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 Ca2+ 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 Ca2+ 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 Ca2+ 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.
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Fu, J., Gao, Z., Shen, B., and Zhu, M. (2015). Canonical transient receptor potential 4 and its small molecule modulators. Sci China Life Sci 58, 39–47.
Furuta, T., Wang, S.S., Dantzker, J.L., Dore, T.M., Bybee, W.J., Callaway, E.M., Denk, W., and Tsien, R.Y. (1999). Brominated 7-hydroxycoumarin-4-ylmethyls: photolabile protecting groups with biologically useful cross-sections for two photon photolysis. Proc Natl Acad Sci USA 96, 1193–1200.
Furuta, T., Takeuchi, H., Isozaki, M., Takahashi, Y., Kanehara, M., Sugimoto, M., Watanabe, T., Noguchi, K., Dore, T.M., Kurahashi, T., Iwamura, M., and Tsien R.Y. (2004). Bhc-cNMPs as either water-soluble or membrane-permeant photoreleasable cyclic nucleotides for both one-and two-hoton excitation. Chem Bio Chem 5, 1119–1128.
Givens, R., Rubina, M., and Wirz, J. (2012). Applications of p-hydroxyphenacyl (pHP) and coumarin-4-ylmethyl photoremovable protecting groups. Photochem Photobiol Sci 11, 472–488.
Goeldner, M., and Givens, R. (2005). Dynamic Studies in Biology: Phototrigges, Photoswitches and Caged Biomolecules (Weinheim: WILEYVCH Verlag GmbH & Co. KGaA).
Hagen, V., Kilic, F., Schaal, J., Dekowski, B., Schmidt, R., and Kotzur, N. (2010). [8-[Bis(carboxymethyl)aminomethyl]-6-bromo-7-hydroxycoumarin-4-yl]methyl moieties as photoremovable protecting groups for compounds with COOH, NH2, OH, and C=O functions. J Org Chem 75, 2790–2797.
Hess, G., Carpenter, B. K., Shembekar, V.R., and Chen, Y. (2007). Protecting group for carboxylic acids that can be photolyzed by visible light. USA Patent, US2007/0243519A1.
Hofmann, T., Obukhov, A.G., Schaefer, M., Harteneck, C., Gudermann, T., and Schultz G. (1999). Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397, 259–263.
Höglinger, D., Nadler, A., and Schultz, C. (2014). Caged lipids as tools for investigating cellular signaling. Biochim Biophys Acta 1841, 1085–1096.
Huang, X.P., Sreekumar, R., Patel, J.R., and Walker, J.W. (1996). Response of cardiac myocytes to a ramp increase of diacylglycerol generated by photolysis of a novel caged diacylglycerol. Biophys J, 70, 2448–2457.
Kamouchi, M., Philipp, S., Flockerzi, V., Wissenbach, U., Mamin, A., Raeymaekers, L., Eggermont, J., Droogmans, G., and Nilius, B. (1999). Properties of heterologously expressed hTRP3 channels in bovine pulmonary artery endothelial cells. J Physiol 2, 345–358.
Kim, Y.A., Ramirez, D.M.C., Costain, W.J., Johnston, L.J., and Bittman, R. (2011). A new tool to assess ceramide bioactivity: 6-bromo-7-hydroxycoumarinylcaged ceramide. Chem Commun 47, 9236–9238.
Lucas, P., Ukhanov, K., Leinders-Zufall, T., and Zufall, F. (2003). A diacylglycerol-gated cation channel in vomeronasal neuron dendrites is impaired in TRPC2 mutant mice: mechanism of pheromone transduction. Neuron 40, 551–61.
Mantulin, W.W., and Song, P.S. (1973). Excited states of skin-sensitizing coumarins and psoralens. Spectroscopic studies. J Am Chem Soc 95, 5122–5129.
Mayer, G., and Heckel, A. (2006). Biologically active molecules with a “Light Switch”. Angew Chem Int Ed 45, 4900–4920.
