Short-chain di-carboxylates as positive allosteric modulators of the pH-dependent 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][1]), 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 summary ### Competing Interest Statement The authors have declared no competing interest. * ASIC : acid-sensing ion channel BAPTA : 1,2-bis( o -aminophenoxy)ethane- N , N , N’ , N’ -tetraacetic acid BHK : baby hamster kidney CAFFE : caffeic acid/caffeate CBX : carboxylic acid/carboxylate DCM : dichloromethane dFBS : heat-inactivated fetal bovine serum ECD : extracellular domain ESI : electrospray ionization FUMAR : fumaric acid/fumarate-/fumarate2- GABA : gamma -aminobutyric acid β3GABAAR : β3 type A GABA receptor GLIC : Gloeobacter violaceus ligand-gated ion channel α1GlyR : alpha 1 glycine receptor HEPES : 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid inter-SU : inter-subunit HPLC : high performance liquid chromatography HRMS : high resolution mass spectrometry Imax : active current extremum value (with largest absolute value) in a concentration-effect plot Ipk : peak current active current extremum value (with largest absolute value) in a segment of recording time MALE : maleic acid/maleate-/maleate2- MES : 2-( N -morpholino) ethanesulfonic acid mM : mmol/L NAM : negative modulator of the allosteric transitions NMDG : N -methyl-D-glucamine NMR : nuclear magnetic resonance OAA : oxaloacetic acid/oxaloacetate-/oxaloacetate2- PDB : Protein Data Bank PAM : positive modulator of the allosteric transitions pHi : pH of solution at the intracellular face of the cell membrane pHo : pH of solution at the extracellular face of the cell membrane pHo50 : extracellular solution pH value producing half of the maximal activation reached using low extracellular pH pHpip : pH of the solution used to fill the recording pipette pLGIC : pentameric ligand-gated ion channel SUCCIN : succinic acid-/succinate-/succinate2- SU : subunit TFA : trifluoroacetic acid. [1]: #ref-8