Glutamate serves as both the mammalian brain's primary excitatory neurotransmitter and as a key neuromodulator to control synapse and circuit function over a wide range of spatial and temporal scales. This functional diversity is decoded by two receptor families: ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). The challenges posed by the complexity and physiological importance of each of these subtypes has limited our appreciation and understanding of how these receptors work in concert. In this review, by comparing both receptor families with a focus on their crosstalk, we argue for a more holistic understanding of neural glutamate signaling.

G-protein coupled receptors (GPCRs), the largest family of membrane signaling proteins, respond to neurotransmitters, hormones and small environmental molecules. The neuronal function of many GPCRs has been difficult to resolve because of an inability to gate them with subtype-specificity, spatial precision, speed and reversibility. To address this, we developed an approach for opto-chemical engineering native GPCRs. We applied this to the metabotropic glutamate receptors (mGluRs) to generate light-agonized and light-antagonized “LimGluRs”. The light-agonized “LimGluR2”, on which we focused, is fast, bistable, and supports multiple rounds of on/off switching. Light gates two of the primary neuronal functions of mGluR2: suppression of excitability and inhibition of neurotransmitter release. The light-antagonized “LimGluR2block” can be used to manipulate negative feedback of synaptically released glutamate on transmitter release. We generalize the optical control to two additional family members: mGluR3 and 6. The system works in rodent brain slice and in zebrafish in vivo, where we find that mGluR2 modulates the threshold for escape behavior. These light-gated mGluRs pave the way for determining the roles of mGluRs in synaptic plasticity, memory and disease.

A reversible structural unlocking reaction, in which the closepacked van der Waals interactions break cooperatively, has been found for the villin headpiece subdomain (HP35) using triplet-triplet-energy transfer to monitor conformational fluctuations from equilibrium. Unlocking is associated with an unfavorable enthalpy change (ΔH 0 ¼ 35 AE 4 kJ∕mol) which is nearly compensated in free energy by the entropy change (ΔS 0 ¼ 112 AE 20 J · mol −1 · K −1 ). The unlocking reaction has a time constant of about 1 μs at 5°C and is enthalpy-limited with an activation energy of 32 AE 1 kJ∕mol and a large Arrhenius preexponential factor of A ¼ 7.5 × 10 11 s −1 . In the unlocked state a fast local conformational fluctuation with a time constant of 170 ns and a low activation barrier of 17 AE 1 kJ∕mol leads to unfolding of the Cterminal helix and to its undocking from the core. On a much slower time scale, global unfolding occurs from the unlocked state. These results suggest that native protein structures are locked into conformations with low amplitude motions. Large scale motions and global unfolding require an initial structural unlocking step leading to a state with properties of a dry molten globule. The experiments additionally yielded information on the dynamics of loop formation between different positions in unfolded HP35. Comparison of the results with dynamics in unstructured model peptides indicates slightly decelerated kinetics of local loop formation in the Cterminal region which points at residual, nonrandom structure. Dynamics of long-range loop formation, in contrast, are not influenced by residual structure, which argues against unfolded state properties as molecular origin for ultrafast folding of HP35.dry molten globule | native-state fluctuations | protein dynamics | protein folding | unfolded state dynamics N ative states of proteins are known to be close-packed but almost nothing is known about at which stage of the folding process packing occurs. Shaknovich and Finkelstein proposed in 1989 (1) that heat-induced protein unfolding begins with a slight expansion of the protein, such that at least some of the close-packing interactions are broken but water cannot yet penetrate into the interior. This dry molten globule state was postulated to be located on the unfolded side of the major folding barrier but it was argued that it should not be experimentally detectable for water soluble proteins (1). Later, GdmCl-induced unfolding experiments on ribonuclease A (2) and dihydrofolate reductase (DHFR) (3) monitored by time resolved NMR spectroscopy observed rapid formation of an intermediate located on the native side of the major unfolding barrier with properties resembling those of the predicted dry molten globule. The observation of an intact hydrogen-bonding network in the unfolding intermediate of ribonuclease A supported the dry nature of this state (4). Recently, FRET-detected unfolding kinetics of monellin showed rapid expansion of the native state prior to global unfolding (5), which is also in line w...

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.

Astrocytes have long been regarded as essentially unexcitable cells that do not contribute to active signaling and information processing in the brain. Contrary to this classical view, it is now firmly established that astrocytes can specifically respond to glutamate released from neurons. Astrocyte glutamate signaling is initiated upon binding of glutamate to ionotropic and/or metabotropic receptors, which can result in calcium signaling, a major form of glial excitability. Release of so-called gliotransmitters like glutamate, ATP and D-serine from astrocytes in response to activation of glutamate receptors has been demonstrated to modulate various aspects of neuronal function in the hippocampus. In addition to receptors, glutamate binds to high-affinity, sodium-dependent transporters, which results in rapid buffering of synaptically-released glutamate, followed by its removal from the synaptic cleft through uptake into astrocytes. The degree to which astrocytes modulate and control extracellular glutamate levels through glutamate transporters depends on their expression levels and on the ionic driving forces that decrease with ongoing activity. Another major determinant of astrocytic control of glutamate levels could be the precise morphological arrangement of fine perisynaptic processes close to synapses, defining the diffusional distance for glutamate, and the spatial proximity of transporters in relation to the synaptic cleft. In this review, we will present an overview of the mechanisms and physiological role of glutamate-induced ion signaling in astrocytes in the hippocampus as mediated by receptors and transporters. Moreover, we will discuss the relevance of astroglial glutamate uptake for extracellular glutamate homeostasis, focusing on how activity-induced dynamic changes of perisynaptic processes could shape synaptic transmission at glutamatergic synapses.

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 (MAG0 460 ). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform lightevoked firing when LiGluR-MAG0 460 was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0 460 was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0 460 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 rodcone dystrophy dog model of blindness, LiGluR-MAG0 460 in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0 460 was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation.retinal gene therapy | visual prosthetics | retinitis pigmentosa | optogenetic pharmacology | azobenzene photoswitches

Coupling fast triplet-triplet energy transfer (TTET) between xanthone and naphthylalanine to the helix-coil equilibrium in alanine-based peptides allowed the observation of local equilibrium fluctuations in ␣-helices on the nanoseconds to microseconds time scale. The experiments revealed faster helix unfolding in the terminal regions compared with the central parts of the helix with time constants varying from 250 ns to 1.4 s at 5°C. Local helix formation occurs with a time constant of Ϸ400 ns, independent of the position in the helix. Comparing the experimental data with simulations using a kinetic Ising model showed that the experimentally observed dynamics can be explained by a 1-dimensional boundary diffusion with positionindependent elementary time constants of Ϸ50 ns for the addition and of Ϸ65 ns for the removal of an ␣-helical segment. The elementary time constant for helix growth agrees well with previously measured time constants for formation of short loops in unfolded polypeptide chains, suggesting that helix elongation is mainly limited by a conformational search.␣-helix-coil transition ͉ protein dynamics ͉ protein folding ͉ triplet-triplet energy transfer

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.