A Red-Shifted, Fast-Relaxing Azobenzene Photoswitch for Visible Light Control of an Ionotropic Glutamate Receptor

Abstract

The use of azobenzene photoswitches has become a dependable method for rapid and exact modulation of biological processes and material science systems. The requirement of ultraviolet light for azobenzene isomerization is not ideal for biological systems due to poor tissue penetration and potentially damaging effects. While modified azobenzene cores with a red-shifted cis-to-trans isomerization have been previously described, they have not yet been incorporated into a powerful method to control protein function: the photoswitchable tethered ligand (PTL) approach. We report the synthesis and characterization of a red-shifted PTL, L-MAG0460, for the light-gated ionotropic glutamate receptor LiGluR. In cultured mammalian cells, the LiGluR +L-MAG0460 system is activated rapidly by illumination with 400–520 nm light to generate a large ionic current. The current rapidly turns off in the dark as the PTL relaxes thermally back to the trans configuration. The visible light excitation and single-wavelength behavior considerably simplify use and should improve utilization in tissue.

Restoration of visual function by expression of a light-gated mammalian ion channel in retinal ganglion cells or ON-bipolar cells

“…Stimulation with broad-spectrum (white) light triggered similar responses (Fig. S2G), as demonstrated previously in HEK cells (24 Fig. 3 A and B), representing an ∼10-fold improvement in sensitivity compared with the published values for the first-generation MAG photoswitch (23).…”

“…Second, LiGluR-MAG0 was bistable, requiring a second longer wavelength pulse of light to reset the system after each activating event (19,41), which would necessitate additional hardware for potential clinical applications. We solved these problems with our photoswitch, MAG0 460 , which is activated by blue light similar to blue cone photoreceptors, responds well to broad-spectrum visible light, and rapidly and spontaneously turns off in the dark (24). LiGluR-MAG0 460 responds dynamically to incremental changes in light intensity and supports reliable retinal output with intensity modulations at moderate frequencies of 4-10 Hz.…”

“…We recently developed a second-generation photoswitch, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0 460 ), to overcome these problems (24). MAG0 460 is activated by white light and spontaneously turns off in the dark.…”

“…1). Expression and labeling in human embryonic kidney (HEK) cells yielded robust LiGluR-MAG0 460 photocurrents that were stable and reversible over hundreds of switching cycles (24). Importantly, LiGluR-MAG0 460 photocurrents scaled with light intensity, yielding a sigmoid intensity-response curve spanning three orders of magnitude (Fig.…”

Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics

“…Two families of MAGs have been synthesized to date: First generation MAGs have a symmetrically-substituted azobenzene core, e.g., L-MAG0 (21) and D-MAG0 (30). Second generation MAGs have an asymmetrically-substituted azobenzene core, e.g., L-MAG0 460 (31) and MAG 2p (32). The latter MAGs are single-color photoswitches incorporating an electron-donating amine and an electron-withdrawing amide substitution, creating a "push-pull" system that both red-shifts the trans isomer absorption spectrum and reduces the thermal stability of the cis isomer (31,33).…”