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Science (New York, N.Y.) Mar 2023Glycine is a major neurotransmitter involved in several fundamental neuronal processes. The identity of the metabotropic receptor mediating slow neuromodulatory effects...
Glycine is a major neurotransmitter involved in several fundamental neuronal processes. The identity of the metabotropic receptor mediating slow neuromodulatory effects of glycine is unknown. We identified an orphan G protein-coupled receptor, GPR158, as a metabotropic glycine receptor (mGlyR). Glycine and a related modulator, taurine, directly bind to a Cache domain of GPR158, and this event inhibits the activity of the intracellular signaling complex regulator of G protein signaling 7-G protein β5 (RGS7-Gβ5), which is associated with the receptor. Glycine signals through mGlyR to inhibit production of the second messenger adenosine 3',5'-monophosphate. We further show that glycine, but not taurine, acts through mGlyR to regulate neuronal excitability in cortical neurons. These results identify a major neuromodulatory system involved in mediating metabotropic effects of glycine, with implications for understanding cognition and affective states.
Topics: Glycine; GTP-Binding Proteins; Receptors, G-Protein-Coupled; Receptors, Glycine; Signal Transduction; Humans; HEK293 Cells; GTP-Binding Protein beta Subunits; RGS Proteins; Protein Domains
PubMed: 36996198
DOI: 10.1126/science.add7150 -
Cell Feb 2021Ligand-gated ion channels mediate signal transduction at chemical synapses and transition between resting, open, and desensitized states in response to neurotransmitter...
Ligand-gated ion channels mediate signal transduction at chemical synapses and transition between resting, open, and desensitized states in response to neurotransmitter binding. Neurotransmitters that produce maximum open channel probabilities (Po) are full agonists, whereas those that yield lower than maximum Po are partial agonists. Cys-loop receptors are an important class of neurotransmitter receptors, yet a structure-based understanding of the mechanism of partial agonist action has proven elusive. Here, we study the glycine receptor with the full agonist glycine and the partial agonists taurine and γ-amino butyric acid (GABA). We use electrophysiology to show how partial agonists populate agonist-bound, closed channel states and cryo-EM reconstructions to illuminate the structures of intermediate, pre-open states, providing insights into previously unseen conformational states along the receptor reaction pathway. We further correlate agonist-induced conformational changes to Po across members of the receptor family, providing a hypothetical mechanism for partial and full agonist action at Cys-loop receptors.
Topics: Animals; Binding Sites; Cell Line; Cryoelectron Microscopy; Glycine; HEK293 Cells; Humans; Imaging, Three-Dimensional; Ion Channel Gating; Maleates; Models, Molecular; Mutant Proteins; Mutation; Neurotransmitter Agents; Protein Domains; Receptors, Glycine; Styrene; Zebrafish; gamma-Aminobutyric Acid
PubMed: 33567265
DOI: 10.1016/j.cell.2021.01.026 -
Nature Communications Jul 2020Glycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyRs) are key players in mediating fast...
Glycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyRs) are key players in mediating fast inhibitory neurotransmission at these synapses. While previous high-resolution structures have provided insights into the molecular architecture of GlyR, several mechanistic questions pertaining to channel function are still unanswered. Here, we present Cryo-EM structures of the full-length GlyR protein complex reconstituted into lipid nanodiscs that are captured in the unliganded (closed), glycine-bound (open and desensitized), and allosteric modulator-bound conformations. A comparison of these states reveals global conformational changes underlying GlyR channel gating and modulation. The functional state assignments were validated by molecular dynamics simulations, and the observed permeation events are in agreement with the anion selectivity and conductance of GlyR. These studies provide the structural basis for gating, ion selectivity, and single-channel conductance properties of GlyR in a lipid environment.
Topics: Allosteric Regulation; Animals; Binding Sites; Glycine; Ion Channel Gating; Lipids; Molecular Dynamics Simulation; Nanoparticles; Neurotransmitter Agents; Protein Conformation; Receptors, Glycine; Xenopus; Zebrafish Proteins
PubMed: 32719334
DOI: 10.1038/s41467-020-17364-5 -
Nature Communications Mar 2023Glycine Receptors (GlyRs) provide inhibitory neuronal input in the spinal cord and brainstem, which is critical for muscle coordination and sensory perception. Synaptic...
