Potent, selective, and subunit‐dependent activation of TRPC5 channels by a xanthine derivative

Background and purpose

TRPC1, TRPC4 and TRPC5 can form homo‐ or heterotetrameric, calcium‐permeable cation channels that have been implicated in various disease states. Recent research has yielded specific and potent xanthine‐based TRPC1/4/5 inhibitors. Here we investigated the possibility of xanthine‐based activation.

Experimental approach

An analogue of the TRPC1/4/5 inhibitor Pico145, AM237, was synthesised and its activity was investigated using HEK cells over‐expressing TRPC4, TRPC5, TRPC4–C1, TRPC5–C1, TRPC1:C4 or TRPC1:C5, and in A498 cells expressing native TRPC1:C4 channels. TRPC1/4/5 ion channel activities were measured by [Ca²⁺]i measurements and by patch‐clamp electrophysiology. Selectivity of AM237 was tested against TRPC3, TRPC6, TRPV4 or TRPM2.

Key results

AM237 potently activated TRPC5:C5 channels in [Ca²⁺]i measurements (EC₅₀ 15‐20 nM), and potentiated TRPC5:C5 activation by sphingosine‐1‐phosphate, yet suppressed TRPC5:C5 channel activity evoked by (‐)‐englerin A (EA). In patch‐clamp studies AM237 also activated TRPC5:C5 channels, with greater effect at positive voltages, but it was a weaker activator than EA. Pico145 competitively inhibited AM237‐induced TRPC5:C5 activation. In contrast, AM237 did not activate TRPC4:C4, TRPC4–C1, TRPC5–C1, TRPC1:C5 and TRPC1:C4 channels, nor native TRPC1:C4 channels in A498 cells, but potently inhibited EA‐dependent activation of these channels with IC50 values ranging from 0.9 to 7 nM. AM237 (300 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 xanthine‐based small molecules can activate, potentiate or inhibit these channels depending on subunit composition.