Binding-site mutations in the α1 subunit of the inhibitory glycine receptor convert the inhibitory metal ion Cu into a positive modulator

The divalent cation copper (Cu²⁺) has been shown to inhibit chloride currents mediated by the inhibitory glycine receptor (GlyR). Here, we analyzed Cu²⁺ inhibition of homo- and hetero-oligomeric GlyRs expressed in Xenopus oocytes. No significant differences in Cu²⁺ inhibitory potency were found between α1, α2 and α3 GlyRs as well as heteromeric α1β receptors. Furthermore, GlyR α1 mutations known to reduce inhibition or potentiation of GlyR currents by Zn²⁺ had no effect on Cu²⁺ inhibition. However, Cu²⁺ was found to competitively antagonize glycine binding, suggesting that Cu²⁺ binds at the agonist-binding site. Mutations within the glycine-binding site of the GlyR α1 subunit reduced the inhibitory potency of Cu²⁺ and led to an up to 4-fold potentiation of glycine-elicited currents by Cu²⁺. Molecular dynamics simulation suggests this to be due to increased Cu²⁺ binding energies. Our data show that GlyR binding-site mutations can convert inhibitors of agonist binding into highly effective positive modulators.     

Glycine and glycine receptor signaling in hippocampal neurons: Diversity,function and regulation

Glycine is a primary inhibitory neurotransmitter in the spinal cord and brainstem. It acts at glycine receptor (GlyR)-chloride channels, as well as a co-agonist of NMDA receptors (NMDARs). In the hippocampus, the study of GlyRs has largely been under-appreciated due to the apparent absence of glycinergic synaptic transmission. Emerging evidence has shown the presence of extrasynaptic GlyRs in the hippocampus, which exert a tonic inhibitory role, and can be highly regulated under many pathophysiological conditions. On the other hand, besides d-serine, glycine has also been shown to modulate NMDAR function in the hippocampus. The simultaneous activation of excitatory NMDARs and inhibitory GlyRs may provide a homeostatic regulation of hippocampal network function. Furthermore, glycine can regulate hippocampal neuronal activity through GlyR-mediated cross-inhibition of GABAergic inhibition, or through the glycine binding site-dependent internalization of NMDARs. Therefore, hippocampal glycine and its receptors may operate in concert to finely regulate hippocampus-dependent high brain function such as learning and memory. Finally, dysfunction of hippocampal glycine signaling is associated with neuropsychiatric disorders. We speculate that further studies of hippocampal glycine-mediated regulation may help develop novel glycine-based approaches for therapeutic developments.     

Hyperekplexia mutation of glycine receptors: decreased gating efficacy with altered binding thermodynamics

[(3)H]Strychnine binding was studied to recombinant human alpha(1) and the hyperekplexia mutant alpha(1)R271L glycine receptors (GlyRs) transiently expressed in human embryonic kidney (HEK)-293 cell cultures at 0, 18 and 37 degrees. The alpha(1)R271L mutation did not affect the linear van't Hoff plots of the exothermic binding of the antagonist [3H]strychnine while it turned taurine into an antagonist with exothermic binding. The inhibition constants of the agonist glycine showed opposite temperature dependence on alpha(1) GlyRs, corresponding to endothermic binding driven by large entropic increases. The temperature dependence of displacement by the partial agonists taurine on alpha(1) GlyRs and glycine on alpha(1)R271L GlyRs was biphasic reflecting negative heat capacity changes, dehydration changes and/or a complex binding mechanism. The thermodynamic discrimination of efficacy is valid for native rat spinal and recombinant human GlyRs. The alpha(1)R271L mutation impairs the transduction mechanism and distorts gating of GlyRs. Thereby it reduces the potency and efficacy of agonists and affects their thermodynamic parameters of binding. The hyperekplexia mutation offers a model system to demonstrate the correlation among pathophysiology, gating efficacy and binding thermodynamics of GlyRs.     

