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

Abstract

AbstractGloeobacter violaceus ligand‐gated ion channel (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 dicarboxylate 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 dicarboxylate 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. imageKey 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 interdependent 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 for eukaryote receptors. We show that short‐chain dicarboxylate compounds are positive modulators of the Gloeobacter violaceus ligand‐gated ion channel (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.