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

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.caged diacylglycerols, TRPC channels, Ca 2+ signaling, optopharmacology Citation:Tiapko, O., Bacsa, B., de la Cruz, G.G., Glasnov, T., and Groschner, K. (2016). Optopharmacological control of TRPC channels by coumarin-caged lipids is associated with a phototoxic membrane effect. Sci China Life Sci 59, 802 -810.

TRPC channels: Structure, function, regulation and recent advances in small molecular probes

“…Moreover, the substrate of PLC, PIP 2 , also exerts complex effects on TRPC channels, including both some levels of dependence on the presence of PIP 2 for channel activation and also some inhibition by the lipid (Imai et al, 2012;Kim et al, 2008;Lemonnier et al, 2008;Otsuguro et al, 2008;Shi et al, 2012;Thakur et al, 2016). Among these factors, only DAGs have been exploited in drug development to create new analogs that activate TRPC3 channels (Lichtenegger et al, 2018;Tiapko, Bacsa, de la Cruz, Glasnov, & Groschner, 2016). Furthermore, the receptor-operated activation of TRPC4/5 channels also includes G i/o -coupled receptors and the PTX-sensitive G i and G o proteins (Thakur et al, 2016).…”

Lipid-independent control of endothelial and neuronal TRPC3 channels by light

“…Azobenzene photoswitches are both suitable and valuable for the high precision control of TRPC channels as has recently been demonstrated by reports on the spatial and temporal precision of TRPC activation achieved with photoconvertible diacylglycerols 4–6. Although exceptionally effective in terms of channel activation, this latter approach suffers from two inherent limitations, namely its general lack of selectivity and a certain temporal inaccuracy due to delay and frequency dependence of optical control based on cooperativity of lipid gating 5.…”

TRPC3 as a Target of Novel Therapeutic Interventions

“…Photorelease of caged 1,2-DOG was found to generate an artifact caused by UV light application in the presence of the generated free coumarin moiety, whereas the UV light itself did not affect membrane conductance. Hence, off-target effects, due to the phototoxicity of the caging structure, requires consideration and is expected to limit the therapeutic value of caged ligand strategies [ 75 ].…”