The beta subunit increases the ginkgolide B sensitivity of inhibitory glycine receptors

We investigated the effect of ginkgolide B (GB), a component of the extract from the leaves of the Ginkgo biloba tree, on recombinant glycine receptors (GlyRs) expressed in Xenopus oocytes by using voltage-clamp recording. GB (0.01-10 microM) inhibited glycine-induced currents of homo-oligomeric alpha1, alpha2 and alpha 3 GlyRs, with the highest potency being found at the alpha1 GlyR (IC(50) value=0.61+/-0.1 microM). Coexpression of the alpha subunits with the beta subunit resulted in a shift of the IC(50) value of GB to nanomolar values, indicating selectivity of GB for beta subunit containing GlyRs. We also analyzed the mechanism of GB inhibition and the effect of point mutations introduced into the alpha1 subunit. Our results are consistent with a channel blocking effect, since (i) GB inhibited glycine currents non-competitively, and (ii) a point mutation in the pore forming M2 domain reduced GB potency. In conclusion, GB is a potent blocker of beta subunit containing GlyR channels and hence can be used to discriminate homo- from hetero-oligomeric GlyRs. As hetero-oligomeric GlyRs are known to be synaptically localized, GB represents a channel blocker that may be employed to separate extrasynaptic from synaptic glycine currents.     

Glycine transporter-1 inhibitors: a patent review (2011–2016)

Introduction: Numerous research groups have developed GlyT-1 inhibitors in the pursuit of providing a novel antipsychotic treatment for schizophrenia. Despite multiple compounds advancing into clinical trials, a GlyT-1 inhibitor has yet to emerge to treat patients. However, the approach remains heavily investigated as it presents potential therapeutic utility for several other CNS and non-CNS-related indications.

Areas covered: This review discusses various GlyT-1 inhibitor chemotypes identified and provides an overview of patent applications filed and published during the period of 2011–2016. The review largely focuses on composition of matter patent applications, although two recently disclosed method of use patents are discussed. Clinical reports are also disseminated.

Expert opinion: Mounting clinical failures with schizophrenic patients have blunted enthusiasm for GlyT-1 inhibition as an approach to treat the disease. However, research in the area remains quite active, as therapeutic potential for several additional indications has emerged. There are numerous and diverse GlyT-1 chemotypes now available that exhibit differentiating modes of binding and ligand-target binding kinetics, and this rich diversity of chemical matter may help further elucidate the target’s pharmacological role in various indications and lead to the identification of a compound with optimal properties that may someday become a drug.     

Gelsemine,a principal alkaloid from Gelsemium sempervirens Ait., exhibits potent and specific antinociception in chronic pain by acting at spinal α3 glycine receptors

The present study examined the antinociceptive effects of gelsemine, the principal alkaloid in Gelsemium sempervirens Ait. A single intrathecal injection of gelsemine produced potent and specific antinociception in formalin-induced tonic pain, bone cancer-induced mechanical allodynia, and spinal nerve ligation-induced painful neuropathy. The antinociception was dose-dependent, with maximal inhibition of 50% to 60% and ED₅₀ values of 0.5 to 0.6 μg. Multiple daily intrathecal injections of gelsemine for 7 days induced no tolerance to antinociception in the rat model of bone cancer pain. Spinal gelsemine was not effective in altering contralateral paw withdrawal thresholds, and had only a slight inhibitory effect on formalin-induced acute nociception. The specific antinociception of gelsemine in chronic pain was blocked dose-dependently by the glycine receptor (GlyR) antagonist strychnine with an apparent ID₅₀ value of 3.8 μg. Gelsemine concentration-dependently displaced H³-strychnine binding to the membrane fraction of rat spinal cord homogenates, with a 100% displacement and a K_i of 21.9 μM. Gene ablation of the GlyR α3 subunit (α3 GlyR) but not α1 GlyR, by a 7-day intrathecal injection of small interfering RNA (siRNA) targeting α3 GlyR or α1 GlyR, nearly completely prevented gelsemine-induced antinociception in neuropathic pain. Our results demonstrate that gelsemine produces potent and specific antinociception in chronic pain states without induction of apparent tolerance. The results also suggest that gelsemine produces antinociception by activation of spinal α3 glycine receptors, and support the notion that spinal α3 glycine receptors are a potential therapeutic target molecule for the management of chronic pain.     

