Synthesis, Characterization, and Testing of Red-Shifted Glutamate Photoswitches

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DESCRIPTION (provided by applicant): Promising new tools which enable an individual protein's function to be controlled with light have been developed and applied in the nascent field of optogenetics. Many of these tools are based on light-modulated ion channels which provide unrivaled temporal and spatial control. Recently, a two-component chemical and biological approach based on a Photoswitchable Tethered Ligand (PTL) and a modified ionotropic glutamate receptor 6 (LiGluR) was developed. The PTL Maleimide-Azobenzene-Glutamate (MAG) takes advantage of the remarkable photochemistry of azobenzene, changes its shape (between the cis and trans isomers) depending on the irradiation wavelength used. When MAG covalently binds via the maleimide moiety to an engineered cysteine residue near the ligand binding domain of LiGluR, it allows for the precise control of channel gating. When expressed in neurons, this light-induced current allows for the induction of action potentials with light. Despite the success of LiGluR, the photochemical properties of this optical switch offer many opportunities for improvement. The most significant of which is the requirement of irradiation with UV light (380 nm), which is problematic because it is both toxic to cells and strongly scattered by many tissues, making penetration in larger organisms nearly impossible. The initial goal of this proposal is to synthesize and characterize the photophysical properties of a red- shifted (visible light modulated) MAG analog. Due to extensive use of azobenzene compounds in the dye industry, the literature describes a variety of methods which can be used for the synthesis of red-shifted MAG. The least complicated method involved synthesizing a "push-pull" type azobenzene core by installing an electron donating amine functionality ("push") at one end of the core counterbalanced by an electron withdrawing amide bond ("pull") at the other. The synthesis of such a red-shifted MAG has been completed and preliminary traces from whole-cell patch clamp experiments in HEK293 cells expressing LiGluR have established that the new MAGs function as state dependant tethered agonists. Further synthesis of a related MAG for testing on light-activated metabotropic glutamate receptors (LimGluR2 and 3) is planned. The next stage will be whole-cell patch clamping to test the red- shifted MAG for ability to elicit action potential firing in dissociated rat hippocampal neurons expressing LiGluR and inhibition of action potential firing and neurotransmitter release with LimGluR2. In addition to further developing a useful tool for basic neuroscience research, the creation of visible light activated versions of LiGluR and LimGluR has applications in the retina. Indeed, these visible-light activated tools will be tested for the ability to restore vision in mouse models of inherited blindness by expressing them in retinal ganglion cells and ON-bipolar cells.