Optopharmacological control of TRPC channels by coumarin-caged lipids is associated with a phototoxic membrane effect.

Oleksandra Tiapko,Toma Glasnov,Bernadett Bacsa,Gema Guedes De La Cruz,Klaus Groschner

doi:10.1007/s11427-016-5095-5

Oleksandra Tiapko, Toma Glasnov + Show 3 more

Open Access

https://doi.org/10.1007/s11427-016-5095-5

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Abstract

Photouncaging of second messengers has been successfully employed to gain mechanistic insight of cellular signaling pathways. One of the most enigmatic processes of ion channel regulation is lipid recognition and lipid-gating of TRPC channels, which represents pivotal mechanisms of cellular Ca(2+) homeostasis. Recently, optopharmacological tools including caged lipid mediators became available, enabling an unprecedented level of temporal and spatial control of the activating lipid species within a cellular environment. Here we tested a commonly used caged ligand approach for suitability to investigate TRPC signaling at the level of membrane conductance and cellular Ca(2+) handling. We report a specific photouncaging artifact that is triggered by the cage structure coumarin at UV illumination. Electrophysiological characterization identified a light-dependent membrane effect of coumarin. UV light (340 nm) as used for photouncaging, initiated a membrane conductance specifically in the presence of coumarin as low as 30 μmol L(-1) concentrations. This conductance masked the TRPC3 conductance evoked by photouncaging, while TRPC-mediated cellular Ca(2+) responses were largely preserved. The observed light-induced membrane effects of the released caging moiety may well interfere with certain cellular functions, and prompt caution in using coumarin-caged second messengers in cellular studies.

Highlights

Diacylglycerol (DAG) is an important and multifunctional second messenger, which plays a key role in signal transduction
Caged diacylglycerols, a high level of specificity in terms of TRPC channels have been suggested from experiments measuring intracellular free Ca2+ via common fluorescence probes
We show clear inhibition of the photouncaging-induced Ca2+ signal by the TRPC3 inhibitor SKF 96365 and no significant light-induced changes in intracellular Ca2+ of wild type Human embryonic kidney 293 (HEK293) cells

Summary

Introduction

Diacylglycerol (DAG) is an important and multifunctional second messenger, which plays a key role in signal transduction. The release of this regulatory lipid is controlled by phospholipase C driven hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) (Ohanian and Ohanian, 2001). TRPC3/6/7 channels are mammalian homologues of light sensitive Drosophila TRP channels and are widely distributed in the central nervous system, kidney, digestive tract and blood vessels (Fu et al, 2015). These channels are classified as non-selective cation channels, which favor transport of Ca2+ over other cations (Kamouchi et al, 1999)

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