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

Abstract

Background and Purpose The TRPC1, TRPC4, and TRPC5 proteins form homotetrameric or heterotetrameric, calcium‐permeable cation channels that are involved 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 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, TRPC4–C1, TRPC5–C1, 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 patch‐clamp electrophysiology. Selectivity of AM237 was tested against TRPC3, TRPC6, TRPV4, or TRPM2 channels. Key Results AM237 potently activated TRPC5:C5 channels (EC50 15–20 nM in [Ca2+]i assay) and potentiated their activation by sphingosine‐1‐phosphate but suppressed activation evoked by (−)‐englerin A (EA). In patch‐clamp studies, AM237 activated TRPC5:C5 channels, with greater effect at positive voltages, but with lower efficacy than EA. Pico145 competitively inhibited AM237‐induced TRPC5:C5 activation. AM237 did not activate TRPC4:C4, TRPC4–C1, TRPC5–C1, TRPC1:C5, and TRPC1:C4 channels, or 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 compounds can activate, potentiate, or inhibit these channels depending on subunit composition.

Cryo-EM structures of human TRPC5 reveal interaction of a xanthine-based TRPC1/4/5 inhibitor with a conserved lipid binding site

“…23,24 However, two closely related xanthine derivatives, AM237 30 and the photoaffinity probe Pico145-DAAlk, 31 are partial agonists of the homomeric TRPC5:C5 channel that inhibit other TRPC1/4/5 channels. Pico145 is a competitive antagonist of AM237 30 and concentration-dependently inhibits both activation and photoaffinity labelling of TRPC5:C5 by Pico145-DAAlk. 31 These data are consistent with the hypothesis that xanthine-based TRPC1/4/5 modulators all bind to the same lipid binding site of TRPC1/4/5 channels, where they can stabilise either open or closed channel conformations depending on xanthine substituent patterns and the composition of the specific TRPC tetramer.…”

“…Mutations to the xanthine binding site were introduced into TRPC5-SYFP2 in pcDNA4. 30 Bacmids were produced according to the Bac-to-Bac protocol (Invitrogen).…”

“…This could explain why Pico145 is an inhibitor of TRPC5, whereas AM237 is a partial agonist. 30 To gain insight into the potent inhibition of TRPC4 channels by Pico145, 23 we next docked Pico145 into the published mTRPC4 apo structure (PDB 5z96; the phospholipid was removed before docking). The docking suggests that Pico145 adopts a similar pose in TRPC4 and TRPC5, making similar interactions with conserved residues, particularly residues equivalent to the TRPC5 residues Q573, F576 and W577 (Supplementary Figure 9).…”

“…The inhibitory potency of Pico145 on TRPC1/4/5 channels is dependent on the EA concentration used in assays, 23 which was expected to complicate analysis of Pico145 responses of TRPC5 variants. Therefore, we instead tested TRPC5 responses to AM237, which is thought to occupy the same binding site as Pico145 (see above and 30,31 ). Mutations that resulted in TRPC5 activation by EA, but not by AM237, were likely to reveal residues contributing to xanthine binding.…”

“…23 Pico145 is orally bioavailable and has been used successfully for studies of TRPC1/4/5 channels in vivo. 25,28,29 Detailed characterisation of Pico145 and the related xanthine AM237a partial TRPC5 agonist that inhibits other TRPC1/4/5 channels 30revealed that: 1) Pico145 and AM237 act rapidly and reversibly in outside-out excised patch recordings, highlighting a membrane-delimited effect; 23,30 2) Pico145 is a competitive antagonist of (-)-englerin A (EA) 23 and AM237, 30 and also inhibits channel activation by sphingosine-1-phosphate (S1P), carbachol and Gd 3+ , 23,24 although picomolar concentrations of Pico145 can potentiate Gd 3+ -induced TRPC4 currents as well; 23 3) Pico145 concentration-dependently inhibits photoaffinity labelling of TRPC5 by the xanthine-based photoaffinity probes Pico145-DAAlk and Pico145-DAAlk2, with IC50 values in the same range as inhibition of TRPC5-mediated calcium influx. 31 These results provide evidence that these xanthines modulate TRPC1/4/5 channels through a direct molecular interaction with the channels and are consistent with the hypothesis that Pico145 and AM237 bind to a well-defined, high-affinity binding site essential to TRPC1/4/5 channel gating.…”