Optocontrol of glutamate receptor activity by single side-chain photoisomerization.

Engineering light-sensitivity into proteins has wide ranging applications in molecular studies and neuroscience. Commonly used tethered photoswitchable ligands, however, require solvent-accessible protein labeling, face structural constrains, and are bulky. Here, we designed a set of optocontrollable NMDA receptors by directly incorporating single photoswitchable amino acids (PSAAs) providing genetic encodability, reversibility, and site tolerance. We identified several positions within the multi-domain receptor endowing robust photomodulation. PSAA photoisomerization at the GluN1 clamshell hinge is sufficient to control glycine sensitivity and activation efficacy. Strikingly, in the pore domain, flipping of a M3 residue within a conserved transmembrane cavity impacts both gating and permeation properties. Our study demonstrates the first detection of molecular rearrangements in real-time due to the reversible light-switching of single amino acid side-chains, adding a dynamic dimension to protein site-directed mutagenesis. This novel approach to interrogate neuronal protein function has general applicability in the fast expanding field of optopharmacology.

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

Mammalian neurotransmitter-gated receptors can be conjugated to photoswitchable tethered ligands (PTLs) to enable photoactivation, or photoantagonism, while preserving normal function at neuronal synapses. "MAG" PTLs for ionotropic and metabotropic glutamate receptors (GluRs) are based on an azobenzene photoswitch that is optimally switched into the liganding state by blue or near-UV light, wavelengths that penetrate poorly into the brain. To facilitate deep-tissue photoactivation with near-infrared light, we measured the efficacy of two-photon (2P) excitation for two MAG molecules using nonlinear spectroscopy. Based on quantitative characterization, we find a recently designed second generation PTL, L-MAG0460, to have a favorable 2P absorbance peak at 850 nm, enabling efficient 2P activation of the GluK2 kainate receptor, LiGluR. We also achieve 2P photoactivation of a metabotropic receptor, LimGluR3, with a new mGluR-specific PTL, D-MAG0460. 2P photoswitching is efficiently achieved using digital holography to shape illumination over single somata of cultured neurons. Simultaneous Ca(2+)-imaging reports on 2P photoswitching in multiple cells with high temporal resolution. The combination of electrophysiology or Ca(2+) imaging with 2P activation by optical wavefront shaping should make second generation PTL-controlled receptors suitable for studies of intact neural circuits.

Long wavelength optical control of glutamate receptor ion channels using a tetra-ortho-substituted azobenzene derivative.

A tetra-ortho-chloro substituted azobenzene unit was incorporated into a photoswitchable tethered ligand for ionotropic glutamate receptors. This compound confers the modified protein with the unusual optical responses of the substituted azo scaffold permitting channel opening with yellow and red light and channel closing with blue light.

Allosteric control of an ionotropic glutamate receptor with an optical switch.

The precise regulation of protein activity is fundamental to life. The allosteric control of an active site by a remote regulatory binding site is a mechanism of regulation found across protein classes, from enzymes to motors to signaling proteins. We describe a general approach for manipulating allosteric control using synthetic optical switches. Our strategy is exemplified by a ligand-gated ion channel of central importance in neuroscience, the ionotropic glutamate receptor (iGluR). Using structure-based design, we have modified its ubiquitous clamshell-type ligand-binding domain to develop a light-activated channel, which we call LiGluR. An agonist is covalently tethered to the protein through an azobenzene moiety, which functions as the optical switch. The agonist is reversibly presented to the binding site upon photoisomerization, initiating clamshell domain closure and concomitant channel gating. Photoswitching occurs on a millisecond timescale, with channel conductances that reflect the photostationary state of the azobenzene at a given wavelength. Our device has potential uses not only in biology but also in bioelectronics and nanotechnology.

The network-selective actions of quinoxalines on the neurocircuitry operations of the rabbit retina.

We examined the contribution of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxalole-4-propionic acid (AMPA)/kainate (KA) receptors to the light-responses of rabbit retinal neurons. In the outer retina, bath application of the AMPA/KA receptor antagonists 6,7-dinitro-quinoxaline-2,3-dione (DNQX) and 2,3,dihydroxy-6-nitro-7-sulfamoyl-benzo-f-quinoxaline (NBQX) blocked the light-responses of horizontal cells. Application of quinoxalines enhanced ON-bipolar cell light-responses, and was associated with a hyperpolarization of their resting potentials. In the inner retina, application of both AMPA/KA and NMDA antagonists to AII amacrine-like cells only partially blocked their light-responses. Their residual responses may reflect electrical coupling to neighboring ON-center cone bipolar cells, and can inhibit OFF-center ganglion cells. ON-sustained ganglion cells were highly sensitive to the quinoxalines, which reduced their light-evoked firing, while the firing of ON-transient cells remained as NMDA-mediated light-responses. Quinoxalines had differential effects on the firing rates of ON- and OFF-center ganglion cells: ON-cells were reduced, while OFF-cells were increased. In contrast, firing rates of ON-OFF ganglion cells were not excited by NBQX, and showed a recovered light-response mediated by NMDA receptors. The receptive field surround was lost in ganglion cells. For comparison, NMDA antagonists had only moderate effects on all ganglion cell light-responses. Our results indicate that NMDA and AMPA/KA receptors both contribute to ganglion cell light-responses. However, AMPA/KA receptors also significantly effect the light-response of neurons presynaptic to retinal ganglion cells, altering the observed pharmacology at the ganglion cell level.