Glycine Receptor Subtypes and Their Roles in Nociception and Chronic Pain.

Disruption of the inhibitory control provided by the glycinergic system is one of the major mechanisms underlying chronic pain. In line with this concept, recent studies have provided robust proof that pharmacological intervention of glycine receptors (GlyRs) restores the inhibitory function and exerts anti-nociceptive effects on preclinical models of chronic pain. A targeted regulation of the glycinergic system requires the identification of the GlyR subtypes involved in chronic pain states. Nevertheless, the roles of individual GlyR subunits in nociception and in chronic pain are yet not well defined. This review aims to provide a systematic outline on the contribution of GlyR subtypes in chronic pain mechanisms, with a particular focus on molecular pathways of spinal glycinergic dis-inhibition mediated by post-translational modifications at the receptor level. The current experimental evidence has shown that phosphorylation of synaptic α1ß and α3ß GlyRs are involved in processes of spinal glycinergic dis-inhibition triggered by chronic inflammatory pain. On the other hand, the participation of α2-containing GlyRs and of ß subunits in pain signaling have been less studied and remain undefined. Although many questions in the field are still unresolved, future progress in GlyR research may soon open new exciting avenues into understanding and controlling chronic pain.

Modulation of GABAA receptors and of GABAergic synapses by the natural alkaloid gelsemine.

The Gelsemium elegans plant preparations have shown beneficial activity against common diseases, including chronic pain and anxiety. Nevertheless, their clinical uses are limited by their toxicity. Gelsemine, one of the most abundant alkaloids in the Gelsemium plants, have replicated these therapeutic and toxic actions in experimental behavioral models. However, the molecular targets underlying these biological effects remain unclear. The behavioral activity profile of gelsemine suggests the involvement of GABAA receptors (GABAARs), which are the main biological targets of benzodiazepines (BDZs), a group of drugs with anxiolytic, hypnotic, and analgesic properties. Here, we aim to define the modulation of GABAARs by gelsemine, with a special focus on the subtypes involved in the BDZ actions. The gelsemine actions were determined by electrophysiological recordings of recombinant GABAARs expressed in HEK293 cells, and of native receptors in cortical neurons. Gelsemine inhibited the agonist-evoked currents of recombinant and native receptors. The functional inhibition was not associated with the BDZ binding site. We determined in addition that gelsemine diminished the frequency of GABAergic synaptic events, likely through a presynaptic modulation. Our findings establish gelsemine as a negative modulator of GABAARs and of GABAergic synaptic function. These pharmacological features discard direct anxiolytic or analgesic actions of gelsemine through GABAARs but support a role of GABAARs on the alkaloid induced toxicity. On the other hand, the presynaptic effects of the alkaloid provide an additional mechanism to explain their beneficial effects. Collectively, our results contribute novel information to improve understanding of gelsemine actions in the mammalian nervous system.

Inhibition of the Glycine Receptor alpha 3 Function by Colchicine.

Colchicine is a plant alkaloid that is widely used as a therapeutic agent. It is widely accepted that colchicine reduces the production of inflammatory mediators mainly by altering cytoskeleton dynamics due to its microtubule polymerization inhibitory activity. However, other lines of evidence have shown that colchicine exerts direct actions on the function of ion channels, which are independent of cytoskeleton alterations. Colchicine is able to modify the function of several pentameric ligand-gated ion channels, including glycine receptors (GlyRs). Previous electrophysiological studies have shown that colchicine act as an antagonist of GlyRs composed by the α 1 subunit. In addition, it was recently demonstrated that colchicine directly bind to the α 3 subunit of GlyRs. Interestingly, other studies have shown a main role of α 3GlyRs on chronic inflammatory pain. Nevertheless, the functional effects of colchicine on the α 3GlyR function are still unknown. Here, by using electrophysiological techniques and bioinformatics, we show that colchicine inhibited the function of the α 3GlyRs. Colchicine elicited concentration-dependent inhibitory effects on α 3GlyRs at micromolar range and decreased the apparent affinity for glycine. Single-channel recordings show that the colchicine inhibition is associated with a decrease in the open probability of the ion channel. Molecular docking assays suggest that colchicine preferentially bind to the orthosteric site in the closed state of the ion channel. Altogether, our results suggest that colchicine is a competitive antagonist of the α 3GlyRs.

Modulation of glycine receptor single-channel conductance by intracellular phosphorylation.

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.

Inhibitory Actions of Tropeines on the α3 Glycine Receptor Function.

Glycine receptors (GlyRs) are chloride-permeable pentameric ligand-gated ion channels. The inhibitory activity of GlyRs is essential for many physiological processes, such as motor control and respiration. In addition, several pathological states, such as hyperekplexia, epilepsy, and chronic pain, are associated with abnormal glycinergic inhibition. Recent studies have pointed out that positive allosteric modulators targeting the GlyR α3 subunit (α3GlyR) displayed beneficial effects in chronic pain models. Interestingly, previous electrophysiological studies have shown that tropeines, which are a family of synthetic antagonists of the serotonin type 3 receptors (5-HT3Rs), potentiate the activity of GlyRs conformed by α1 subunits. However, despite its importance as a pharmacological target in chronic pain, it is currently unknown whether the α3GlyR function is modulated by tropeines. Using electrophysiological techniques and molecular docking simulations, here we show that tropeines are inhibitors of the α3GlyR function. Tropisetron, a prototypical tropeine, exerted concentration-dependent inhibitory effects on α3GlyRs at the low micromolar range. In addition, three other tropeines showed similar effects. Single-channel recordings show that tropisetron inhibition is associated with a decrease in the open probability of the ion channel. Molecular docking assays suggest that tropeines preferentially bind to an agonist-free, closed state of the ion channel. The tropeine binding occurs in a discrete pocket around the vicinity of the orthosteric site within the extracellular domain of α3GlyR. Thus, our results describe the pharmacological modulation of tropeines on α3GlyRs. These findings may contribute to the development of GlyR-selective tropeine derivatives for basic and/or clinical applications.

Functional modulation of glycine receptors by the alkaloid gelsemine.

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.