Glycine Receptors (GlyRs) provide inhibitory neuronal input in the spinal cord and brainstem, which is critical for muscle coordination and sensory perception. Synaptic GlyRs are a heteromeric assembly of α and β subunits. Here we present cryo-EM structures of full-length zebrafish α1βGlyR in the presence of an antagonist (strychnine), agonist (glycine), or agonist with a positive allosteric modulator (glycine/ivermectin). Each structure shows a distinct pore conformation with varying degrees of asymmetry. Molecular dynamic simulations found the structures were in a closed (strychnine) and desensitized states (glycine and glycine/ivermectin). Ivermectin binds at all five interfaces, but in a distinct binding pose at the β-α interface. Subunit-specific features were sufficient to solve structures without a fiduciary marker and to confirm the 4α:1β stoichiometry recently observed. We also report features of the extracellular and intracellular domains. Together, our results show distinct compositional and conformational properties of αβGlyR and provide a framework for further study of this physiologically important channel.
Topics: Animals; Receptors, Glycine; Strychnine; Zebrafish; Ivermectin; Glycine
PubMed: 36914669
DOI: 10.1038/s41467-023-37106-7 -
ELife Aug 2022To clarify the determinants of agonist efficacy in pentameric ligand-gated ion channels, we examined a new compound, aminomethanesulfonic acid (AMS), a molecule...
To clarify the determinants of agonist efficacy in pentameric ligand-gated ion channels, we examined a new compound, aminomethanesulfonic acid (AMS), a molecule intermediate in structure between glycine and taurine. Despite wide availability, to date there are no reports of AMS action on glycine receptors, perhaps because AMS is unstable at physiological pH. Here, we show that at pH 5, AMS is an efficacious agonist, eliciting in zebrafish α1 glycine receptors a maximum single-channel open probability of 0.85, much greater than that of β-alanine (0.54) or taurine (0.12), and second only to that of glycine itself (0.96). Thermodynamic cycle analysis of the efficacy of these closely related agonists shows supra-additive interaction between changes in the length of the agonist molecule and the size of the anionic moiety. Single particle cryo-electron microscopy structures of AMS-bound glycine receptors show that the AMS-bound agonist pocket is as compact as with glycine, and three-dimensional classification demonstrates that the channel populates the open and the desensitized states, like glycine, but not the closed intermediate state associated with the weaker partial agonists, β-alanine and taurine. Because AMS is on the cusp between full and partial agonists, it provides a new tool to help us understand agonist action in the pentameric superfamily of ligand-gated ion channels.
Topics: Animals; Cryoelectron Microscopy; Glycine; Receptors, Glycine; Sulfonic Acids; Taurine; Zebrafish; beta-Alanine
PubMed: 35975975
DOI: 10.7554/eLife.79148 -
Cells Apr 2022Mechanical allodynia (pain to normally innocuous tactile stimuli) is a widespread symptom of inflammatory and neuropathic pain. Spinal or medullary dorsal horn (SDH or...
Mechanical allodynia (pain to normally innocuous tactile stimuli) is a widespread symptom of inflammatory and neuropathic pain. Spinal or medullary dorsal horn (SDH or MDH) circuits mediating tactile sensation and pain need to interact in order to evoke mechanical allodynia. PKCγ-expressing (PKCγ) interneurons and inhibitory controls within SDH/MDH inner lamina II (II) are pivotal in connecting touch and pain circuits. However, the relative contribution of GABA and glycine to PKCγ interneuron inhibition remains unknown. We characterized inhibitory inputs onto PKCγ interneurons by combining electrophysiology to record spontaneous and miniature IPSCs (sIPSCs, mIPSCs) and immunohistochemical detection of GABARα2 and GlyRα1 subunits in adult rat MDH. While GlyR-only- and GABAR-only-mediated mIPSCs/sIPSCs are predominantly recorded from PKCγ interneurons, immunohistochemistry reveals that ~80% of their inhibitory synapses possess both GABARα2 and GlyRα1. Moreover, nearly all inhibitory boutons at gephyrin-expressing synapses on these cells contain glutamate decarboxylase and are therefore GABAergic, with around half possessing the neuronal glycine transporter (GlyT2) and therefore being glycinergic. Thus, while GABA and glycine are presumably co-released and GABARs and GlyRs are present at most inhibitory synapses on PKCγ interneurons, these interneurons exhibit almost exclusively GABAR-only and GlyR-only quantal postsynaptic inhibitory currents, suggesting a pharmacological specialization of their inhibitory synapses.
