Molecular Pharmacology of Gelsemium Alkaloids on Inhibitory Receptors.

Indole alkaloids are the main bioactive molecules of the Gelsemium genus plants. Diverse reports have shown the beneficial actions of Gelsemium alkaloids on the pathological states of the central nervous system (CNS). Nevertheless, Gelsemium alkaloids are toxic for mammals. To date, the molecular targets underlying the biological actions of Gelsemium alkaloids at the CNS remain poorly defined. Functional studies have determined that gelsemine is a modulator of glycine receptors (GlyRs) and GABAA receptors (GABAARs), which are ligand-gated ion channels of the CNS. The molecular and physicochemical determinants involved in the interactions between Gelsemium alkaloids and these channels are still undefined. We used electrophysiological recordings and bioinformatic approaches to determine the pharmacological profile and the molecular interactions between koumine, gelsemine, gelsevirine, and humantenmine and these ion channels. GlyRs composed of α1 subunits were inhibited by koumine and gelsevirine (IC50 of 31.5 ± 1.7 and 40.6 ± 8.2 µM, respectively), while humantenmine did not display any detectable activity. The examination of GlyRs composed of α2 and α3 subunits showed similar results. Likewise, GABAARs were inhibited by koumine and were insensitive to humantenmine. Further assays with chimeric and mutated GlyRs showed that the extracellular domain and residues within the orthosteric site were critical for the alkaloid effects, while the pharmacophore modeling revealed the physicochemical features of the alkaloids for the functional modulation. Our study provides novel information about the molecular determinants and functional actions of four major Gelsemium indole alkaloids on inhibitory receptors, expanding our knowledge regarding the interaction of these types of compounds with protein targets of the CNS.

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.

Pentameric Ligand-Gated Ion Channels as Pharmacological Targets Against Chronic Pain.

Chronic pain is a common detrimental condition that affects around 20% of the world population. The current drugs to treat chronic pain states, especially neuropathic pain, have a limited clinical efficiency and present significant adverse effects that complicates their regular use. Recent studies have proposed new therapeutic strategies focused on the pharmacological modulation of G-protein-coupled receptors, transporters, enzymes, and ion channels expressed on the nociceptive pathways. The present work intends to summarize recent advances on the pharmacological modulation of pentameric ligand-gated ion channels, which plays a key role in pain processing. Experimental data have shown that novel allosteric modulators targeting the excitatory nicotinic acetylcholine receptor, as well as the inhibitory GABAA and glycine receptors, reverse chronic pain-related behaviors in preclinical assays. Collectively, these evidences strongly suggest the pharmacological modulation of pentameric ligand-gated ion channels is a promising strategy towards the development of novel therapeutics to treat chronic pain states in humans.

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.

Large Intracellular Domain-Dependent Effects of Positive Allosteric Modulators on Glycine Receptors.

Glycine receptors (GlyRs) are members of the pentameric ligand-gated ionic channel family (pLGICs) and mediate fast inhibitory neurotransmission in the brain stem and spinal cord. The function of GlyRs can be modulated by positive allosteric modulators (PAMs). So far, it is largely accepted that both the extracellular (ECD) and transmembrane (TMD) domains constitute the primary target for many of these PAMs. On the other hand, the contribution of the intracellular domain (ICD) to the PAM effects on GlyRs remains poorly understood. To gain insight about the role of the ICD in the pharmacology of GlyRs, we examined the contribution of each domain using a chimeric receptor. Two chimeras were generated, one consisting of the ECD of the prokaryotic homologue Gloeobacter violaceus ligand-gated ion channel (GLIC) fused to the TMD of the human α1GlyR lacking the ICD (Lily) and a second with the ICD (Lily-ICD). The sensitivity to PAMs of both chimeric receptors was studied using electrophysiological techniques. The Lily receptor showed a significant decrease in the sensitivity to four recognized PAMs. Remarkably, the incorporation of the ICD into the Lily background was sufficient to restore the wild-type α1GlyR sensitivity to these PAMs. Based on these data, we can suggest that the ICD is necessary to form a pLGIC having full sensitivity to positive allosteric modulators.

Phosphorylation state-dependent modulation of spinal glycine receptors alleviates inflammatory pain.

Diminished inhibitory neurotransmission in the superficial dorsal horn of the spinal cord is thought to contribute to chronic pain. In inflammatory pain, reductions in synaptic inhibition occur partially through prostaglandin E2- (PGE2-) and PKA-dependent phosphorylation of a specific subtype of glycine receptors (GlyRs) that contain α3 subunits. Here, we demonstrated that 2,6-di-tert-butylphenol (2,6-DTBP), a nonanesthetic propofol derivative, reverses inflammation-mediated disinhibition through a specific interaction with heteromeric αßGlyRs containing phosphorylated α3 subunits. We expressed mutant GlyRs in HEK293T cells, and electrophysiological analyses of these receptors showed that 2,6-DTBP interacted with a conserved phenylalanine residue in the membrane-associated stretch between transmembrane regions 3 and 4 of the GlyR α3 subunit. In native murine spinal cord tissue, 2,6-DTBP modulated synaptic, presumably αß heteromeric, GlyRs only after priming with PGE2. This observation is consistent with results obtained from molecular modeling of the α-ß subunit interface and suggests that in α3ßGlyRs, the binding site is accessible to 2,6-DTBP only after PKA-dependent phosphorylation. In murine models of inflammatory pain, 2,6-DTBP reduced inflammatory hyperalgesia in an α3GlyR-dependent manner. Together, our data thus establish that selective potentiation of GlyR function is a promising strategy against chronic inflammatory pain and that, to our knowledge, 2,6-DTBP has a unique pharmacological profile that favors an interaction with GlyRs that have been primed by peripheral inflammation.

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.