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

GABA_A receptors (GABA_ARs) are pentameric ligand-gated ion channels that mediate synaptic inhibition throughout the central nervous system. The α₁β₂γ₂ receptor is the major subtype in the brain; GABA binds at the β₂(+)α₁(-) interface. The structure of the homomeric β₃ GABA_AR, 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 β₃(+)α₁(-) heteromeric interface in the homomeric human β₃ GABA_AR 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 β₃ GABA_AR variants containing Y87F, Q89R, and G152T. Reversion of Phe⁸⁷ to tyrosine abolished GABA's inhibitory effect and partially recovered direct activation by propofol. This tyrosine is conserved in homomeric GABA_ARs 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 β₃ GABA_AR are sufficient to reconstitute GABA-mediated activation and suggests that Tyr⁸⁷ prevents inhibitory effects of GABA.