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Glycine receptors (GlyR) are regulated by small-molecule binding at several allosteric sites. Cannabinoids like tetrahydrocannabinol (THC) and N-arachidonyl-ethanol-amide (AEA) potentiate the GlyR response but their mechanism of action is not fully established. By combining millisecond coarse-grained (CG) MD simulations powered by Martini 3 with backmapping to all-atom representations, we have characterized the cannabinoid-binding site(s) at the zebrafish GlyR-α1 active state with atomic resolution. Based on hundreds of thousand ligand-binding events, we find that cannabinoids bind to the transmembrane domain of the receptor at both intrasubunit and intersubunit sites. For THC, the intrasubunit binding mode predicted in simulation is in excellent agreement with recent cryo-EM structures, while intersubunit binding recapitulates in full previous mutagenesis experiments. Intriguingly, AEA is predicted to bind at the same intersubunit site despite the strikingly different chemistry. Statistical analyses of the ligand-receptor interactions highlight potentially relevant residues for GlyR potentiation, offering experimentally testable predictions. The predictions for AEA have been validated by electrophysiology recordings of rationally designed mutants. The results highlight the existence of multiple cannabinoid-binding sites for the allosteric regulation of GlyR and put forward an effective strategy for the identification and structural characterization of allosteric binding sites., (© 2024. The Author(s).)

Glycine receptors (GlyRs) are members of the Cys-loop receptors that constitute a major portion of mammalian neurotransmitter receptors. Recent resolution of heteromeric GlyR structures in multiple functional states raised fundamental questions regarding the gating mechanism of GlyR, and generally the Cys-loop family receptors. Here, we characterized in detail equilibrium properties as well as the transition kinetics between functional states. We show that, while all allosteric sites bind cooperatively to glycine, occupation of 2 sites at the α-α interfaces is sufficient for activation and necessary for high-efficacy gating. Differential glycine concentration dependence of desensitization rate, extent, and its recovery suggests separate but concerted roles of ligand-binding and ionophore reorganization. Based on these observations and available structural information, we developed a quantitative gating model that accurately predicts both equilibrium and kinetical properties throughout the glycine gating cycle. This model likely applies generally to the Cys-loop receptors and informs on pharmaceutical endeavors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Glycine receptors (GlyR) conduct inhibitory glycinergic neurotransmission in the spinal cord and the brainstem. They play an important role in muscle tone, motor coordination, respiration, and pain perception. However, the mechanism underlying GlyR activation remains unclear. There are five potential glycine binding sites in α1 GlyR, and different binding patterns may cause distinct activation or desensitization behaviors. In this study, we investigated the coupling of protein conformational changes and glycine binding events to elucidate the influence of binding patterns on the activation and desensitization processes of α1 GlyRs. Subsequently, we explored the energetic distinctions between the apical and lateral pathways during α1 GlyR conduction to identify the pivotal factors in the ion conduction pathway preference. Moreover, we predicted the mutational effects of the key residues and verified our predictions using electrophysiological experiments. For the mutants that can be activated by glycine, the predictions of the mutational directions were all correct. The strength of the mutational effects was assessed using Pearson's correlation coefficient, yielding a value of -0.77 between the calculated highest energy barriers and experimental maximum current amplitudes. These findings contribute to our understanding of GlyR activation, identify the key residues of GlyRs, and provide guidance for mechanistic studies on other pLGICs.

Glycine receptors are pentameric ligand-gated ion channels that conduct chloride ions across postsynaptic membranes to facilitate fast inhibitory neurotransmission. In addition to gating by the glycine agonist, interactions with lipids and other compounds in the surrounding membrane environment modulate their function, but molecular details of these interactions remain unclear, in particular, for cholesterol. Here, we report coarse-grained simulations in a model neuronal membrane for three zebrafish glycine receptor structures representing apparent resting, open, and desensitized states. We then converted the systems to all-atom models to examine detailed lipid interactions. Cholesterol bound to the receptor at an outer-leaflet intersubunit site, with a preference for the open and desensitized versus resting states, indicating that it can bias receptor function. Finally, we used short atomistic simulations and iterative amino acid perturbations to identify residues that may mediate allosteric gating transitions. Frequent cholesterol contacts in atomistic simulations clustered with residues identified by perturbation analysis and overlapped with mutations influencing channel function and pathology. Cholesterol binding at this site was also observed in a recently reported pig heteromeric glycine receptor. These results indicate state-dependent lipid interactions relevant to allosteric transitions of glycine receptors, including specific amino acid contacts applicable to biophysical modeling and pharmaceutical design.

Startle disease (SD) is characterized by enhanced startle responses, generalized muscle stiffness, unexpected falling, and fatal apnea episodes due to disturbed feedback inhibition in the spinal cord and brainstem of affected individuals. Mutations within the glycine receptor (GlyR) subunit and glycine transporter 2 (GlyT2) genes have been identified in individuals with SD. Impaired inhibitory neurotransmission in SD is due to pre- and/or postsynaptic GlyR or presynaptic GlyT2 dysfunctions. Previous research has focused on mutated GlyRs and GlyT2 that impair ion channel/transporter function or trafficking. With insights provided by recently solved cryo-electron microscopy and X-ray structures of GlyRs, a detailed picture of structural transitions important for receptor gating has emerged, allowing a deeper understanding of SD at the molecular level. Moreover, studies on novel SD mutations have demonstrated a higher complexity of SD, with identification of additional clinical signs and symptoms and interaction partners representing key players for fine-tuning synaptic processes. Although our knowledge has steadily improved during the last years, changes in synaptic localization and GlyR or GlyT2 homeostasis under disease conditions are not yet completely understood. Combined proteomics, interactomics, and high-resolution microscopy techniques are required to reveal alterations in receptor dynamics at the synaptic level under disease conditions., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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.

The recognition of Cannabis as a source of new compounds suitable for medical use has attracted strong interest from the scientific community in its research, and substantial progress has accumulated regarding cannabinoids' activity; however, a thorough description of their molecular mechanisms of action remains a task to complete. Highlighting their complex pharmacology, the list of cannabinoids' interactors has vastly expanded beyond the canonical cannabinoid receptors. Among those, we have focused our study on the glycine receptor (GlyR), an ion channel involved in the modulation of nervous system responses, including, to our interest, sensitivity to peripheral pain. Here, we report the use of computational methods to investigate possible binding modes between the GlyR and Δ 9 -tetrahydrocannabinol (THC). After obtaining a first pose for the THC binding from a biased molecular docking simulation and subsequently evaluating it by molecular dynamic simulations, we found a dynamic system with an identifiable representative binding mode characterized by the specific interaction with two transmembrane residues (Phe293 and Ser296). Complementarily, we assessed the role of membrane cholesterol in this interaction and positively established its relevance for THC binding to GlyR. Lastly, the use of restrained molecular dynamics simulations allowed us to refine the description of the binding mode and of the cholesterol effect. Altogether, our findings contribute to the current knowledge about the GlyR-THC mode of binding and propose a new starting point for future research on how cannabinoids in general, and THC in particular, modulate pain perception in view of its possible clinical applications., (© 2022 Wiley Periodicals LLC.)

