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

GABA A receptors (GABA A Rs) are pentameric ligand-gated ion channels that mediate synaptic inhibition throughout the central nervous system. The α 1 β 2 γ 2 receptor is the major subtype in the brain; GABA binds at the β 2 (+)α 1 (−) interface. The structure of the homomeric β 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 β 3 (+)α 1 (−) heteromeric interface in the homomeric human β 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 β 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 β 3 GABA A R are sufficient to reconstitute GABA-mediated activation and suggests that Tyr 87 prevents inhibitory effects of GABA.