)-thiazole-4-carboxamide [2] inhibited TRPC3 and TRPC6 currents with respective IC50 values of 1.4 ± 1.1 and 0.9 ± 0.2 μM (Mean ± SD, n=3). These values are in good agreement with the published data.This study demonstrates that TRPC3, TRPC6 and TRPC7 currents can be measured on automated electrophysiology platforms. For TRPC channels, the QPatch is suitable to profile channel biophysics whereas the Quattro is more applicable for compound profiling.[1] Patent WO2011/107474 A1.[2] Patent WO2012/037349 A2.