Glycine receptors (GlyRs) are pentameric, 'Cys-loop' receptors that form chloride-permeable channels and mediate fast inhibitory signalling throughout the central nervous system 1,2 . In the spinal cord and brainstem, GlyRs regulate locomotion and cause movement disorders when mutated 2,3 . However, the stoichiometry of native GlyRs and the mechanism by which they are assembled remain unclear, despite extensive investigation 4-8 . Here we report cryo-electron microscopy structures of native GlyRs from pig spinal cord and brainstem, revealing structural insights into heteromeric receptors and their predominant subunit stoichiometry of 4α:1β. Within the heteromeric pentamer, the β(+)-α(-) interface adopts a structure that is distinct from the α(+)-α(-) and α(+)-β(-) interfaces. Furthermore, the β-subunit contains a unique phenylalanine residue that resides within the pore and disrupts the canonical picrotoxin site. These results explain why inclusion of the β-subunit breaks receptor symmetry and alters ion channel pharmacology. We also find incomplete receptor complexes and, by elucidating their structures, reveal the architectures of partially assembled α-trimers and α-tetramers., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The strychnine-sensitive pentameric glycine receptor (GlyR) mediates fast inhibitory neurotransmission in the mammalian nervous system. Only heteromeric GlyRs mediate synaptic transmission, as they contain the β subunit that permits clustering at the synapse through its interaction with scaffolding proteins. Here, we show that α2 and β subunits assemble with an unexpected 4:1 stoichiometry to produce GlyR with native electrophysiological properties. We determined structures in multiple functional states at 3.6-3.8 Å resolutions and show how 4:1 stoichiometry is consistent with the structural features of α2β GlyR. Furthermore, we show that one single β subunit in each GlyR gives rise to the characteristic electrophysiological properties of heteromeric GlyR, while more β subunits render GlyR non-conductive. A single β subunit ensures a univalent GlyR-scaffold linkage, which means the scaffold alone regulates the cluster properties., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Diminished inhibitory control of spinal nociception is one of the major culprits of chronic pain states. Restoring proper synaptic inhibition is a well-established rational therapeutic approach explored by several pharmaceutical companies. A particular challenge arises from the need for site-specific intervention to avoid deleterious side effects such as sedation, addiction, or impaired motor control, which would arise from wide-range facilitation of inhibition. Specific targeting of glycinergic inhibition, which dominates in the spinal cord and parts of the hindbrain, may help reduce these side effects. Selective targeting of the α3 subtype of glycine receptors (GlyRs), which is highly enriched in the superficial layers of the spinal dorsal horn, a key site of nociceptive processing, may help to further narrow down pharmacological intervention on the nociceptive system and increase tolerability. This review provides an update on the physiological properties and functions of α3 subtype GlyRs and on the present state of related drug discovery programs.

Inhibitory synapses are also known as symmetric synapses due to their lack of prominent postsynaptic densities (PSDs) under a conventional electron microscope (EM). Recent cryo-EM tomography studies indicated that inhibitory synapses also contain PSDs, albeit with a rather thin sheet-like structure. It is not known how such inhibitory PSD (iPSD) sheet might form. Here, we demonstrate that the key inhibitory synapse scaffold protein gephyrin, when in complex with either glycine or GABA A receptors, spontaneously forms highly condensed molecular assemblies via phase separation both in solution and on supported membrane bilayers. Multivalent and specific interactions between the dimeric E-domain of gephyrin and the glycine/GABA A receptor multimer are essential for the iPSD condensate formation. Gephyrin alone does not form condensates. The linker between the G- and E-domains of gephyrin inhibits the iPSD condensate formation via autoinhibition. Phosphorylation of specific residues in the linker or binding of target proteins such as dynein light chain to the linker domain regulates gephyrin-mediated glycine/GABA A receptor clustering. Thus, analogous to excitatory PSDs, iPSDs are also formed by phase separation-mediated condensation of scaffold protein/neurotransmitter receptor complexes.

Pentameric ligand-gated ion channels (pLGICs) are receptor proteins that are sensitive to their membrane environment, but the mechanism for how lipids modulate function under physiological conditions in a state dependent manner is not known. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. Using a realistic model of a neuronal membrane coupled with coarse-grained molecular dynamics simulations, we demonstrate that some key lipid-protein interactions are dependent on the receptor state, suggesting that lipids may regulate the receptor's conformational dynamics. Comparison with existing structural data confirms known lipid binding sites, but we also predict further protein-lipid interactions including a site at the communication interface between the extracellular and transmembrane domain. Moreover, in the active state, cholesterol can bind to the binding site of the positive allosteric modulator ivermectin. These protein-lipid interaction sites could in future be exploited for the rational design of lipid-like allosteric drugs., Competing Interests: The authors have declared that no competing interests exist.

The glycine receptor (GlyR), a member of the pentameric ligand-gated ion channel family (pLGIC), displays remarkable variations in the affinity and efficacy of the full agonist glycine and the partial agonist taurine depending on the cell system used. Despite detailed insights in the GlyR three-dimensional structure and activation mechanism, little is known about conformational rearrangements induced by these agonists. Here, we characterized the conformational states of the α1 GlyR upon binding of glycine and taurine by microscale thermophoresis expressed in HEK293 cells and Xenopus oocytes after solubilization in amphipathic styrene-maleic acid copolymer nanodiscs. Our results show that glycine and taurine induce different conformational transitions of the GlyR upon ligand binding. In contrast, the variability of agonist affinity is not mediated by an altered conformational change. Thus, our data shed light on specific agonist induced conformational features and mechanisms of pLGIC upon ligand binding determining receptor activation in native environments.

GABA and glycine act as inhibitory neurotransmitters in the CNS. Inhibitory neurotransmission is mediated via activation of ionotropic GABA A and glycine receptors. We used a modeling approach to explain the opposite effects of the general anesthetic etomidate (ETM) and fenamate mefenamic acid (MFA) on GABA- and glycine-activated currents recorded in isolated cerebellar Purkinje cells and hippocampal pyramidal neurons, respectively. These drugs potentiated GABA A Rs but blocked GlyRs. We built a homology model of α1β GlyR based on the cryo-EM structure of open α1 GlyR, used the α1β3γ2 GABA A R structure from the PDB, and applied Monte-Carlo energy minimization to optimize models of receptors and ligand-receptor complexes. In silico docking suggests that ETM/MFA bind at the transmembrane β( +)/α( -) intersubunit interface in GABA A R. Our models predict that the bulky side chain of the highly conserved Arg 19' residue at the plus interface side wedges the interface and maintains the conducting receptor state. We hypothesized that MFA/ETM binding at the β( +)/α( -) interface leads to prolongation of receptor life-time in the open state. Having analyzed different GABA A R and GlyR structures available in the PDB, we found that mutual arrangement of the Arg 19' and Gln -26' side chains at the plus and minus interface sides, respectively, plays an important role when the receptor switches from the open to closed state. We show that this process is accompanied by narrowing of the intersubunit interfaces, leading to extrusion of the Arg 19' side chain from the interface. Our models allow us to explain the lack of GlyR potentiation in our electrophysiological experiments.

We report the results of our in silico study of approved drugs as potential treatments for COVID-19. The study is based on the analysis of normal modes of proteins. The drugs studied include chloroquine, ivermectin, remdesivir, sofosbuvir, boceprevir, and α-difluoromethylornithine (DMFO). We applied the tools we developed and standard tools used in the structural biology community. Our results indicate that small molecules selectively bind to stable, kinetically active residues and residues adjoining them on the surface of proteins and inside protein pockets, and that some prefer hydrophobic sites over other active sites. Our approach is not restricted to viruses and can facilitate rational drug design, as well as improve our understanding of molecular interactions, in general.

Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRβ subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels. SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif., (Copyright © 2020 the authors.)

The glycine receptor (GlyR) is by far the best-characterized pentameric ligand-gated ion channel, with several high-resolution structures from X-ray crystallography, cryoelectron microscopy (cryo-EM), and modeling. Nonetheless, the significance of the currently available open-pore conformations is debated due to their diversity in the pore geometry. Here, we discuss the physiological significance of existing models of the GlyR active state based on conductance and selectivity measurements by computational electrophysiology. The results support the conclusion that the original cryo-EM reconstruction of the active state obtained in detergents as well as its subsequent refinement by molecular dynamics simulations are likely to be non-physiological as they feature artificially dilated ion pores. In addition, the calculations indicate that a physiologically relevant open pore should be constricted within a radius of 2.5 and 2.8 Å, which is consistent with previous modeling, electrophysiology measurements, and the most recent cryo-EM structures obtained in a native lipid membrane environment., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cannabinoids are reported to rescue cocaine-induced seizures (CISs), a severe complication in cocaine users. However, the molecular targets for cannabinoid therapy of CISs remain unclear. Here, we report that the systemic administration of cannabinoids alleviates CISs in a CB 1 /CB 2 -receptor-independent manner. In HEK293 cells and cortical neurons, cocaine-induced dysfunction of the glycine receptor (GlyR) is restored by cannabinoids. Such restoration is blocked by GlyRα1 S296A mutation. Consistently, the therapeutic effects of cannabinoids on CISs are also eliminated in GlyRα1 S296A mutant mice. Based on molecular dynamic simulation, the hydrogen-bonding interaction between cocaine and the GlyR is weakened by cannabinoid docking. Without altering cocaine distribution across the brain, cannabinoids significantly suppress cocaine-exaggerated neuronal excitability in the prefrontal cortex (PFC) and hippocampus by rehabilitating extra-synaptic GlyR function. Microinjection of cannabinoids into the PFC and hippocampus restores cocaine-puzzled neural activity and alleviates CISs. These findings suggest that using GlyR-hypersensitive cannabinoids may represent a potential therapeutic strategy for treating CISs., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glycine receptors (GlyRs) are anion-permeable pentameric ligand-gated ion channels (pLGICs). The GlyR activation is critical for the control of key neurophysiological functions, such as motor coordination, respiratory control, muscle tone and pain processing. The relevance of the GlyR function is further highlighted by the presence of abnormal glycinergic inhibition in many pathophysiological states, such as hyperekplexia, epilepsy, autism and chronic pain. In this context, previous studies have shown that the functional inhibition of  GlyRs containing the α3 subunit is a pivotal mechanism of pain hypersensitivity. This pathway involves the activation of EP2 receptors and the subsequent PKA-dependent phosphorylation of α3GlyRs within the intracellular domain (ICD), which decrease the GlyR-associated currents and enhance neuronal excitability. Despite the importance of this mechanism of glycinergic dis-inhibition associated with dysfunctional α3GlyRs, our current understanding of the molecular events involved is limited. Here, we report that the activation of PKA signaling pathway decreases the unitary conductance of α3GlyRs. We show in addition that the substitution of the PKA-targeted serine with a negatively charged residue within the ICD of α3GlyRs and of chimeric receptors combining bacterial GLIC and α3GlyR was sufficient to generate receptors with reduced conductance. Thus, our findings reveal a potential biophysical mechanism of glycinergic dis-inhibition and suggest that post-translational modifications of the ICD, such as phosphorylation, may shape the conductance of other pLGICs.

Pentameric ligand-gated ion channels are key players in mediating fast neurotransmission. Glycine receptors are chloride-selective members of this receptor family that mediate inhibitory synaptic transmission and are implicated in neurological disorders including autism and hyperekplexia. They have been structurally characterized by both X-ray crystallography and cryoelectron microscopy (cryo-EM) studies, with the latter giving rise to what was proposed as a possible open state. However, recent work has questioned the physiological relevance of this open state structure, since it rapidly collapses in molecular dynamics simulations. Here, we show that the collapse can be avoided by a careful equilibration protocol that reconciles the more problematic regions of the original density map and gives a stable open state that shows frequent selective chloride permeation. The protocol developed in this work provides a means to refine open-like structures of the whole pentameric ligand-gated ion channel superfamily and reconciles the previous issues with the cryo-EM structure., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Glycine is a major inhibitory neurotransmitter in the CNS, where it modulates both sensory and motor transduction throughout its binding to glycine receptors (GlyRs), pentameric chloride channels that share structural and functional properties with type A γ-aminobutyric acid receptors (GABA A R). A large number of structurally diverse organic compounds have been identified as GlyR and GABA A R allosteric modulators, making these receptors attractive pharmacological targets. Taking into account the recent resolved crystal structures of GABA A R/neurosteroid complexes, and due to the high sequence identity between the GABA A R and GlyR transmembrane domains, in this work we applied molecular modeling methods to explore the neurosteroid binding to GlyR. Our results indicated that neurosteroid binding sites of GABA A Rs are also conserved in the GlyRs. Furthermore, docking and molecular dynamics simulations predicted that neurosteroids are stably recognized at these sites, providing precise information on the molecular basis of the neurosteroid binding mode to GlyR. The comparison of how allopregnanolone and pregnanolone 3-OH moieties are recognized by the GlyR binding pocket revealed significant differences that may be associated to opposite effects of these isomers on the GlyR response., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Glycine receptors (GlyR) mediate fast inhibitory neurotransmission by switching between discrete states in response to ligand-binding events. Recent high-resolution structures from cryoelectron microscopy (cryo-EM) and X-ray crystallography have provided atomistic models for the open and closed states. Notably, the cryo-EM structure in complex with glycine illuminated a previously unreported wide-open state, whose physiological significance is debated. Here, we present the structure of an ion-conducting state of GlyR α1 captured by molecular dynamics and validate its physiological relevance with computational electrophysiology and polyatomic anion permeation simulations. Our analysis suggests that none of the experimental structures is a true representation of the physiologically active state, although previously characterized open channels in GLIC at pH 4, or GluCl/GlyR with ivermectin bound, provide reasonable models. These results open the door to an original functional annotation and support the conclusion that pore closing by desensitization versus deactivation involves the reorientation of the pore-lining helices in opposite directions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Cys-loop receptors play important roles in fast neuronal signal transmission. Functional receptors are pentamers, with each subunit having an extracellular, transmembrane (TM) and intracellular domain. Each TM domain contains 4 α-helices (M1-M4) joined by loops of varying lengths. Many of the amino acid residues that constitute these α-helices are hydrophobic, and there has been particular interest in aromatic residues, especially those in M4, which have the potential to contribute to the assembly and function of the receptor via a range of interactions with nearby residues., Results: Here we show that many aromatic residues in the M1, M3 and M4 α-helices of the glycine receptor are involved in the function of the receptor. The residues were explored by creating a range of mutant receptors, characterising them using two electrode voltage clamp in Xenopus oocytes, and interpreting changes in receptor parameters using currently available structural information on the open and closed states of the receptor. For 7 residues function was ablated with an Ala substitution: 3 Tyr residues at the extracellular end of M1, 2 Trp residues located towards the centers of M1 and M3, and a Phe and a Tyr residue in M4. For many of these an alternative aromatic residue restored wild-type-like function indicating the importance of the π ring. EC 50 s were increased with Ala substitution of 8 other aromatic residues, with those in M1 and M4 also having reduced currents, indicating a role in receptor assembly. The structure shows many potential interactions with nearby residues, especially between those that form the M1/M3/M4 interface, and we identify those that are supported by the functional data., Conclusion: The data reveal the importance and interactions of aromatic residues in the GlyR M1, M3 and M4 α-helices, many of which are essential for receptor function.

