[Coordination chemogenetics for regulation of glutamate receptors in neuron].

Transmembrane receptors transmit extracellular information into cells. In many cases, protein families are composed of highly homologous subtypes, each of which has unique cellular functions. Therefore, it is highly desired for understanding the physiological roles of the receptor in tissues or animals. However, it is difficult to control the activity of receptors in a cell-type- and subtype-specific manner with high temporal resolution using traditional pharmacological or genetic engineering methods. Recently, chemogenetics has been focused on controlling the cellular signaling in a cell-type-specific manner, which allows for elucidating the function of specific cell types with high temporal resolution. However, conventional chemogenetics are not suitable for understanding the roles of each receptor. Therefore, we have developed a chemogenetic method, termed coordination chemogenetics, in which coordination chemistry and genetic engineering are combined. The coordination chemogenetics enabled artificial activation of ionotropic glutamate receptor (GluA2) and metabotropic glutamate receptor (mGlu1). A palladium (Pd) complex successfully activated mGlu1 in mGlu1(N264H) knock-in mice, demonstrating that endogenous mGlu1 activation is sufficient to evoke a key cellular mechanism of synaptic plasticity that underlies motor learning in the cerebellum. We also expanded the coordination chemogenetics for orthogonal activation of mGlu1 activity using Cu2+, Zn2+, and Pd complexes for analyzing the individual roles of mGlu1 simultaneously. Notably, coordination chemogenetics can be expanded to apply selective inhibition of transmembrane receptors, and the dissociation is much slower than that of conventional inhibitors. Thus, coordination chemogenetics would be a unique method for controlling mGlu1 in a cell-type-specific manner.

Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue.

Direct activation of cell-surface receptors is highly desirable for elucidating their physiological roles. A potential approach for cell-type-specific activation of a receptor subtype is chemogenetics, in which both point mutagenesis of the receptors and designed ligands are used. However, ligand-binding properties are affected in most cases. Here, we developed a chemogenetic method for direct activation of metabotropic glutamate receptor 1 (mGlu1), which plays essential roles in cerebellar functions in the brain. Our screening identified a mGlu1 mutant, mGlu1(N264H), that was activated directly by palladium complexes. A palladium complex showing low cytotoxicity successfully activated mGlu1 in mGlu1(N264H) knock-in mice, revealing that activation of endogenous mGlu1 is sufficient to evoke the critical cellular mechanism of synaptic plasticity, a basis of motor learning in the cerebellum. Moreover, cell-type-specific activation of mGlu1 was demonstrated successfully using adeno-associated viruses in mice, which shows the potential utility of this chemogenetics for clarifying the physiological roles of mGlu1 in a cell-type-specific manner.

[Quantification of AMPA-type glutamate receptors trafficking by ligand-directed two-step labeling].

Glutamate receptors mediate excitatory neurotransmission in the central nervous system, which have essential roles in our learning and memory. Recent studies have revealed that the trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate receptors (AMPA receptors) are dynamically regulated during synaptic plasticity, the cellular basis of learning and memory. Conventionally, biochemical methods such as surface-biotin labeling or genetic incorporation of fluorescent proteins have been utilized to analyze the AMPA receptors dynamics. However, conflicting findings have been reported because of serious issues in these conventional methods. As the alternative, we have developed a new method for labeling AMPA receptors endogenously expressed in neurons by chemical approaches. This is based on a covalent chemical labeling strategy driven by selective ligand-protein recognition to tether small fluorophores to the target receptors, termed ligand-directed acyl imidazole chemistry. This method has successfully visualized AMPA receptors endogenously expressed in neurons. However, the original method required several hours for fluorophore labeling, which hampered analyzing the dynamics of AMPA receptors in detail. As the alternative, we have recently developed an improved strategy for rapid and selective labeling of chemical probes to cell-surface AMPA receptors by combining ligand-directed chemistry and bio-orthogonal click chemistry. This method allowed to quantify their trafficking, which revealed unique features of AMPA receptors such as long lifetime and rapid recycling in neurons. Notably, this method can be expanded to other receptors. Thus, the two-step labeling method would be a useful tool for understanding the physiological or pathophysiological roles of glutamate receptors in neurons.

Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking.

The regulation of glutamate receptor localization is critical for development and synaptic plasticity in the central nervous system. Conventional biochemical and molecular biological approaches have been widely used to analyze glutamate receptor trafficking, especially for α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-type glutamate receptors (AMPARs). However, conflicting findings have been reported because of a lack of useful tools for analyzing endogenous AMPARs. Here, we develop a method for the rapid and selective labeling of AMPARs with chemical probes, by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological temperature in culture medium. This method allows us to quantify AMPAR distribution and trafficking, which reveals some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method is also successfully expanded to selectively label N-methyl-D-aspartate-type glutamate receptors. Thus, bioorthogonal two-step labeling may be a versatile tool for investigating the physiological and pathophysiological roles of glutamate receptors in neurons.

Orthogonal Activation of Metabotropic Glutamate Receptor Using Coordination Chemogenetics.

Cell-surface receptors play a pivotal role as transducers of extracellular input. Although different cell types express the same receptor, the physiological roles of the receptor are highly dependent on cell type. To understand each role, tactics for cell-specific activation of the target receptor are in high demand. Herein, we developed an orthogonal activation method targeting metabotropic glutamate receptor 1 (mGlu1), a G-protein coupled receptor. In this method, direct activation via coordination-based chemogenetics (dA-CBC) was adopted, where activation of mGlu1 was artificially induced by a protein conformational change in response to the coordination of a metal ion or metal-ion complex. Our structure-based protein design and screening approach identified mGlu1 mutants that were directly activated by the coordination of Cu2+ or Zn2+, in addition to our previous Pd-complex-sensitive mGlu1 mutant. Notably, the activation of the mutants was mutually orthogonal, resulting in cell-type selective activation in a model system using HEK293 cells.

Chemogenetic Approach Using Ni(II) Complex-Agonist Conjugates Allows Selective Activation of Class A G-Protein-Coupled Receptors.

Investigating individual G-protein-coupled receptors (GPCRs) involved in various signaling cascades can unlock a myriad of invaluable physiological findings. One of the promising strategies for addressing the activity of each subtype of receptor is to design chemical turn-on switches on the target receptors. However, valid methods to selectively control class A GPCRs, the largest receptor family encoded in the human genome, remain limited. Here, we describe a novel approach to chemogenetically manipulate activity of engineered class A GPCRs carrying a His4 tag, using metal complex-agonist conjugates (MACs). This manipulation is termed coordination tethering. With the assistance of coordination bonds, MACs showed 10-100-fold lower EC50 values in the engineered receptors, compared with wild-type receptors. Such coordination tethering enabled selective activation of ß2-adrenoceptors and muscarinic acetylcholine receptors, without loss of natural receptor responses, in living mammalian cells, including primary cultured astrocytes. Our generalized, modular chemogenetic approach should facilitate more precise control and deeper understanding of individual GPCR signaling pathways in living systems.