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 cis conformation to the trans conformation of azobenzene photolipids is rather slow in the dark and may be modified by ligand–protein interactions. Cis photolipid-induced changes in pure lipid membranes as visualized from the morphological response of giant unilamellar vesicles indicated that thermal cis–trans isomerization of both PhoDAG-1 and OptoDArG is essentially slow in the lipid bilayer environment. While the currents activated by cis PhoDAG remained stable upon termination of UV light exposure (dark, UV-OFF), cis OptoDArG-induced TRPC3/6/7 activity displayed a striking isoform-dependent exponential decay. The deactivation kinetics of cis 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 cis OptoDArG to TRPC channels promotes transition of cis OptoDArG to the trans conformation. This process is suggested to provide valuable information on DAG–ion channel interactions and may enable highly selective photopharmacological interventions.

Photomanipulation of minimal synthetic cells: area increase, softening and interleaflet coupling of membrane models doped with azobenzene-lipid photoswitches

“…Because of their large size, GUVs offer the possibility to directly monitor the membrane under a microscope. Light – triggered changes have been investigated on GUVs to interrogate, among others, (i) light-sensitive proteins embedded in the membrane such as the photoreceptor bacteriorhodopsin [26] , (ii) the photoactivation of channel proteins [27] , (iii) membrane embedded fluorescent dyes which raise the membrane tension and can cause transient poration under irradiation [28] . The effect of azobenzene derivatives have also been studied with giant vesicles.…”

Photo‐Lipids: Light‐Sensitive Nano‐Switches to Control Membrane Properties

“…For eukaryotic mammalian cells, photo‐lipids were primarily used to study signalling pathways leading to apoptosis, [76] cytokine production, [77] lipid signalling, [78,79] and biosynthesis [43,80] or nociception [81,82] . Rather recently, the impact of photo‐lipids on transient receptor potential canonical channels (TRCP) was also reported [83–85] . Although it was widely suggested that TRCP channels are activated by DAG, [86,87] the mechanism of activation of these channels is not fully understood, given the fact that a direct interaction with DAG is yet to be reported [88] .…”

“…[81,82] Rather recently, the impact of photo-lipids on transient receptor potential canonical channels (TRCP) was also reported. [83][84][85] Although it was widely suggested that TRCP channels are activated by DAG, [86,87] the mechanism of activation of these channels is not fully understood, given the fact that a direct interaction with DAG is yet to be reported. [88] Lichtenegger et al [83] demonstrated that the TRCP3 activity could be regulated using OptoDArG in human embryonic kidney 293 cells (HEK293).…”

“…[84] Nonetheless, OptoDArG appears to be more efficient than PhoDAGs to control the TCRP channel activity, probably because of a better stability in the membrane. [85]…”

“…Its isomerization from trans to cis caused channel opening events and altered cellular calcium ion levels [84] . Nonetheless, OptoDArG appears to be more efficient than PhoDAGs to control the TCRP channel activity, probably because of a better stability in the membrane [85] …”