The extracellular domains (ECDs) of Cys-loop receptors contain many aromatic amino acids, but only relatively few have been well studied. Here we explore the roles of Tyr and Trp residues in the ECD of the glycine receptor and show that four such residues that have not been previously studied (Y24, Y58, W170, and Y197) contribute significantly to the function of the protein. The residues were studied by creating mutant receptors, characterizing them using two-electrode voltage clamp in Xenopus oocytes, and interpreting changes in receptor parameters using structural information about the open and closed states of the receptor. Alanine substitution of all these residues ablates function or increases the glycine EC 50 . There are also a number of changes in the relative maximal responses to taurine, a partial agonist, compared to glycine. Further mutations, in combination with structural information, suggest Y24 contributes to an anion-π interaction with a binding loop D residue, Y58 to an S-π interaction stabilizing the Cys loop, W170 to hydrophobic interactions stabilizing the hydrophobic interior of the subunit, and Y197 to a hydrogen bond linking binding loops B and C. These interactions appear to be broadly conserved in other Cys-loop receptors. Thus, we have identified new regions of the glycine receptor that are important contributors to receptor activation and are likely also to contribute to function in other members of this important protein family.

The glycine receptor (GlyR) belongs to a superfamily of pentameric ligand-gated ion channels (pLGICs) that mediate fast neurotransmission. GlyR typically modulates inhibitory transmission by antagonizing membrane depolarization through anion influx. Allosteric interactions between the receptor and its lipid surroundings affect receptor function, and cholesterol is essential for pLGIC activity. Cholesterol at compositions below ∼33 mol percent has been shown to have negligible chemical activity, suggesting that specific interactions between membrane proteins and cholesterol become significant only at concentrations above this stoichiometric threshold. Human α1 GlyR was purified from baculovirus infected insect cells and reconstituted in unilamellar vesicles at cholesterol/lipid ratios above and below the cholesterol activity threshold with equivalent aliquots of azi-cholesterol, a photoactivatable nonspecific cross-linker. After photoactivation, cross-linked cholesterol-GlyR was trypsinized and mass fingerprinted. Mass shifted peptides containing cholesterol were identified by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS), and sites of direct covalent attachment to peptides were refined by targeted MS/MS. Differential patterns of dozens of cholesterol-GlyR cross-links were identified in these comparative studies, with sites of cross-linking found primarily in the fourth transmembrane helix and extramembranous connecting loops and mapping the lipid-accessible surface of the receptor. Unique cross-linking observed in both reduced and elevated cholesterol composition suggests different apo-state structural conformations of GlyR as a function of cholesterol concentration and, in the latter studies, identified potential specific binding sites for cholesterol in the receptor.

Functional impairments or trafficking defects of inhibitory glycine receptors (GlyRs) have been linked to human hyperekplexia/startle disease and autism spectrum disorders. We found that a lack of synaptic integration of GlyRs, together with disrupted receptor function, is responsible for a lethal startle phenotype in a novel spontaneous mouse mutant shaky , caused by a missense mutation, Q177K, located in the extracellular β8-β9 loop of the GlyR α1 subunit. Recently, structural data provided evidence that the flexibility of the β8-β9 loop is crucial for conformational transitions during opening and closing of the ion channel and represents a novel allosteric binding site in Cys-loop receptors. We identified the underlying neuropathological mechanisms in male and female shaky mice through a combination of protein biochemistry, immunocytochemistry, and both in vivo and in vitro electrophysiology. Increased expression of the mutant GlyR α1 Q177K subunit in vivo was not sufficient to compensate for a decrease in synaptic integration of α1 Q177K β GlyRs. The remaining synaptic heteromeric α1 Q177K β GlyRs had decreased current amplitudes with significantly faster decay times. This functional disruption reveals an important role for the GlyR α1 subunit β8-β9 loop in initiating rearrangements within the extracellular-transmembrane GlyR interface and that this structural element is vital for inhibitory GlyR function, signaling, and synaptic clustering. SIGNIFICANCE STATEMENT GlyR dysfunction underlies neuromotor deficits in startle disease and autism spectrum disorders. We describe an extracellular GlyR α1 subunit mutation (Q177K) in a novel mouse startle disease mutant shaky Structural data suggest that during signal transduction, large transitions of the β8-β9 loop occur in response to neurotransmitter binding. Disruption of the β8-β9 loop by the Q177K mutation results in a disruption of hydrogen bonds between Q177 and the ligand-binding residue R65. Functionally, the Q177K change resulted in decreased current amplitudes, altered desensitization decay time constants, and reduced GlyR clustering and synaptic strength. The GlyR β8-β9 loop is therefore an essential regulator of conformational rearrangements during ion channel opening and closing., (Copyright © 2017 Schaefer, Berger et al.)

Ivermectin acts as a positive allosteric modulator of several Cys-loop receptors including the glutamate-gated chloride channels (GluCls), γ-aminobutyric acid receptors (GABA A Rs), glycine receptors (GlyRs), and neuronal α7-nicotinic receptors (α7 nAChRs). The crystal structure of Caenorhabditis elegans GluCl complexed with ivermectin revealed the details of its ivermectin binding site. Although the electron microscopy structure of zebrafish GlyRα1 complexed with ivermectin demonstrated a similar binding orientation, detailed structural information on the ivermectin binding and pore opening for Cys-loop receptors in vertebrates has been elusive. Here we present the crystal structures of human GlyRα3 in complex with ivermectin at 2.85 and 3.08 Å resolution. Our structures allow us to explore in detail the molecular recognition of ivermectin by GlyRs, GABA A Rs, and α7 nAChRs. Comparisons with previous structures reveal how the ivermectin binding expands the ion channel pore. Our results hold promise in structure-based design of GlyR modulators for the treatment of neuropathic pain., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The formation and stability of synapses are key questions in neuroscience. Post-synaptic domains have been classically conceived as resulting from local insertion and turnover of proteins at the synapse. However, insertion is likely to occur outside the post-synaptic domains and advances in single-molecule imaging have shown that proteins diffuse in the plane of the membrane prior to their accumulation at synapses. We quantitatively investigated this scenario using computer simulations and mathematical analysis, taking for definiteness the specific case of inhibitory synapse components, i.e., the glycine receptor (GlyR) and the associated gephyrin scaffolding protein. The observed domain sizes of scaffold clusters can be explained by a dynamic balance between the aggregation of gephyrin proteins diffusing while bound to GlyR and their turnover at the neuron membrane. We also predict the existence of extrasynaptic clusters with a characteristic size distribution that significantly contribute to the size fluctuations of synaptic domains. New super-resolution data for gephyrin proteins established the existence of extrasynaptic clusters the sizes of which are consistent with the model predictions in a range of model parameters. At a general level, our results highlight aggregation with removal as a non-equilibrium phase separation which produces structures of tunable size.

