Direct Activation of TRPC3 Channels by the Antimalarial Agent Artemisinin.

Nicole Urban,Michael Schaefer

doi:10.3390/cells9010202

Abstract

(1) Background: Members of the TRPC3/TRPC6/TRPC7 subfamily of canonical transient receptor potential (TRP) channels share an amino acid similarity of more than 80% and can form heteromeric channel complexes. They are directly gated by diacylglycerols in a protein kinase C-independent manner. To assess TRPC3 channel functions without concomitant protein kinase C activation, direct activators are highly desirable. (2) Methods: By screening 2000 bioactive compounds in a Ca2+ influx assay, we identified artemisinin as a TRPC3 activator. Validation and characterization of the hit was performed by applying fluorometric Ca2+ influx assays and electrophysiological patch-clamp experiments in heterologously or endogenously TRPC3-expressing cells. (3) Results: Artemisinin elicited Ca2+ entry through TRPC3 or heteromeric TRPC3:TRPC6 channels, but did not or only weakly activated TRPC6 and TRPC7. Electrophysiological recordings confirmed the reversible and repeatable TRPC3 activation by artemisinin that was inhibited by established TRPC3 channel blockers. Rectification properties and reversal potentials were similar to those observed after stimulation with a diacylglycerol mimic, indicating that artemisinin induces a similar active state as the physiological activator. In rat pheochromocytoma PC12 cells that endogenously express TRPC3, artemisinin induced a Ca2+ influx and TRPC3-like currents. (4) Conclusions: Our findings identify artemisinin as a new biologically active entity to activate recombinant or native TRPC3-bearing channel complexes in a membrane-confined fashion.

Highlights

Among mammalian transient receptor potential (TRP) channels, the “canonical” or “classical”TRPC3 isoform was the first to become functionally characterized upon heterologous expression [1].Its direct activation by diacylglycerols added a new facet to the spectrum of actions caused by the membrane-resident lipid second messenger [2]
Human embryonic kidney 293 (HEK293) cells were stably transfected with expression plasmids that encode hTRPC3-YFP, hTRPC6-YFP, hTRPC7-YFP, mTRPC4ß-YFP, rTRPV1-CFP, rTRPV2-YFP, rTRPV3-YFP, mTRPV4-YFP, or hTRPM2 as previously described
Upon acute addition of the compounds at a concentration of 20 μM to fluo-4-loaded HEK cells that stably expressed a TRPC3-YFP fusion protein (HEKTRPC3-YFP ), an immediate and transient increase in the fluorescence signal of the Ca2+ indicator became apparent in wells that contained artemisinin and artenimol (Figure S1A)

Summary

Introduction

Among mammalian transient receptor potential (TRP) channels, the “canonical” or “classical”TRPC3 isoform was the first to become functionally characterized upon heterologous expression [1].Its direct activation by diacylglycerols added a new facet to the spectrum of actions caused by the membrane-resident lipid second messenger [2]. One of the most striking effects is an impaired motor coordination that is linked to the strong expression of TRPC3 and its activation by phospholipase C (PLC)-coupled metabotropic glutamate receptors in a specific pool of cerebellar. Purkinje neurons [3,4] and was confirmed by a similar effect in a mutant “moonwalker” mouse strain that carries a point mutation in the TRPC3-encoding gene [5]. Despite these and other data demonstrating that TRPC3 mediates a plethora of effects in the context of G-protein- or receptor tyrosine kinase-mediated signalling, its validation as a pharmacological target is still ongoing [6].

Methods

Results

Conclusion