Amino acid substitutions in the human homomeric β3 GABAA receptor that enable activation by GABA

Abstract

Edited by Roger J. Colbran GABA A receptors (GABA A Rs) are pentameric ligand-gated ion channels that mediate synaptic inhibition throughout the central nervous system. The ␣ 1 ␤ 2 ␥ 2 receptor is the major subtype in the brain; GABA binds at the ␤ 2 (؉)␣ 1 (؊) interface. The structure of the homomeric ␤ 3 GABA A R, which is not activated by GABA, has been solved. Recently, four additional heteromeric structures were reported, highlighting key residues required for agonist binding. Here, we used a protein engineering method, taking advantage of knowledge of the key binding residues, to create a ␤ 3 (؉)␣ 1 (؊) heteromeric interface in the homomeric human ␤ 3 GABA A R that enables GABA-mediated activation. Substitutions were made in the complementary side of the orthosteric binding site in loop D (Y87F and Q89R), loop E (G152T), and loop G (N66D and A70T). The Q89R and G152T combination enabled low-potency activation by GABA and potentiation by propofol but impaired direct activation by higher propofol concentrations. At higher concentrations, GABA inhibited gating of ␤ 3 GABA A R variants containing Y87F, Q89R, and G152T. Reversion of Phe 87 to tyrosine abolished GABA's inhibitory effect and partially recovered direct activation by propofol. This tyrosine is conserved in homomeric GABA A Rs and in the Erwinia chrysanthemi ligand-gated ion channel and may be essential for the absence of an inhibitory effect of GABA on homomeric channels. This work demonstrated that only two substitutions, Q89R and G152T, in ␤ 3 GABA A R are sufficient to reconstitute GABA-mediated activation and suggests that Tyr 87 prevents inhibitory effects of GABA.

GABAA Receptor β2E155 Residue Located at the Agonist-Binding Site Is Involved in the Receptor Gating

“…It is known that β subunits can form the spontaneously active (i.e., without GABA) homomers (Blair et al, 1988;Cestari et al, 1996;Wooltorton et al, 1997), which could contribute to the observed spontaneous activity. However, it needs to be stressed that functional homomers were demonstrated for the β 3 (Connolly et al, 1996;Davies et al, 1997;Wooltorton et al, 1997;Gottschald Chiodi et al, 2019) and β 1 (Sigel et al, 1989;Sanna et al, 1995;Krishek et al, 1996;Miko et al, 2004) subunits, whereas in the present study, β 2 subunits were expressed. Moreover, the expression level of putative β subunit homomers is expected to be the same in the case of WT and the mutants, and the spontaneous activity in the case of the WT receptors was negligible indicating that contribution from the β homomers is low if any.…”

Integrated workflow for the identification of new GABAAR positive allosteric modulators based on the in silico screening with further in vitro validation. Case study using Enamine's stock chemical space

“…An estimation of the binding free energy (scoring function) of the protein‐ligand complex was conducted, along with a comparison of the number of H‐bonds formed between protein and ligand atoms. The quality of the final model was assessed using ICM Molsoft standard docking procedure against non‐benzodiazepine compounds [50, 51]. Since our search algorithm was adapted based on Z‐drugs, we proceeded to screen another segment of the reference set, comprising 90 active compounds (<1 μM) from the ChemblDB, through the docking model to estimate the degree of selectivity [52].…”

Gamma-Aminobutyric Acid Signaling in Damage Response, Metabolism, and Disease

“…Since long-term imbalances in these metabolites can lead to brain diseases [5], an understanding of the regulation of GABA uptake is necessary. The GABA-A receptor is a quintessential ligand-gated ion channel that mediates synaptic inhibition throughout the central nervous system [6]. After GABA performs its action as a neurotransmitter, it is reabsorbed into the presynaptic neurons and neural bridges [7].…”