Mechanisms of photoswitch conjugation and light activation of an ionotropic glutamate receptor

Abstract

The analysis of cell signaling requires the rapid and selective manipulation of protein function. We have synthesized photoswitches that covalently modify target proteins and reversibly present and withdraw a ligand from its binding site due to photoisomerization of an azobenzene linker. We describe here the properties of a glutamate photoswitch that controls an ion channel in cells. Affinity labeling and geometric constraints ensure that the photoswitch controls only the targeted channel, and enables spatial patterns of light to favor labeling in one location over another. Photoswitching to the activating state places a tethered glutamate at a high (millimolar) effective local concentration near the binding site. The fraction of active channels can be set in an analog manner by altering the photostationary state with different wavelengths. The bistable photoswitch can be turned on with millisecond-long pulses at one wavelength, remain on in the dark for minutes, and turned off with millisecond long pulses at the other wavelength, yielding sustained activation with minimal irradiation. The system provides rapid, reversible remote control of protein function that is selective without orthogonal chemistry.azobenzene ͉ ion channel ͉ photoisomerization ͉ optical switch ͉ remote control M uch progress has been made recently in the real-time noninvasive detection of protein function (1), but the development of approaches for remote protein manipulation within the complex environment of the cell has lagged behind. A major advance has been the development of photolysable cages for soluble ligands (2). The caged ligand is allowed to slowly diffuse into tissue in its inert form, and a powerful light flash cleaves a photolabile protecting group, releasing the active ligand rapidly (within microseconds) (3,4). This provides fast on-rates that are complemented with reasonably fast off-rates, which depend on native binding affinity, diffusion, sequestration, and breakdown. However, because native ligands often act on multiple proteins, this approach has limited selectivity. Introducing foreign receptors that bind nonnative ligands, on the other hand, enables cellular stimulation without the activation of endogenous proteins (5, 6) and can even be used in a photolysable form for rapid release (7).Photoisomerizable moieties have also found use in the remote and selective control of native protein function. In this case, reversible photochemically induced changes in the shape or electronic character of functionally important amino acids have been used to control the function of proteins in response to light (8-10) or to alter the backbone structure of peptides (11), thereby controlling their interaction with other biological macromolecules (12). In an alternative strategy, photoisomerization of a tethered ligand can be used to reversibly present, and withdraw, a ligand from a binding site. To date, several ion channel photoswitches have been reported wherein a ligand is tethered to the channel via a linker containing a photoisom...

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

“…It was shown previously that L-MAG0 460 covalently tethered to position L439C activates the channel under blue light, while photocurrent decreases in the dark by thermal isomerization of L-MAG0 460 (22). Here, 1P stimulation was achieved with a 470-nm LED used as a wide-field light source that illuminated the entire neuron (Fig.…”

“…3). Dissociated hippocampal neurons were transfected with an hSyn (human synapsin 1) gene promoter driven LiGluR (GluK2-L439C-K456A), where L439C is known to bind L-MAG0 as a cis-activated agonist (21)(22)(23)(24) (Fig. 3A).…”

“…PTLs have since been developed for a variety of other receptors and channels (2). Maleimideazobenzene-glutamate (MAG) compounds are designed to be conjugated to the extracellular ligand binding domains of genetically engineered mammalian glutamate receptors (GluRs), enabling pharmacological manipulation of signaling pathways dependent on the excitatory neurotransmitter glutamate (21)(22)(23)(24)(25). MAGs have been used to study neurotransmission and neuroplasticity in a variety of preparations (26)(27)(28)(29).…”

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

“…However, the therapeutic utility of the first-generation MAG0 was limited by two properties. First, the spectral sensitivity of the original MAG0 chromophore was outside of the visible range, peaking in the UV light at 380 nm (41), which penetrates the lens poorly and is damaging to corneal, lens epithelial, and retinal cells. 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.…”

“…First, the spectral sensitivity of the original MAG0 chromophore was outside of the visible range, peaking in the UV light at 380 nm (41), which penetrates the lens poorly and is damaging to corneal, lens epithelial, and retinal cells. 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).…”