Fumarate as positive modulator of allosteric transitions in the pentameric ligand-gated ion channel GLIC: Requirement of an intact vestibular pocket.

GLIC is a prokaryotic orthologue of brain pentameric neurotransmitter receptors. Using whole-cell patch-clamp electrophysiology in a host cell line, we show that short-chain di-carboxylate compounds are positive modulators of pHo 5-evoked GLIC activity, with a rank order of action fumarate > succinate > malonate > glutarate. Potentiation by fumarate depends on intracellular pH, mainly as a result of a strong decrease of the pHo 5-evoked current when intracellular pH decreases. The modulating effect of fumarate also depends on extracellular pH, as fumarate is a weak inhibitor at pHo 6 and shows no agonist action at neutral pHo. A mutational analysis of residue-dependency for succinate and fumarate effects, based on two carboxylate-binding pockets previously identified by crystallography (Fourati et al. 2020), shows that positive modulation involves both the inter-subunit pocket, homologous to the neurotransmitter-binding orthotopic site, and the intra-subunit (also called vestibular) pocket. An almost similar pattern of mutational impact is observed for the effect of caffeate, a known negative modulator. We propose, for both di-carboxylate compounds and caffeate, a model where the inter-subunit pocket is the actual binding site, and the region corresponding to the vestibular pocket is required either for inter-subunit binding itself, or for binding-to-gating coupling during the allosteric transitions involved in pore gating modulation. KEY POINTS: Using a bacterial orthologue of brain pentameric neurotransmitter receptors, we show that the orthotopic/orthosteric agonist site and the adjacent vestibular region are functionally inter-dependent in mediating compound-elicited modulation. We propose that the two sites in the extracellular domain are involved "in series", a mechanism which may have relevance to Eukaryote receptors. We show that short-chain di-carboxylate compounds are positive modulators of GLIC. The most potent compound identified is fumarate, known to occupy the orthotopic/orthosteric site in previously published crystal structures. We show that intracellular pH modulates GLIC allosteric transitions, as previously known for extracellular pH. We report a caesium to sodium permeability ratio (PCs/PNa) of 0.54 for GLIC ion pore. Abstract figure legends We show that a low-pH intracellular solution negatively modulates allosteric transitions (see Legend to Fig. 3E for details), in the Prokaryote pentameric ligand-gated ion channel (pLGIC) GLIC, known to be activated by the agonist action of low-pH extracellular solutions. We use this property in the characterization of fumarate, and other compounds, as positive modulators of allosteric transitions. Data from a mutational analysis in two carboxylate binding pockets, together with published crystallographic data, shows that the modulator action of compounds binding at the conserved orthotopic site (homologous to the neurotransmitter / agonist binding site in brain pLGICs) is under the strict control of the adjacent intra-subunit vestibular region. We propose that both positive and negative modulators of allosteric transitions act by binding to the orthotopic main binding site. The 2-site "in series" mechanism proposed may have some relevance to Eukaryote pLGICs. This article is protected by copyright. All rights reserved.