Topics: Animals; Glycine; Hyperalgesia; Interneurons; Pain; Rats; Receptors, Glycine; Substantia Gelatinosa; Synaptic Transmission; gamma-Aminobutyric Acid
PubMed: 35456035
DOI: 10.3390/cells11081356 -
BMC Neuroscience Jun 2023The glycinergic system plays an important inhibitory role in the mouse central nervous system, where glycine controls the excitability of spinal itch- and pain-mediating...
BACKGROUND
The glycinergic system plays an important inhibitory role in the mouse central nervous system, where glycine controls the excitability of spinal itch- and pain-mediating neurons. Impairments of the glycine receptors can cause motor and sensory deficits. Glycine exerts inhibition through interaction with ligand-gated ion channels composed of alpha and beta subunits. We have investigated the mRNA expression of the glycine receptor alpha 3 (Glra3) subunit in the nervous system as well as in several peripheral organs of female and male mice.
RESULTS
Single-cell RNA sequencing (scRNA-seq) data analysis on the Zeisel et al. (2018) dataset indicated widespread but low expression of Glra3 in vesicular glutamate transporter 2 (Vglut2, Slc17a6) positive and vesicular inhibitory amino acid transporter (Viaat, Slc32a1)positive neurons of the mouse central nervous system. Highest occurrence of Glra3 expression was identified in the cortex, amygdala, and striatal regions, as well as in the hypothalamus, brainstem and spinal cord. Bulk quantitative real-time-PCR (qRT-PCR) analysis demonstrated Glra3 expression in cortex, amygdala, striatum, hypothalamus, thalamus, pituitary gland, hippocampus, cerebellum, brainstem, and spinal cord. Additionally, male mice expressed higher levels of Glra3 in all investigated brain areas compared with female mice. Lastly, RNAscope spatially validated Glra3 expression in the areas indicated by the single-cell and bulk analyses. Moreover, RNAscope analysis confirmed co-localization of Glra3 with Slc17a6 or Slc32a1 in the central nervous system areas suggested from the single-cell data.
CONCLUSIONS
Glra3 expression is low but widespread in the mouse central nervous system. Clear sex-dependent differences have been identified, indicating higher levels of Glra3 in several telencephalic and diencephalic areas, as well as in cerebellum and brainstem, in male mice compared with female mice.
Topics: Mice; Male; Female; Animals; Receptors, Glycine; Glycine; Neurons; Brain; Spinal Cord; RNA, Messenger
PubMed: 37264306
DOI: 10.1186/s12868-023-00800-9 -
The Journal of Pharmacology and... Apr 2017The glycine receptor is a pentameric ligand-gated ion channel that is involved in fast inhibitory neurotransmission in the central nervous system. Zinc is an allosteric...
The glycine receptor is a pentameric ligand-gated ion channel that is involved in fast inhibitory neurotransmission in the central nervous system. Zinc is an allosteric modulator of glycine receptor function, enhancing the effects of glycine at nanomolar to low-micromolar concentrations and inhibiting its effects at higher concentrations. Low-nanomolar concentrations of contaminating zinc in electrophysiological buffers are capable of synergistically enhancing receptor modulation by other compounds, such as ethanol. This suggests that, unless accounted for, previous studies of glycine receptor modulation were measuring the effects of modulator plus comodulation by zinc on receptor function. Since zinc is present in vivo at a variety of concentrations, it will influence glycine receptor modulation by other pharmacologic agents. We investigated the utility of previously described "zinc-enhancement-insensitive" 1 glycine receptor mutants D80A, D80G, and W170S to probe for interactions between zinc and other allosteric modulators at the glycine receptor. We found that only the W170S mutation conferred complete abolishment of zinc enhancement across a variety of agonist and zinc concentrations. Using 1 W170S receptors, we established that, in addition to ethanol, zinc interacts with inhalants, but not volatile anesthetics, to synergistically enhance channel function. Additionally, we determined that this interaction is abolished at higher zinc concentrations when receptor-enhancing binding sites are saturated, suggesting a mechanism by which modulators such as ethanol and inhalants are capable of increasing receptor affinity for zinc, in addition to enhancing channel function on their own.