Glycine receptors (GlyR) belong to the pentameric ligand-gated ion channel (pLGIC) superfamily and mediate fast inhibitory transmission in the vertebrate CNS. Disruption of glycinergic transmission by inherited mutations produces startle disease in man. Many startle mutations are in GlyRs and provide useful clues to the function of the channel domains. E103K is one of few startle mutations found in the extracellular agonist binding site of the channel, in loop A of the principal side of the subunit interface. Homology modeling shows that the side chain of Glu-103 is close to that of Arg-131, in loop E of the complementary side of the binding site, and may form a salt bridge at the back of the binding site, constraining its size. We investigated this hypothesis in recombinant human α1 GlyR by site-directed mutagenesis and functional measurements of agonist efficacy and potency by whole cell patch clamp and single channel recording. Despite its position near the binding site, E103K causes hyperekplexia by impairing the efficacy of glycine, its ability to gate the channel once bound, which is very high in wild type GlyR. Mutating Glu-103 and Arg-131 caused various degrees of loss-of-function in the action of glycine, whereas mutations in Arg-131 enhanced the efficacy of the slightly bigger partial agonist sarcosine ( N -methylglycine). The effects of the single charge-swapping mutations of these two residues were largely rescued in the double mutant, supporting the possibility that they interact via a salt bridge that normally constrains the efficacy of larger agonist molecules., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Partial agonists have lower efficacies than compounds considered 'full agonists', eliciting submaximal responses even at saturating concentrations. Taurine is a partial agonist at the glycine receptor (GlyR), a member of the cys-loop ligand-gated ion channel superfamily. The molecular mechanisms responsible for agonism are not fully understood but evidence suggests that efficacy at these receptors is determined by conformational changes that occur early in the process of receptor activation. We previously identified a residue located near the human α1 glycine binding site (aspartate-97; D97) that, when mutated to arginine (D97R), results in GlyR channels opening spontaneously with a high open probability, mimicking the effects of saturating glycine concentrations on wildtype GlyR. This D97 residue is hypothesized to form an electrostatic interaction with arginine-119 on an adjacent subunit, stabilizing the channel in a shut state. Here we demonstrate that the disruption of this putative bond increases the efficacy of partial agonists including taurine, as well as two other β-amino acid partial agonists, β-aminobutyric acid (β-ABA) and β-aminoisobutyric acid (β-AIBA). Even the subtle charge-conserving mutation of D97 to glutamate (D97E) markedly affects partial agonist efficacy. Mutation to the neutral alanine residue in the D97A mutant mimics the effects seen with D97R, indicating that charge repulsion does not significantly affect these findings. Our findings suggest that the determination of efficacy following ligand binding to the glycine receptor may involve the disruption of an intersubunit electrostatic interaction occurring near the agonist binding site., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Current therapies to treat persistent pain and neuropathic pain are limited by poor efficacy, side effects and risk of addiction. Here, we present a novel class of potent selective, central nervous system (CNS)-penetrant potentiators of glycine receptors (GlyRs), ligand-gated ion channels expressed in the CNS. AM-1488 increased the response to exogenous glycine in mouse spinal cord and significantly reversed mechanical allodynia induced by nerve injury in a mouse model of neuropathic pain. We obtained an X-ray crystal structure of human homopentameric GlyRα3 in complex with AM-3607, a potentiator of the same class with increased potency, and the agonist glycine, at 2.6-Å resolution. AM-3607 binds a novel allosteric site between subunits, which is adjacent to the orthosteric site where glycine binds. Our results provide new insights into the potentiation of cysteine-loop receptors by positive allosteric modulators and hold promise in structure-based design of GlyR modulators for the treatment of neuropathic pain.

Glycine receptors (GlyR) are inhibitory Cys-loop ion channels that contribute to the control of excitability along the central nervous system (CNS). GlyR are found in the spinal cord and brain stem, and more recently they were reported in higher regions of the CNS such as the hippocampus and nucleus accumbens. GlyR are involved in motor coordination, respiratory rhythms, pain transmission, and sensory processing, and they are targets for relevant physiologic and pharmacologic modulators. Several studies with protein crystallography and cryoelectron microscopy have shed light on the residues and mechanisms associated with the activation, blockade, and regulation of pentameric Cys-loop ion channels at the atomic level. Initial studies conducted on the extracellular domain of acetylcholine receptors, ion channels from prokaryote homologs-Erwinia chrysanthemi ligand-gated ion channel (ELIC), Gloeobacter violaceus ligand-gated ion channel (GLIC)-and crystallized eukaryotic receptors made it possible to define the overall structure and topology of the Cys-loop receptors. For example, the determination of pentameric GlyR structures bound to glycine and strychnine have contributed to visualizing the structural changes implicated in the transition between the open and closed states of the Cys-loop receptors. In this review, we summarize how the new information obtained in functional, mutagenesis, and structural studies have contributed to a better understanding of the function and regulation of GlyR., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Our study reports computer software that simulates the work of a single glycine receptor (GlyR). GlyRs have been found in various types of tissues, but their most important role seems to be in neurons, where they hyperpolarise membranes by opening chloride transmembrane channels. The software is based on a combination of two blocks. One block describes the Brownian dynamics of charged particle motion in a dielectric medium, and the other block determines the probability and timing of receptor activation. Using this software, the voltage-current dependencies and time curves of the transmembrane current were obtained. The mean value of the simulated anion current (4.5 ± 0.3 pA) is in good agreement with measured values under identical conditions ([Formula: see text] pA). It was shown that there is a condition under which the GlyR anion channel remains active despite a negligible chloride gradient. Virtual experiments allow evaluation of the value of half maximal effective concentration (EC[Formula: see text]) of the GlyR ([Formula: see text] [Formula: see text]M) and confirm that this receptor activates according to a mechanism involving three ligand binding sites. The advantage of the model is the ability to adjust parameters to the precise demands of experimental researchers. Moreover, the introduced algorithm has low computational power demands; therefore, it can be used as a research tool for assistance with structural experiments and applied aspects of neurophysiology.

Background and Purpose: Gelsemine is one of the principal alkaloids produced by the Gelsemium genus of plants belonging to the Loganiaceae family. The extracts of these plants have been used for many years, for a variety of medicinal purposes. Coincidentally, recent studies have shown that gelsemine exerts anxiolytic and analgesic effects on behavioural models. Several lines of evidence have suggested that these beneficial actions were dependent on glycine receptors, which are inhibitory neurotransmitter-gated ion channels of the CNS. However, it is currently unknown whether gelsemine can directly modulate the function of glycine receptors., Experimental Approach: We examined the functional effects of gelsemine on glycine receptors expressed in transfected HEK293 cells and in cultured spinal neurons by electrophysiological techniques., Key Results: Gelsemine directly modulated recombinant and native glycine receptors and exerted conformation-specific and subunit-selective effects. Gelsemine modulation was voltage-independent and was associated with differential changes in the apparent affinity for glycine and in the open probability of the ion channel. In addition, the alkaloid preferentially targeted glycine receptors in spinal neurons and showed only minor effects on GABAA and AMPA receptors. Furthermore, gelsemine significantly diminished the frequency of glycinergic and glutamatergic synaptic events without altering the amplitude., Conclusions and Implications: Our results provide a pharmacological basis to explain, at least in part, the glycine receptor-dependent, beneficial and toxic effects of gelsemine in animals and humans. In addition, the pharmacological profile of gelsemine may open new approaches to the development of subunit-selective modulators of glycine receptors., (© 2016 The British Pharmacological Society.)

