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 alpha(1)beta(2)gamma(2) receptor is the major subtype in the brain; GABA binds at the beta(2)(+)alpha(1)(-) interface. The structure of the homomeric beta(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 beta(3)(+)alpha(1)(-) heteromeric interface in the homomeric human beta(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 beta(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 beta(3) GABA(A)R are sufficient to reconstitute GABA-mediated activation and suggests that Tyr(87) prevents inhibitory effects of GABA.