A System for Assessing Dual Action Modulators of Glycine Transporters and Glycine Receptors.

Casey I Gallagher,Susan Shimmon,Diba Sheipouri,Tristan Rawling,Robert J Vandenberg

doi:10.3390/biom10121618

Casey I Gallagher, Susan Shimmon + Show 3 more

Open Access

https://doi.org/10.3390/biom10121618

Copy DOI

Abstract

Reduced inhibitory glycinergic neurotransmission is implicated in a number of neurological conditions such as neuropathic pain, schizophrenia, epilepsy and hyperekplexia. Restoring glycinergic signalling may be an effective method of treating these pathologies. Glycine transporters (GlyTs) control synaptic and extra-synaptic glycine concentrations and slowing the reuptake of glycine using specific GlyT inhibitors will increase glycine extracellular concentrations and increase glycine receptor (GlyR) activation. Glycinergic neurotransmission can also be improved through positive allosteric modulation (PAM) of GlyRs. Despite efforts to manipulate this synapse, no therapeutics currently target it. We propose that dual action modulators of both GlyTs and GlyRs may show greater therapeutic potential than those targeting individual proteins. To show this, we have characterized a co-expression system in Xenopus laevis oocytes consisting of GlyT1 or GlyT2 co-expressed with GlyRα1. We use two electrode voltage clamp recording techniques to measure the impact of GlyTs on GlyRs and the effects of modulators of these proteins. We show that increases in GlyT density in close proximity to GlyRs diminish receptor currents. Reductions in GlyR mediated currents are not observed when non-transportable GlyR agonists are applied or when Na+ is not available. GlyTs reduce glycine concentrations across different concentration ranges, corresponding with their ion-coupling stoichiometry, and full receptor currents can be restored when GlyTs are blocked with selective inhibitors. We show that partial inhibition of GlyT2 and modest GlyRα1 potentiation using a dual action compound, is as useful in restoring GlyR currents as a full and potent single target GlyT2 inhibitor or single target GlyRα1 PAM. The co-expression system developed in this study will provide a robust means for assessing the likely impact of GlyR PAMs and GlyT inhibitors on glycine neurotransmission.

Highlights

Glycine is an inhibitory neurotransmitter in the central nervous system
We show that a dual action lipid, N-oleoyl-glycine, targeting GlyRα1 and GlyT2 with relatively low affinity and efficacy is just as useful as single target lipids and suggest that modulating the glycinergic synapse to improve inhibitory neurotransmission without severe side-effect profiles may benefit from a multi-target approach
Human GlyT1b and GlyT2a Wild Type (WT) cDNA were subcloned into the plasmid oocyte transcription vector and human GlyRα1, GlyRα3 and GlyRβ into pGEMHE

Summary

Introduction

Glycine is an inhibitory neurotransmitter in the central nervous system. Presynaptic release of glycine from inhibitory neurons activates strychnine-sensitive glycine receptors (GlyRs), causing an influx of Cl− to hyperpolarize postsynaptic neurons [1]. There are four known GlyR α subunits (α1-α4), and one β subunit, which can assemble as α homopentamers, or as heteropentamers in a 2α:3β or 3α:2β arrangement [2]. Glycine transporters (GlyTs) control extracellular concentrations of glycine and influence the dynamics of glycinergic signaling [3]. Two subtypes have been identified in humans, GlyT1 and GlyT2 [4]. In addition to clearing glycine from the synapse, GlyT2 serves to recycle glycine for accumulation in presynaptic terminals of glycinergic neurons and subsequent exocytosis [5]

Objectives

Methods

Results

Conclusion