Background and Purpose
The TRPC1, TRPC4, and TRPC5 proteins form homotetrameric or heterotetrameric, calciumâpermeable cation channels that are involved in various disease states. Recent research has yielded specific and potent xanthineâbased TRPC1/4/5 inhibitors. Here, we investigated the possibility of xanthineâbased activators of these channels.
Experimental Approach
An analogue of the TRPC1/4/5 inhibitor Pico145, AM237, was synthesized and its activity was investigated using HEK cells overexpressing TRPC4, TRPC5, TRPC4âC1, TRPC5âC1, TRPC1:C4 or TRPC1:C5 channels, and in A498 cells expressing native TRPC1:C4 channels. TRPC1/4/5 channel activities were assayed by measuring intracellular concentration of Ca2+ ([Ca2+]i) and by patchâclamp electrophysiology. Selectivity of AM237 was tested against TRPC3, TRPC6, TRPV4, or TRPM2 channels.
Key Results
AM237 potently activated TRPC5:C5 channels (EC50 15â20Â nM in [Ca2+]i assay) and potentiated their activation by sphingosineâ1âphosphate but suppressed activation evoked by (â)âenglerin A (EA). In patchâclamp studies, AM237 activated TRPC5:C5 channels, with greater effect at positive voltages, but with lower efficacy than EA. Pico145 competitively inhibited AM237âinduced TRPC5:C5 activation. AM237 did not activate TRPC4:C4, TRPC4âC1, TRPC5âC1, TRPC1:C5, and TRPC1:C4 channels, or native TRPC1:C4 channels in A498 cells, but potently inhibited EAâdependent activation of these channels with IC50 values ranging from 0.9 to 7Â nM. AM237 (300Â nM) did not activate or inhibit TRPC3, TRPC6, TRPV4, or TRPM2 channels.
Conclusions and Implications
This study suggests the possibility for selective activation of TRPC5 channels by xanthine derivatives and supports the general principle that xanthineâbased compounds can activate, potentiate, or inhibit these channels depending on subunit composition.