Xanthine-based photoaffinity probes allow assessment of ligand engagement by TRPC5 channels

Abstract

Diazirine-containing photoaffinity probes, based on the potent and selective TRPC1/4/5 channel inhibitor Pico145, allowed the development of an assay to probe cellular interactions between TRPC5 protein and xanthine-based TRPC5 channel modulators.

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

“…Therefore, we decided to test TRPC5 responses to AM237, which is thought to occupy the same binding site as Pico145 (see above and refs. 31 , 32 ). Mutations that resulted in TRPC5 activation by EA, but not by AM237, were likely to reveal residues contributing to xanthine binding.…”

“…The xanthines Pico145 and HC-070 are the most potent and selective TRPC1/4/5 inhibitors to date 24 , 25 . However, three closely related xanthine derivatives, AM237 31 and the photoaffinity probes Pico145-DA and Pico145-DAAlk 32 , are partial agonists of the homomeric TRPC5:C5 channel that inhibit other TRPC1/4/5 channels. Pico145 is a competitive antagonist of AM237 31 and concentration-dependently inhibits both activation and photoaffinity labelling of TRPC5:C5 by Pico145-DAAlk 32 .…”

“…However, three closely related xanthine derivatives, AM237 31 and the photoaffinity probes Pico145-DA and Pico145-DAAlk 32 , are partial agonists of the homomeric TRPC5:C5 channel that inhibit other TRPC1/4/5 channels. Pico145 is a competitive antagonist of AM237 31 and concentration-dependently inhibits both activation and photoaffinity labelling of TRPC5:C5 by Pico145-DAAlk 32 . 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 (this paper and refs.…”

“…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 (this paper and refs. 31 , 32 ), where they can stabilise either open or closed channel conformations depending on xanthine substituent patterns and the composition of the specific TRPC tetramer. Therefore, targeting the xanthine-binding site of TRPC1/4/5 channels may allow the development of modulators of specific TRPC channel tetramers, a major challenge in TRPC1/4/5 pharmacology.…”

“…Pico145 is orally bioavailable and has been used successfully for studies of TRPC1/4/5 channels in vivo 26 , 29 , 30 . Detailed characterisation of Pico145 and the related xanthine AM237—a partial TRPC5 agonist that inhibits other TRPC1/4/5 channels 31 —revealed that: (1) Pico145 and AM237 act rapidly and reversibly in outside-out excised patch recordings, highlighting a membrane-delimited effect 24 , 31 ; (2) Pico145 is a competitive antagonist of (−)-englerin A (EA) 24 and AM237 31 , and also inhibits channel activation by sphingosine-1-phosphate (S1P), carbachol and Gd 3+ 24 , 25 , although picomolar concentrations of Pico145 can potentiate Gd 3+ -induced TRPC4 currents as well 24 ; (3) Pico145 concentration-dependently inhibits photoaffinity labelling of TRPC5 by the xanthine-based photoaffinity probes Pico145-DAAlk and Pico145-DAAlk2, with IC 50 values in the same range as inhibition of TRPC5-mediated calcium influx 32 . These results provide evidence that these xanthines modulate TRPC1/4/5 channels through 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.…”