| Summary: | Azobenzene-based photochromic lipids are valuable probes for the analysis of ion channel–lipid interactions. Rapid photoisomerization of these molecules enables the analysis of lipid gating kinetics and provides information on lipid sensing. Thermal relaxation of the metastable <i>cis</i> conformation to the <i>trans</i> conformation of azobenzene photolipids is rather slow in the dark and may be modified by ligand–protein interactions. <i>Cis</i> photolipid-induced changes in pure lipid membranes as visualized from the morphological response of giant unilamellar vesicles indicated that thermal <i>cis–trans</i> isomerization of both PhoDAG-1 and OptoDArG is essentially slow in the lipid bilayer environment. While the currents activated by <i>cis</i> PhoDAG remained stable upon termination of UV light exposure (dark, UV-OFF), <i>cis</i> OptoDArG-induced TRPC3/6/7 activity displayed a striking isoform-dependent exponential decay. The deactivation kinetics of <i>cis</i> OptoDArG-induced currents in the dark was sensitive to mutations in the L2 lipid coordination site of TRPC channels. We conclude that the binding of <i>cis</i> OptoDArG to TRPC channels promotes transition of <i>cis</i> OptoDArG to the <i>trans</i> conformation. This process is suggested to provide valuable information on DAG–ion channel interactions and may enable highly selective photopharmacological interventions.
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