The biological role of the glycinergic synapse in early zebrafish motility

Glycine mediates fast inhibitory neurotransmission in the spinal cord, brainstem and retina. Loss of synaptic glycinergic transmission in vertebrates leads to a severe locomotion defect characterized by an exaggerated startle response accompanied by transient muscle rigidity in response to sudden acoustic or tactile stimuli. Several molecular components of the glycinergic synapse have been characterized as an outcome of genetic and physiological analyses of synaptogenesis in mammals. Recently, the glycinergic synapse has been studied using a forward genetic approach in zebrafish. This review aims to discuss molecular components of the glycinergic synapse, such as glycine receptor subunits, gephyrin, gephyrin-binding proteins and glycine transporters, as well as recent studies relevant to the genetic analysis of the glycinergic synapse in zebrafish.     

Emerging themes in GABAergic synapse development

Glutamatergic synapse development has been rigorously investigated for the past two decades at both the molecular and cell biological level yet a comparable intensity of investigation into the cellular and molecular mechanisms of GABAergic synapse development has been lacking until relatively recently. This review will provide a detailed overview of the current understanding of GABAergic synapse development with a particular emphasis on assembly of synaptic components, molecular mechanisms of synaptic development, and a subset of human disorders which manifest when GABAergic synapse development is disrupted. An unexpected and emerging theme from these studies is that glutamatergic and GABAergic synapse development share a number of overlapping molecular and cell biological mechanisms that will be emphasized in this review.     

What we have learned from crystal structures of proteins to receptor function

The activity of ligand gated channels is crucial for proper brain function and dysfunction of a single receptor subtype have led to neurological impairments ranging from benign to major diseases such as epilepsy, startle diseases, etc. Molecular biology and crystallography allowed the characterization at the atomic scale of the first four transmembrane ligand gated channels and of proteins sharing a high degree of homology with the neurotransmitter-binding domain.Gaining an adequate knowledge of the structural features of the ligand binding pocket led to the possibilities of developing virtual screening based approaches and probing in silico the docking of very large numbers of molecules. Development of new computing tools further extended such possibilities and rendered possible the screening of the chemical universe database GDB-11, which contains all possible organic molecules up to 11 atoms of C, N, O and F. In the case of the nicotinic acetylcholine receptors molecules identified using such screening methods were synthesized and characterized in binding assays and their pose determined in crystal structure with the acetylcholine binding protein. However, in spite of these thorough approaches, functional studies revealed that these molecules had a greater affinity for the pore domain of the channel and acted as open channel blocker rather than binding site antagonist.In this work, we discuss the potential and current limitations of how progresses made with the crystal structures of ligand gated channels, or ligand binding proteins, can be used in combination with virtual screening and functional assays, to identify novel compounds.     

Associated proteins: The universal toolbox controlling ligand gated ion channel function

Ligand gated ion channels are integral multimeric membrane proteins that can detect with high sensitivity the presence of a specific transmitter in the extracellular space and transduce this signal into an ion flux. While these receptors are widely expressed in the nervous system, their expression is not limited to neurons or their postsynaptic targets but extends to non-neuronal cells where they participate in many physiological responses. Cells have developed complex regulatory mechanisms allowing for the precise control and modulation of ligand gated ion channels. In this overview the roles of accessory subunits and associated proteins in these regulatory mechanisms are reviewed and their relevance illustrated by examples at different ligand gated ion channel types, with emphasis on nicotinic acetylcholine receptors. Dysfunction of ligand gated ion channels can result in neuromuscular, neurological or psychiatric disorders. A better understanding of the precise function of associated proteins and how they impact on ligand gated ion channels will provide new therapeutic opportunities for clinical intervention.