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)

Research output: Contribution to journalArticle

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

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GABA-A Receptors
Amino Acid Substitution
gamma-Aminobutyric Acid
Substitution reactions
Chemical activation
Amino Acids
Propofol
Ligand-Gated Ion Channels
Tyrosine
Pectobacterium chrysanthemi
GABA Agents
Protein Engineering
Neurology
Central Nervous System
Binding Sites
Brain

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

Cite this

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title = "Amino acid substitutions in the human homomeric β3 GABAA receptor that enable activation by GABA",
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.",
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",
author = "{Gottschald Chiodi}, Carla and Daniel Baptista-Hon and William Hunter and Tim Hales",
note = "CGC thanks Capes Science without Borders scheme (BEX 0321/13-3) for funding.",
year = "2019",
month = "2",
day = "15",
doi = "10.1074/jbc.RA118.006229",
language = "English",
volume = "294",
pages = "2375--2385",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology",
number = "7",

}

TY - JOUR

T1 - Amino acid substitutions in the human homomeric β3 GABAA receptor that enable activation by GABA

AU - Gottschald Chiodi, Carla

AU - Baptista-Hon, Daniel

AU - Hunter, William

AU - Hales, Tim

N1 - CGC thanks Capes Science without Borders scheme (BEX 0321/13-3) for funding.

PY - 2019/2/15

Y1 - 2019/2/15

N2 - 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.

AB - 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.

KW - Amino Acid Substitution

KW - Catalytic Domain

KW - HEK293 Cells

KW - Humans

KW - Ion Channel Gating

KW - Mutation, Missense

KW - Pectobacterium chrysanthemi/chemistry

KW - Propofol/pharmacology

KW - Protein Structure, Secondary

KW - Receptors, GABA-B/chemistry

KW - gamma-Aminobutyric Acid/chemistry

UR - http://www.scopus.com/inward/record.url?scp=85061581158&partnerID=8YFLogxK

U2 - 10.1074/jbc.RA118.006229

DO - 10.1074/jbc.RA118.006229

M3 - Article

VL - 294

SP - 2375

EP - 2385

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 7

ER -