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

Carla Gottschald Chiodi, Daniel Baptista-Hon, William Hunter, Tim Hales (Lead / Corresponding author)

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Abstract

GABAA receptors (GABAARs) 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 GABAAR, 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 GABAAR 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 GABAAR 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 GABAARs 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 GABAAR are sufficient to reconstitute GABAmediated activation, and suggests that Tyr87 prevents inhibitory effects of GABA.
Original languageEnglish
Pages (from-to)2375-2385
Number of pages11
JournalJournal of Biological Chemistry
Volume294
Issue number7
Early online date13 Dec 2018
DOIs
Publication statusPublished - 15 Feb 2019

Keywords

  • Amino Acid Substitution
  • Catalytic Domain
  • HEK293 Cells
  • Humans
  • Ion Channel Gating
  • Mutation, Missense
  • Pectobacterium chrysanthemi/chemistry
  • Propofol/pharmacology
  • Protein Structure, Secondary
  • Receptors, GABA-B/chemistry
  • gamma-Aminobutyric Acid/chemistry

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