Characterization of DAG Binding to TRPC Channels by Target-Dependent cis–trans Isomerization of OptoDArG

Abstract

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.