Permeation of ions through open channels and their accessibility to pore-targeting drugs depend on the pore cross-sectional dimensions, which are known only for static X-ray and cryo-EM structures. Here, we have built homology models of the closed, open and desensitized α1β2γ2 GABAA receptor (GABAAR). The models are based, respectively, on the X-ray structure of α3 glycine receptor (α3 GlyR), cryo-EM structure of α1 GlyR and X-ray structure of β3 GABAAR. We employed Monte Carlo energy minimizations to explore how the pore lumen may increase due to repulsions of flexible side chains from a variable-diameter electroneutral atom (an expanding sphere) pulled through the pore. The expanding sphere computations predicted that the pore diameter averaged along the permeation pathway is larger by approximately 3 Å than that computed for the models with fixed sidechains. Our models predict three major pore constrictions located at the levels of -2', 9' and 20' residues. Residues around the -2' and 9' rings are known to form the desensitization and activation gates of GABAAR. Our computations predict that the 20' ring may also serve as GABAAR gate whose physiological role is unclear. The side chain flexibility of residues -2', 9' and 20' and hence the dimensions of the constrictions depend on the GABAAR functional state.

Key Points: Hyperekplexia or startle disease is a serious neurological condition affecting newborn children and usually involves dysfunctional glycinergic neurotransmission. Glycine receptors (GlyRs) are major mediators of inhibition in the spinal cord and brainstem. A missense mutation, replacing asparagine (N) with lysine (K), at position 46 in the GlyR α1 subunit induced hyperekplexia following a reduction in the potency of the transmitter glycine; this resulted from a rapid deactivation of the agonist current at mutant GlyRs. These effects of N46K were rescued by mutating a juxtaposed residue, N61 on binding Loop D, suggesting these two asparagines may interact. Asparagine 46 is considered to be important for the structural stability of the subunit interface and glycine binding site, and its mutation represents a new mechanism by which GlyR dysfunction induces startle disease., Abstract: Dysfunctional glycinergic inhibitory transmission underlies the debilitating neurological condition, hyperekplexia, which is characterised by exaggerated startle reflexes, muscle hypertonia and apnoea. Here we investigated the N46K missense mutation in the GlyR α1 subunit gene found in the ethylnitrosourea (ENU) murine mutant, Nmf11, which causes reduced body size, evoked tremor, seizures, muscle stiffness, and morbidity by postnatal day 21. Introducing the N46K mutation into recombinant GlyR α1 homomeric receptors, expressed in HEK cells, reduced the potencies of glycine, β-alanine and taurine by 9-, 6- and 3-fold respectively, and that of the competitive antagonist strychnine by 15-fold. Replacing N46 with hydrophobic, charged or polar residues revealed that the amide moiety of asparagine was crucial for GlyR activation. Co-mutating N61, located on a neighbouring β loop to N46, rescued the wild-type phenotype depending on the amino acid charge. Single-channel recording identified that burst length for the N46K mutant was reduced and fast agonist application revealed faster glycine deactivation times for the N46K mutant compared with the WT receptor. Overall, these data are consistent with N46 ensuring correct alignment of the α1 subunit interface by interaction with juxtaposed residues to preserve the structural integrity of the glycine binding site. This represents a new mechanism by which GlyR dysfunction induces startle disease., (© 2016 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The glycine receptor is a member of the Cys-loop receptor superfamily of ligand-gated ion channels and is implicated as a possible therapeutic target for the treatment of diseases such as alcoholism and inflammatory pain. In humans, four glycine receptor subtypes (α1, α2, α3, and β) co-assemble to form pentameric channel proteins as either α homomers or αβ heteromers. To date, few agents have been identified that can selectively modulate the glycine receptor, especially those possessing subtype specificity. We used a cell-based method of phage display panning, coupled with two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, to identify novel heptapeptide modulators of the α1β glycine receptor. This involved a panning procedure in which the phage library initially underwent subtractive panning against Human Embryonic Kidney (HEK) 293 cells expressing alternative glycine receptor subtypes before panning the remaining library over HEK 293 cells expressing the target, the α1β glycine receptor. Peptides were identified that act with selectivity on α1β and α3β, compared to α2β, glycine receptors. In addition, peptide activity at the glycine receptor decreased when zinc was chelated by tricine, similar to previous observations of a decrease in ethanol's enhancing actions at the receptor in the absence of zinc. Comparisons of the amino acid sequences of heptapeptides capable of potentiating glycine receptor function revealed several consensus sequences that may be predictive of a peptide's enhancing ability., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Key Points: Cellular stimuli can modulate the ion selectivity of some anion channels, such as CFTR, ANO1 and the glycine receptor (GlyR), by changing pore size. Ion selectivity of CFTR, ANO1 and GlyR is critically affected by the electric permittivity and diameter of the channel pore. Pore size change affects the energy barriers of ion dehydration as well as that of size-exclusion of anion permeation. Pore dilatation increases the bicarbonate permeability (P HC O3/ Cl ) of CFTR, ANO1 and GlyR. Dynamic change in P HC O3/ Cl may mediate many physiological and pathological processes., Abstract: Chloride (Cl(-) ) and bicarbonate (HCO3 (-) ) are two major anions and their permeation through anion channels plays essential roles in our body. However, the mechanism of ion selection by the anion channels is largely unknown. Here, we provide evidence that pore dilatation increases the bicarbonate permeability (P HC O3/ Cl ) of anion channels by reducing energy barriers of size-exclusion and ion dehydration of HCO3 (-) permeation. Molecular, physiological and computational analyses of major anion channels, such as cystic fibrosis transmembrane conductance regulator (CFTR), anoctamin-1(ANO1/TMEM16A) and the glycine receptor (GlyR), revealed that the ion selectivity of anion channels is basically determined by the electric permittivity and diameter of the pore. Importantly, cellular stimuli dynamically modulate the anion selectivity of CFTR and ANO1 by changing the pore size. In addition, pore dilatation by a mutation in the pore-lining region alters the anion selectivity of GlyR. Changes in pore size affected not only the energy barriers of size exclusion but that of ion dehydration by altering the electric permittivity of water-filled cavity in the pore. The dynamic increase in P HC O3/ Cl by pore dilatation may have many physiological and pathophysiological implications ranging from epithelial HCO3 (-) secretion to neuronal excitation., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

