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 lipi...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.展开更多
基金supported by the Austrian Science Fund(FWFW 1226-B18)
文摘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.