Topics: Allosteric Regulation; Animals; Dose-Response Relationship, Drug; Ethanol; Female; Glycine; Protein Binding; Receptors, Glycine; Xenopus laevis; Zinc
PubMed: 28087784
DOI: 10.1124/jpet.116.239152 -
Molecular Medicine (Cambridge, Mass.) Apr 2023Pain, although unpleasant, is an essential warning mechanism against injury and damage of the organism. An intricate network of specialised sensors and transmission... (Review)
Review
Pain, although unpleasant, is an essential warning mechanism against injury and damage of the organism. An intricate network of specialised sensors and transmission systems contributes to reception, transmission and central sensitization of pain. Here, we briefly introduce some of the main aspects of pain signal transmission, including nociceptors and nociceptive signals, mechanisms of inflammatory and neuropathic pain, and the situation of diabetes-associated neuropathic pain. The role of glia-astrocytes, microglia, satellite glia cells-and their specific channels, transporters and signaling pathways is described. A focus is on the contribution of inhibitory synaptic signaling to nociception and a possible role of glycine receptors in glucose-mediated analgesia and treatment-induced diabetic neuropathy. Inhibitory receptors such as GABA- and glycine receptors are important contributors to nociceptive signaling; their contribution to altered pain sensation in diabetes may be of clinical relevance, and they could be promising therapeutic targets towards the development of novel analgesics.
Topics: Humans; Receptors, Glycine; Diabetic Neuropathies; Neuralgia; Signal Transduction; Neuroglia; Diabetes Mellitus
PubMed: 37069517
DOI: 10.1186/s10020-023-00647-0 -
Proceedings of the National Academy of... Aug 2017Neuronal inhibition can occur via synaptic mechanisms or through tonic activation of extrasynaptic receptors. In spinal cord, glycine mediates synaptic inhibition...
Neuronal inhibition can occur via synaptic mechanisms or through tonic activation of extrasynaptic receptors. In spinal cord, glycine mediates synaptic inhibition through the activation of heteromeric glycine receptors (GlyRs) composed primarily of α1 and β subunits. Inhibitory GlyRs are also found throughout the brain, where GlyR α2 and α3 subunit expression exceeds that of α1, particularly in forebrain structures, and coassembly of these α subunits with the β subunit appears to occur to a lesser extent than in spinal cord. Here, we analyzed GlyR currents in several regions of the adolescent mouse forebrain (striatum, prefrontal cortex, hippocampus, amygdala, and bed nucleus of the stria terminalis). Our results show ubiquitous expression of GlyRs that mediate large-amplitude currents in response to exogenously applied glycine in these forebrain structures. Additionally, tonic inward currents were also detected, but only in the striatum, hippocampus, and prefrontal cortex (PFC). These tonic currents were sensitive to both strychnine and picrotoxin, indicating that they are mediated by extrasynaptic homomeric GlyRs. Recordings from mice deficient in the GlyR α3 subunit () revealed a lack of tonic GlyR currents in the striatum and the PFC. In animals, GlyR tonic currents were preserved; however, the amplitudes of current responses to exogenous glycine were significantly reduced. We conclude that functional α2 and α3 GlyRs are present in various regions of the forebrain and that α3 GlyRs specifically participate in tonic inhibition in the striatum and PFC. Our findings suggest roles for glycine in regulating neuronal excitability in the forebrain.
Topics: Animals; Female; Glycine; Glycine Agents; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Picrotoxin; Prosencephalon; Receptors, Glycine; Strychnine
PubMed: 28784756
DOI: 10.1073/pnas.1703839114