Michael A. Kienzler

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

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.

Tuning Photochromic Ion Channel Blockers

Photochromic channel blockers provide a conceptually simple and convenient way to modulate neuronal activity with light. We have recently described a family of azobenzenes that function as tonic blockers of K(v) channels but require UV-A light to unblock and need to be actively switched by toggling between two different wavelengths. We now introduce red-shifted compounds that fully operate in the visible region of the spectrum and quickly turn themselves off in the dark. Furthermore, we have developed a version that does not block effectively in the dark-adapted state, can be switched to a blocking state with blue light, and reverts to the inactive state automatically. Photochromic blockers of this type could be useful for the photopharmacological control of neuronal activity under mild conditions.

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

Significance We restored visual function to animal models of human blindness using a chemical compound that photosensitizes a mammalian ion channel. Virus-mediated expression of this light sensor in surviving retinal cells of blind mice restored light responses in vitro, reanimated innate light avoidance, and enabled learned visually guided behavior. The treatment also restored light responses to the retina of blind dogs. Patients that might benefit from this treatment would need to have intact ganglion cell and nerve fiber layers. In general, these are patients diagnosed with retinitis pigmentosa and some forms of Leber congenital amaurosis. Patients diagnosed with other types of blindness, for example, age-related macular degeneration or diabetic retinopathy, would not be candidates for this treatment. Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0460). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0460 was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0460 was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0460 in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0460 in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0460 was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation.

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

Significance MAGs (maleimide-azobenzene-glutamate) are photoswitches that covalently bind to genetically engineered glutamate receptors (GluRs) and, under the control of light, mimic or block the action of the excitatory neurotransmitter glutamate. However the blue and near-UV light that optimally photoswitch MAGs do not penetrate well into the brain. In this paper, we show how MAGs can instead be photoswitched by two-photon (2P) absorption of near-infrared light, which penetrates deeper into tissue. We demonstrate 2P control of MAG-dependent ionic currents in neurons, and synthesize a new MAG photoswitch to enable 2P activation of a G protein coupled receptor signaling cascade through a metabotropic GluR. These optogenetic tools bring exceptional spatiotemporal resolution and pharmacological specificity to the study of synaptic transmission and plasticity in intact neural circuits. 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 Ca2+-imaging reports on 2P photoswitching in multiple cells with high temporal resolution. The combination of electrophysiology or Ca2+ imaging with 2P activation by optical wavefront shaping should make second generation PTL-controlled receptors suitable for studies of intact neural circuits.

Vinyl Quinones as Diels−Alder Dienes: Concise Synthesis of (−)-Halenaquinone

A concise asymmetric synthesis of (-)-halenaquinone is described. The synthesis features a diastereoselective Heck cyclization to set a quaternary center as well as a novel intramolecular inverse-electron-demand Diels-Alder reaction involving a vinyl quinone. The synthesis is highly convergent and features a minimal amount of protecting group manipulations.

Exploring the Pharmacology and Action Spectra of Photochromic Open- Channel Blockers

Ions enter or leavea voltage-gated ion channel through selectivity filter on theextracellular side, which is connected to a water-filled innercavity. The path from this inner cavity to the intracellularmilieu is controlled by a voltage gate, which opens and closesin response to changes in the membrane potential. Local anes-thetics such as lidocaine or procaine bind in the inner cavitybelow the selectivity filter, preventing the permeation of cat-ions along their electrochemical gradients.

Phospholipase D2 specifically regulates TREK potassium channels via direct interaction and local production of phosphatidic acid

Significance Our work provides evidence for a mechanism for the formation of membrane microdomains in which the local concentration of a phospholipid can change independently of the bulk membrane to confer selectivity on membrane protein regulation. We found that, despite the fact that all TWIK-related K channel (TREK) family members are sensitive to phosphatidic acid (PA), only TREK1 and TREK2 are potentiated by phospholipase D2 (PLD2) (which produces PA), but not by PLD1. This surprising specificity is due to the direct binding of PLD2 to TREK. This binding allows a local PA production that tonically activates the channel. Furthermore, we found the local signaling via PA to have a secondary focusing effect for primary alcohols, which inhibit the channel by altering the PA microdomain. Membrane lipids serve as second messengers and docking sites for proteins and play central roles in cell signaling. A major question about lipid signaling is whether diffusible lipids can selectively target specific proteins. One family of lipid-regulated membrane proteins is the TWIK-related K channel (TREK) subfamily of K2P channels: TREK1, TREK2, and TWIK-related arachdonic acid stimulated K+ channel (TRAAK). We investigated the regulation of TREK channels by phosphatidic acid (PA), which is generated by phospholipase D (PLD) via hydrolysis of phosphatidylcholine. Even though all three of the channels are sensitive to PA, we found that only TREK1 and TREK2 are potentiated by PLD2 and that none of these channels is modulated by PLD1, indicating surprising selectivity. We found that PLD2, but not PLD1, directly binds to the C terminus of TREK1 and TREK2, but not to TRAAK. The results have led to a model for selective lipid regulation by localization of phospholipid enzymes to specific effector proteins. Finally, we show that regulation of TREK channels by PLD2 occurs natively in hippocampal neurons.

Precise modulation of neuronal activity with synthetic photoswitchable ligands

To tackle some of the most fundamental questions in neurobiology at the molecular level, classical genetics and pharmacology have been effective tools to perturb the activity of neuronal receptors and ion channels. However, conventional pharmacology has several drawbacks that are especially problematic when studying neural circuits and synapses. First, standard diffusion and partitioning of the ligand mean poor spatial and temporal control of ligand activity. Second, even in cases of high specificity for ligand action, it is relatively common for the receptor or channel of interest to be expressed on multiple nearby cell types, or even on both sides of a synapse, making it difficult to isolate the effects of the target receptor within the circuit or synapse of interest. Light-based techniques that operate at the intersection of chemistry, biology, and neuroscience have been developed to overcome these challenges. We focus here on systems that contain a synthetic photoswitch, a small molecule that absorbs light to reversibly change its shape. The most commonly used photoswitch in biological applications is azobenzene due to its synthetic tractability, tunable photochemical properties, and biological compatibility (Fig. 1a). The lowest energy isomer, the straight trans-azobenzene, isomerizes to the bent cis-azobenzene configuration upon irradiation with near-UV light. Subsequent irradiation with longer wavelength visible light, or thermal relaxation, leads the metastable cis-azobenzene to revert to the trans-azobenzene isomer. Open in a separate window Figure 1 Strategies for incorporating synthetic photoswitches into neuroscience tools. A) The trans and cis isomers of azobenzene can be interconverted with different wavelengths of light. Cartoons show how the core azobenzene structure can be elaborated into photoswitchable tools. B) Photochromic ligands (PCLs) switch between active and inactive compounds that freely diffuse and function with endogenous channels and receptors. C) Photoswitchable Tethered Ligands (PTLs) covalently attach to an engineered protein to provide photocontrol. D) Photoswitchable orthogonal remotely tethered ligands (PORTLs) are conceptually a cross between PCLs and PTLs, they use a self-labeling protein fused to the target protein and a photoswitch ligand that has long linker to accommodate attachment farther from the ligand binding site.