N-methyl-d-aspartate (NMDA) receptors composed of glycine-binding GluN1 and GluN3 subunits function as excitatory glycine receptors that respond to agonist application only with a very low efficacy. Binding of glycine to the high-affinity GluN3 subunits triggers channel opening, whereas glycine binding to the low-affinity GluN1 subunits causes an auto-inhibition of the maximal glycine-inducible receptor current (Imax). Hence, competitive antagonists of the GluN1 subunit strongly potentiate glycine responses of wild type (wt) GluN1/GluN3 receptors. Here, we show that co-expression of N-terminal domain (NTD) deleted GluN1 (GluN1(ΔNTD)) and GluN3 (GluN3(ΔNTD)) subunits in Xenopus oocytes generates GluN1/GluN3 receptors with a large increase in the glycine-inducible Imax accompanied by a strongly impaired GluN1 antagonist-mediated potentiation. Affinity purification after metabolic or surface labeling revealed no differences in subunit stoichiometry and surface expression between wt GluN1/GluN3A and mutant GluN1(ΔNTD)/GluN3A(ΔNTD) receptors, indicating a specific effect of NTD deletions on the efficacy of receptor opening. Notably, GluN1/GluN3A(ΔNTD) receptors showed a similar increase in Imax and a greatly reduced GluN1 antagonist-mediated current potentiation as GluN1(ΔNTD)/GluN3A(ΔNTD) receptors, whereas the glycine-induced currents of GluN1(ΔNTD)/GluN3A receptors resembled those of wt GluN1/GluN3A receptors. Furthermore, oxidative crosslinking of the homophilic GluN3A NTD intersubunit interface in mutant GluN1/GluN3A(R319C) receptors caused both a decrease in the glycine-induced Imax concomitantly with a marked increase in GluN1 antagonist-mediated current potentiation, whilst mutations within the intrasubunit region linking the GluN3A NTD to the ligand binding domain had opposite effects. Together these results show that the GluN3A NTD constitutes a crucial regulatory determinant of GluN1/GluN3A receptor function., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cys-loop receptors mediate fast synaptic transmission in the nervous system, and their dysfunction is associated with a number of diseases. While some sequence variability is essential to ensure specific recognition of a chemically diverse set of ligands, other parts of the underlying amino acid sequences show a high degree of conservation, possibly to preserve the overall structural fold across the protein family. In this study, we focus on the only two absolutely conserved residues across the Cys-loop receptor family, two Trp side chains in the WXD motif of Loop D and in the WXPD motif of Loop A. Using a combination of conventional mutagenesis, unnatural amino acid incorporation, immunohistochemistry and MD simulations, we demonstrate the crucial contributions of these two Trp residues to receptor expression and function in two prototypical Cys-loop receptors, the anion-selective GlyR α1 and the cation-selective nAChR α7. Specifically, our results rule out possible electrostatic contributions of these Trp side chains and instead suggest that the overall size and shape of this aromatic pair is required in stabilizing the Cys-loop receptor extracellular domain.

The Cys-loop receptors play prominent roles in the nervous system. They include γ-aminobutyric acid type A receptors, nicotinic acetylcholine receptors, 5-hydroxytryptamine type-3 receptors, and glycine receptors. Proteostasis represents an optimal state of the cellular proteome in normal physiology. The proteostasis network regulates the folding, assembly, degradation, and trafficking of the Cys-loop receptors, ensuring their efficient functional cell surface expressions. Here, we summarize current advances about the protein biogenesis process of the Cys-loop receptors. Because operating on individual biogenesis steps influences the receptor cell surface level, manipulating the proteostasis network components can regulate the function of the receptors, representing an emerging therapeutic strategy for corresponding channelopathies., (© 2016 Elsevier Inc. All rights reserved.)

Previously, we have shown that ginkgolic acid has an ability to potentiate currents, mediated by αl subunits of glycine receptor. In order to define the mechanism of subunit specific action of ginkgolic acid we have performed comparative analysis of the amino acid sequences of at and α2 subunits of glycine receptor. Amino acids that contribute to the different action of ginkgolic acid on glycine receptors formed by these subunits were determined. Using whole-cell configuration of patch-clamp recording, we have demonstrated that mutation of three residues in α2 subunit for corresponding ones from αl subunit makes α2 receptors sensitive to the potentiation by ginkgolic acid. Currents, mediated by α2 mutant receptors, increased by 89±14% after application of ginkgolic acid. Similarly to ai receptors α2 mutant receptors have shown a decrease in EC₅₀. for glycine under the action of ginkgolic acid. Thus, subunit selectivity of the ginkgolic acid is in strong connection with three amino acid residues that are different in α1 And α2 subunits of glycine receptor.

Ligand-gated ion channels (LGICs) are cell surface integral proteins that mediate the fast neurotransmission in the nervous system. LGICs require auxiliary subunits for their trafficking, assembly and pharmacological modulation. Auxiliary subunits do not form functional homomeric receptors, but are reported to assemble with the principal subunits in order to modulate their pharmacological profiles. For example, nACh receptors are built at least by co-assemble of α and β subunits, and the neuronal auxiliary subunits β3 and α5 and muscle type β, δ, γ, and ϵ determine the agonist affinity of these receptors. Serotonergic 5-HT3B, 5-HT3C, 5-HT3D and 5-HT3E are reported to assemble with the 5-HT3A subunit to modulate its pharmacological profile. Functional studies evaluating the role of γ2 and δ auxiliary subunits of GABAA receptors have made important advances in the understanding of the action of benzodiazepines, ethanol and neurosteroids. Glycine receptors are composed principally by α1-3 subunits and the auxiliary subunit β determines their synaptic location and their pharmacological response to propofol and ethanol. NMDA receptors appear to be functional as heterotetrameric channels. So far, the existence of NMDA auxiliary subunits is controversial. On the other hand, Kainate receptors are modulated by NETO 1 and 2. AMPA receptors are modulated by TARPs, Shisa 9, CKAMP44, CNIH2-3 auxiliary proteins reported that controls their trafficking, conductance and gating of channels. P2X receptors are able to associate with auxiliary Pannexin-1 protein to modulate P2X7 receptors. Considering the pharmacological relevance of different LGICs auxiliary subunits in the present work we will highlight the therapeutic potential of these modulator proteins., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

It is well accepted that ethanol is able to produce major health and economic problems associated to its abuse. Because of its intoxicating and addictive properties, it is necessary to analyze its effect in the central nervous system. However, we are only now learning about the mechanisms controlling the modification of important membrane proteins such as ligand-activated ion channels by ethanol. Furthermore, only recently are these effects being correlated to behavioral changes. Current studies show that the glycine receptor (GlyR) is a susceptible target for low concentrations of ethanol (5-40mM). GlyRs are relevant for the effects of ethanol because they are found in the spinal cord and brain stem where they primarily express the α1 subunit. More recently, the presence of GlyRs was described in higher regions, such as the hippocampus and nucleus accumbens, with a prevalence of α2/α3 subunits. Here, we review data on the following aspects of ethanol effects on GlyRs: (1) direct interaction of ethanol with amino acids in the extracellular or transmembrane domains, and indirect mechanisms through the activation of signal transduction pathways; (2) analysis of α2 and α3 subunits having different sensitivities to ethanol which allows the identification of structural requirements for ethanol modulation present in the intracellular domain and C-terminal region; (3) Genetically modified knock-in mice for α1 GlyRs that have an impaired interaction with G protein and demonstrate reduced ethanol sensitivity without changes in glycinergic transmission; and (4) GlyRs as potential therapeutic targets., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The role of the outermost transmembrane α-helix in both the maturation and function of the prokaryotic pentameric ligand-gated ion channels, GLIC and ELIC, was examined by Ala scanning mutagenesis, deletion mutations, and mutant cycle analyses. Ala mutations at the M4-M1/M3 interface lead to loss-of-function phenotypes in GLIC, with the largest negative effects occurring near the M4 C terminus. In particular, two aromatic residues at the M4 C terminus form a network of π-π and/or cation-π interactions with residues on M3 and the β6-β7 loop that is essential for both maturation and function. M4-M1/M3 interactions appear to be optimized in GLIC with even subtle structural changes at this interface leading to detrimental effects. In contrast, mutations along the M4-M1/M3 interface of ELIC typically lead to gain-of-function phenotypes, suggesting that these interactions in ELIC are not optimized for channel function. In addition, no cluster of interacting residues involving the M4 C terminus, M3, and the β6-β7 loop was found, suggesting that the M4 C terminus plays little role in ELIC maturation or function. This study shows that M4 makes distinct contributions to the maturation and gating of these two closely related homologs, suggesting that GLIC and ELIC exhibit divergent features of channel function., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Neurotransmitter-gated ion channels of the Cys-loop receptor family are essential mediators of fast neurotransmission throughout the nervous system and are implicated in many neurological disorders. Available X-ray structures of prokaryotic and eukaryotic Cys-loop receptors provide tremendous insights into the binding of agonists, the subsequent opening of the ion channel, and the mechanism of channel activation. Yet the mechanism of inactivation by antagonists remains unknown. Here we present a 3.0 Å X-ray structure of the human glycine receptor-α3 homopentamer in complex with a high affinity, high-specificity antagonist, strychnine. Our structure allows us to explore in detail the molecular recognition of antagonists. Comparisons with previous structures reveal a mechanism for antagonist-induced inactivation of Cys-loop receptors, involving an expansion of the orthosteric binding site in the extracellular domain that is coupled to closure of the ion pore in the transmembrane domain.

