A Red-Shifted, Fast-Relaxing Azobenzene Photoswitch for Visible Light Control of an Ionotropic Glutamate Receptor
ABSTRACT Azobenzene photoswitches have become a dependable method for rapid and exact modulation of biological processes and material science systems. The requirement of ultraviolet (UV) light for azobenzene isomerization is not ideal for biological systems due to poor tissue penetration and potentially damaging effects. While modified azo-benzene 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 trans. The visible light excitation and single wavelength behavior considerably simplifies use and should improve utilization in tissue.
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ABSTRACT: Cell signaling is often mediated by the binding of multiple ligands to multisubunit receptors. The probabilistic nature and sometimes slow rate of binding encountered with diffusible ligands can impede attempts to determine how the ligand occupancy controls signaling in such protein complexes. We describe a solution to this problem that uses a photoswitched tethered ligand as a 'ligand clamp' to induce rapid and stable binding and unbinding at defined subsets of subunits. We applied the approach to study gating in ionotropic glutamate receptors (iGluRs), ligand-gated ion channels that mediate excitatory neurotransmission and plasticity at glutamatergic synapses in the brain. We probed gating in two kainate-type iGluRs, GluK2 homotetramers and GluK2-GluK5 heterotetramers. Ultrafast (submillisecond) photoswitching of an azobenzene-based ligand on specific subunits provided a real-time measure of gating and revealed that partially occupied receptors can activate without desensitizing. The findings have implications for signaling by locally released and spillover glutamate.Nature Chemical Biology 02/2014; 10(4). DOI:10.1038/nchembio.1458 · 13.22 Impact Factor
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ABSTRACT: The effect of aryl substitution on various aspects of the interconversion of ortho-hydroxychalcones and flavanones has been studied using multiple spectroscopic techniques. Derivatization of the core scaffold predictably alters the midpoint pH of this equilibration process suggesting its viability for application as a functional colorimetric molecular switch.Organic & Biomolecular Chemistry 05/2014; 12(28). DOI:10.1039/c4ob00398e · 3.49 Impact Factor
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ABSTRACT: Synthetic photochromic compounds can be designed to control a variety of proteins and their biochemical functions in living cells, but the high spatiotemporal precision and tissue penetration of two-photon stimulation has never been investigated in these molecules. Here we demonstrate two-photon excitation of azobenzene-based protein switches, and versatile strategies to enhance their photochemical responses. This enables new applications to control the activation of neurons and astrocytes with cellular and subcellular resolution.Journal of the American Chemical Society 05/2014; 136(24). DOI:10.1021/ja5026326 · 11.44 Impact Factor