Mentel, M., Laketa, V., Subramanian, D., Gillandt, H., and Schultz, C. (2011). Photoactivatable and cell-membranepermeable phosphatidylinositol 3,4,5-tris-phosphate. Angew Chem Int Ed Engl 50, 3811–3814.
Nadler, A., Reither, G., Feng, S., Stein, F., Reither, S., Müller, R., and Schultz, C. (2013). The fatty acid composition of diacylglycerols determines local signaling patterns. Angew Chemie Int Ed 52, 6330–6334.
Newton, A.C. (2010). Regulation of conventional and novel protein kinase C isozymes by phosphorylation and lipids. In Protein Kinase C in Cancer Signaling and Therapy, Current Cancer Research, M. G. Kazanietz, ed. (Berlin: Springer), pp. 9–23.
Ohanian, J., and Ohanian, V. (2001). Lipid second messenger regulation: the role of diacylglycerol kinases and their relevance to hypertension. J Hum Hypertens 15, 93–98.
Park, S.C., Goodrich, R.P., Yerram, N., Sowemino-Coker, S.O., Platz, M.S., and Aquila, B. (1996). Photodinamic inactivation of viral and bacterial blood contaminants with halogenated coumarin and furocoumarin sensitizers. USA Patent, WO9608965A1.
Pelliccioli, A.P., and Wirz, J. (2002). Photoremovable protecting groups: reaction mechanisms and applications. Photochem Photobiol Sci 1, 441–458.
Rosker, C., Graziani, A., Lukas, M., Eder, P., Zhu, M.X., Romanin, C., and Groschner, K. (2004). Ca2+ signaling by TRPC3 involves Na+ entry and local coupling to the Na+/Ca2+ exchanger. J Biol Chem 279, 13696–13704.
van Rossum, D.B., Oberdick, D., Rbaibi, Y., Bhardwaj, G., Barrow, R.K., Nikolaidis, N., Snyder, S.H., Kiselyov K., and Patterson R.L. (2008). TRP_2, a lipid trafficking domain that mediates diacylglycerol-induced vesicle fusion. J Biol Chem 283, 34384–34392.
Singh, A., Hildebrand, M.E., Garcia, E., and Snutch, T.P. (2010). The transient receptor potential channel antagonist SKF 96365 is a potent blocker of low-voltage-activated T-type calcium channels. Br J Pharmacol 160, 1464–1475.
Subramanian, D., Laketa, V., Muller, R., Tischer, C., Zarbakhsh, S., Pepperkok, R., and Schultz, C. (2010). Activation of membranepermeant caged PtdIns(3)P induces endosomal fusion in cells. Nat Chem Biol 6, 324–326.
Suzuki, A.Z., Watanabe, T., Kawamoto, M., Nishiyama, K., Yamashita, H., Ishii, M., Iwamura, M., and Furuta, T. (2003). Coumarin-4-ylmethoxycarbonyls as phototriggers for alcohols and phenols. Org Lett 5, 4867–4870.
Tsien, R.Y., and Furuta, T. (2000). Protecting groups with increased photosensitivities. USA Patent, WO/00/31588.
Yang, C., and Kazanietz, M.G. (2003). Divergence and complexities in DAG signaling: looking beyond PKC. Trends Pharmacol Sci 24, 602–608.
Zou, Q., Fanga, Y., Zhao, Y., Zhao, H., Wang, Y., Gub, Y., and Wu, F. (2013). Synthesis and in vitro photocytotoxicity of coumarin derivatives for one-and two-photon excited photodynamic therapy. J Med Chem 56, 5288–5294.
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Tiapko, O., Bacsa, B., de la Cruz, G.G. et al. Optopharmacological control of TRPC channels by coumarin-caged lipids is associated with a phototoxic membrane effect. Sci. China Life Sci. 59, 802–810 (2016). https://doi.org/10.1007/s11427-016-5095-5
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DOI: https://doi.org/10.1007/s11427-016-5095-5