Recent studies on the molecular determinants important for glycine receptor biogenesis and function mechanisms indicate an important role of basic residues within the intracellular loop between transmembrane domains (TM) 3 and 4. We investigate the role of loop length and loop exchange in combination with the presence or absence of basic stretches (318)RRKRR and (385)KKIDK of the human glycine receptor α1 using expression in transfected cell lines. Exchanges of the large intracellular loop between members of the Cys-loop receptor family have been shown to keep functionality of the host receptor. Here, constructs were generated with deletion of the intracellular loop of the glycine receptor α1, insertion of the loop from the prokaryotic Cys-loop receptor of Gloeobacter violaceus both with and without leaving the basic stretches at the N-terminal and C-terminal part of the intracellular domain. All receptor constructs were expressed at the cell surface with the significantly lowest expression of the construct with a deletion of the glycine receptor α1 TM3-4 loop, except the two basic stretches adjoined. Functionality of the inhibitory glycine receptor chimeras was demonstrated with whole cell recordings from transfected cells. Chimeras lacking the basic stretches result in non-functionality. An analysis of receptor desensitization demonstrated that close proximity of both basic stretches resulted in large fractions of desensitizing currents. We conclude that the TM3-4 loop length is critical for glycine receptor α1 desensitization and a direct neighborhood of both basic stretches changes receptor properties from non-desensitizing to desensitizing., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

We recently developed ultra-sensitive ethanol receptors (USERs) as a novel tool for investigation of single receptor subunit populations sensitized to extremely low ethanol concentrations that do not affect other receptors in the nervous system. To this end, we found that mutations within the extracellular Loop 2 region of glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs) can significantly increase receptor sensitivity to micro-molar concentrations of ethanol resulting in up to a 100-fold increase in ethanol sensitivity relative to wild-type (WT) receptors. The current study investigated: (1) Whether structural manipulations of Loop 2 in α1 GlyRs could similarly increase receptor sensitivity to other anesthetics; and (2) If mutations exclusive to the C-terminal end of Loop 2 are sufficient to impart these changes. We expressed α1 GlyR USERs in Xenopus oocytes and tested the effects of three classes of anesthetics, isoflurane (volatile), propofol (intravenous), and lidocaine (local), known to enhance glycine-induced chloride currents using two-electrode voltage clamp electrophysiology. Loop 2 mutations produced a significant 10-fold increase in isoflurane and lidocaine sensitivity, but no increase in propofol sensitivity compared to WT α1 GlyRs. Interestingly, we also found that structural manipulations in the C-terminal end of Loop 2 were sufficient and selective for α1 GlyR modulation by ethanol, isoflurane, and lidocaine. These studies are the first to report the extracellular region of α1 GlyRs as a site of lidocaine action. Overall, the findings suggest that Loop 2 of α1 GlyRs is a key region that mediates isoflurane and lidocaine modulation. Moreover, the results identify important amino acids in Loop 2 that regulate isoflurane, lidocaine, and ethanol action. Collectively, these data indicate the commonality of the sites for isoflurane, lidocaine, and ethanol action, and the structural requirements for allosteric modulation on α1 GlyRs within the extracellular Loop 2 region., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Mutagenesis and labeling studies have identified amino acids from the human α1 glycine receptor (GlyR) extracellular, transmembrane (TM), and intracellular domains in mediating ethanol (EtOH) potentiation. However, limited high-resolution structural data for physiologically relevant receptors in this Cys-loop receptor superfamily have made pinpointing the critical amino acids difficult. Homologous ion channels from lower organisms provide conserved models for structural and functional properties of Cys-loop receptors. We previously demonstrated that a single amino acid variant of the Gloeobacter violaceus ligand-gated ion channel (GLIC) produced EtOH and anesthetic sensitivity similar to that of GlyRs and provided crystallographic evidence for EtOH binding to GLIC., Methods: We directly compared EtOH modulation of the α1 GlyR and GLIC to a chimera containing the TM domain from human α1 GlyRs and the ligand-binding domain of GLIC using 2-electrode voltage-clamp electrophysiology of receptors expressed in Xenopus laevis oocytes., Results: EtOH potentiated α1 GlyRs in a concentration-dependent manner in the presence of zinc-chelating agents, but did not potentiate GLIC at pharmacologically relevant concentrations. The GLIC/GlyR chimera recapitulated the EtOH potentiation of GlyRs, without apparent sensitivity to zinc chelation. For chimera expression in oocytes, it was essential to suppress leakage current by adding 50 μM picrotoxin to the media, a technique that may have applications in expression of other ion channels., Conclusions: Our results are consistent with a TM mechanism of EtOH modulation in Cys-loop receptors. This work highlights the relevance of bacterial homologs as valuable model systems for studying ion channel function of human receptors and demonstrates the modularity of these channels across species., (Copyright © 2015 by the Research Society on Alcoholism.)

The human glycine receptors (hGlyRs) are chloride-selective ion channels that mediate inhibitory neurotransmission in the brain stem and spinal cord. They are also targets for compounds of potential use in analgesic therapies. Here, we develop a strategy to discover analgesic drugs via structure-based virtual screening based on the recently published NMR structure of the hGlyR-α1 transmembrane domain (PDB ID: 2M6I ) and the critical role of residue S296 in hGlyR-α1 potentiation by Δ(9)-tetrahydrocannabinol (THC). We screened 1549 FDA-approved drugs in the DrugBank database on an ensemble of 180 hGlyR-α1 structures generated from molecular dynamics simulations of the NMR structure of the hGlyR-α1 transmembrane domain in different lipid environments. Thirteen hit compounds from the screening were selected for functional validation in Xenopus laevis oocytes expressing hGlyR-α1. Only one compound showed no potentiation effects; seven potentiated hGlyR-α1 at a level greater than THC at 1 μM. Our virtual screening protocol is generally applicable to drug targets with lipid-facing binding sites.