Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Tiapko, O.
Exploring lipid-mediated TRPC signaling by light
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Graz Medical University; 2018. pp. 72
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- Autor*innen der Med Uni Graz:
- Tiapko Oleksandra
- Betreuer*innen:
- Graier Wolfgang
- Groschner Klaus
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- Abstract:
- Introduction: TRPC3 channels have been attracting much attention through the years with its unique mechanism of activation, which results in Ca2+ influx in excitable and non-excitable cells. Deciphering the principle of ligand recognition by TRPC channels and clarifying their exact role in native cells demands specific tools. Photopharmacology as a novel and highly precise technique for manipulation of signaling molecules opens a venue for targeting TRPC3 with high temporal and spacious precision. The technique offers two strategies, which are suitable for optical control of TRPC3 channels: caged and photochromic second messengers. Light-controlled activation of channel activating ligands may provide mechanistic insights into the signaling function of TRPC3 and advance their therapeutic targeting. Aim: we set out to develop specific photopharmacological tools for manipulation of TRPC3 channel activity and function in vitro and in vivo. Materials and methods: In this work we characterized photoswitchable ligands for suitability to control TRPC3 channels by light. We combined electrophysiological and Ca2+ imagine techniques to detect activity of TRPC3 overexpressed in HEK293 cells during photoactivation of lipid- and non-lipid activators. Additionally, two cell models (endothelial cells and neurons) with endogenously expressed TRPC channels were used to characterize the selectivity of these photopharmacological tools. Results and conclusion: Lipid uncaging induced Ca2+ transients in TRPC3 transfected cells. However, the uncaging-induced conductance was identified as an unexpected UV illumination-dependent artifact, which was produced by the cage structure. Therefore, caging structures in photopharmacological experiments have to be used with caution. Photochromic moieties like azobenzene allows for control of the conformational state by light dependent cis-trans isomerization and, thereby of the biological activity of a ligand. Azobenzene incorporation into lipids was employed as an alternative to the uncaging strategy. A mutagenesis screen, guided by homology modeling of TRPC3 in combination with utilization of photosensitive lipids revealed an essential lipid-protein interaction site within the pore complex of TRPC3. To exert a more specific control over endogenous channels, we synthetized a photochromic derivative of the TRPC3/6 agonist GSK1702934A and assigned this benzimidazole derivative as OptoBI-1. Light-stimulation of OptoBI-1 enabled precise control over TRPC channels in endothelial cells and neurons. In summary, this work provides a basis for a development of future strategies to explore TRPC channels in vitro and in vivo.