Dataset Information

Novel mechanism of modulation at a ligand-gated ion channel; action of 5-Cl-indole at the 5-HT₃ A receptor.

ABSTRACT:

Background and purpose

The 5-HT₃ receptor is a prototypical member of the Cys-loop ligand-gated ion channel (LGIC) superfamily and an established therapeutic target. In addition to activation via the orthosteric site, receptor function can be modulated by allosteric ligands. We have investigated the pharmacological action of Cl-indole upon the 5-HT₃ A receptor and identified that this positive allosteric modulator possesses a novel mechanism of action for LGICs.

Experimental approach

The impact of Cl-indole upon the 5-HT₃ receptor was assessed using single cell electrophysiological recordings and [³ H]-granisetron binding in HEK293 cells stably expressing the 5-HT₃ receptor.

Key results

Cl-indole failed to evoke 5-HT₃ A receptor-mediated responses (up to 30 ?M) or display affinity for the [³ H]-granisetron binding site. However, in the presence of Cl-indole, termination of 5-HT application revealed tail currents mediated via the 5-HT₃ A receptor that were independent of the preceding 5-HT concentration but were antagonized by the 5-HT₃ receptor antagonist, ondansetron. These tail currents were absent in the 5-HT₃ AB receptor. Furthermore, the presence of 5-HT revealed a concentration-dependent increase in the affinity of Cl-indole for the orthosteric binding site of the human 5-HT₃ A receptor.

Conclusions and implications

Cl-indole acts as both an orthosteric agonist and an allosteric modulator, but the presence of an orthosteric agonist (e.g. 5-HT) is a prerequisite to reveal both actions. Precedent for ago-allosteric action is available, yet the essential additional presence of an orthosteric agonist is now reported for the first time. This widening of the pharmacological mechanisms to modulate LGICs may offer further therapeutic opportunities.

SUBMITTER: Powell AD

PROVIDER: S-EPMC5120152 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Novel mechanism of modulation at a ligand-gated ion channel; action of 5-Cl-indole at the 5-HT<sub>3</sub> A receptor.

Powell Andrew D AD Grafton Gillian G Roberts Alexander A Larkin Shannon S O'Neill Nathanael N Palandri Josephine J Otvos Reka R Cooper Alison J AJ Ulens Chris C Barnes Nicholas M NM

British journal of pharmacology 20161101 24

<h4>Background and purpose</h4>The 5-HT<sub>3</sub> receptor is a prototypical member of the Cys-loop ligand-gated ion channel (LGIC) superfamily and an established therapeutic target. In addition to activation via the orthosteric site, receptor function can be modulated by allosteric ligands. We have investigated the pharmacological action of Cl-indole upon the 5-HT<sub>3</sub> A receptor and identified that this positive allosteric modulator possesses a novel mechanism of action for LGICs.<h4> ...[more]

PMID: 27677804

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Project description:The superfamily of pentameric ligand-gated ion channels includes neurotransmitter receptors that mediate fast synaptic transmission in vertebrates, and are targets for drugs including alcohols, anesthetics, benzodiazepines, and anticonvulsants. However, the mechanisms of ion channel opening, gating, and modulation in these receptors leave many open questions, despite their pharmacological importance. Subtle conformational changes in both the extracellular and transmembrane domains are likely to influence channel opening, but have been difficult to characterize given the limited structural data available for human membrane proteins. Recent crystal structures of a modified Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in multiple states offer an appealing model system for structure-function studies. However, the pharmacology of the crystallographic GluCl construct is not well established. To establish the functional relevance of this system, we used two-electrode voltage-clamp electrophysiology in Xenopus oocytes to characterize activation of crystallographic and native-like GluCl constructs by L-glutamate and ivermectin. We also tested modulation by ethanol and other anesthetic agents, and used site-directed mutagenesis to explore the role of a region of Loop F which was implicated in ligand gating by molecular dynamics simulations. Our findings indicate that the crystallographic construct functionally models concentration-dependent agonism and allosteric modulation of pharmacologically relevant receptors. Specific substitutions at residue Leu174 in loop F altered direct L-glutamate activation, consistent with computational evidence for this region's role in ligand binding. These insights demonstrate conservation of activation and modulation properties in this receptor family, and establish a framework for GluCl as a model system, including new possibilities for drug discovery. In this study, we elucidate the validity of a modified glutamate-gated chloride channel (GluClcryst ) as a structurally accessible model for GABAA receptors. In contrast to native-like controls, GluClcryst exhibits classical activation by its neurotransmitter ligand L-glutamate. The modified channel is also sensitive to allosteric modulators associated with human GABAA receptors, and to site-directed mutations predicted to alter channel opening.

Dataset Information

Novel mechanism of modulation at a ligand-gated ion channel; action of 5-Cl-indole at the 5-HT₃ A receptor.

Background and purpose

Experimental approach

Key results

Conclusions and implications

Publications

Novel mechanism of modulation at a ligand-gated ion channel; action of 5-Cl-indole at the 5-HT<sub>3</sub> A receptor.

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Dataset Information

Novel mechanism of modulation at a ligand-gated ion channel; action of 5-Cl-indole at the 5-HT3 A receptor.

Background and purpose

Experimental approach

Key results

Conclusions and implications

Publications

Novel mechanism of modulation at a ligand-gated ion channel; action of 5-Cl-indole at the 5-HT<sub>3</sub> A receptor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Novel mechanism of modulation at a ligand-gated ion channel; action of 5-Cl-indole at the 5-HT₃ A receptor.