Expression and purification of a functional heteromeric GABAA receptor for structural studies

Derek P. Claxton, Eric Gouaux

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

The GABA-gated chloride channels of the Cys-loop receptor family, known as GABAA receptors, function as the primary gatekeepers of fast inhibitory neurotransmission in the central nervous system. Formed by the pentameric arrangement of five identical or homologous subunits, GABAA receptor subtypes are defined by the subunit composition that shape ion channel properties. An understanding of the structural basis of distinct receptor properties has been hindered by the absence of high resolution structural information for heteromeric assemblies. Robust heterologous expression and purification protocols of high expressing receptor constructs are vital for structural studies. Here, we describe a unique approach to screen for well-behaving and functional GABAA receptor subunit assemblies by using the Xenopus oocyte as an expression host in combination with fluorescence detection size exclusion chromatography (FSEC). To detect receptor expression, GFP fusions were introduced into the α1 subunit isoform. In contrast to expression of α1 alone, co-expression with the β subunit promoted formation of monodisperse assemblies. Mutagenesis experiments suggest that the α and β subunits can tolerate large truncations in the non-conserved M3/M4 cytoplasmic loop without compromising oligomeric assembly or GABA-gated channel activity, although removal of N-linked glycosylation sites is negatively correlated with expression level. Additionally, we report methods to improve GABAA receptor expression in mammalian cell culture that employ recombinant baculovirus transduction. From these methods we have identified a well-behaving minimal functional construct for the α1/β1 GABAA receptor subtype that can be purified in milligram quantities while retaining high affinity agonist binding activity.

Original languageEnglish (US)
Article numbere0201210
JournalPLoS One
Volume13
Issue number7
DOIs
StatePublished - Jul 1 2018

Fingerprint

GABA-A Receptors
Purification
receptors
gamma-Aminobutyric Acid
Cysteine Loop Ligand-Gated Ion Channel Receptors
Glycosylation
Mutagenesis
Chloride Channels
Size exclusion chromatography
Baculoviridae
Neurology
Xenopus
Ion Channels
Cell culture
Synaptic Transmission
Oocytes
Gel Chromatography
Protein Isoforms
Fusion reactions
Central Nervous System

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Expression and purification of a functional heteromeric GABAA receptor for structural studies. / Claxton, Derek P.; Gouaux, Eric.

In: PLoS One, Vol. 13, No. 7, e0201210, 01.07.2018.

Research output: Contribution to journalArticle

@article{5d757ce9c86c4a84a6d1395d13353df6,
title = "Expression and purification of a functional heteromeric GABAA receptor for structural studies",
abstract = "The GABA-gated chloride channels of the Cys-loop receptor family, known as GABAA receptors, function as the primary gatekeepers of fast inhibitory neurotransmission in the central nervous system. Formed by the pentameric arrangement of five identical or homologous subunits, GABAA receptor subtypes are defined by the subunit composition that shape ion channel properties. An understanding of the structural basis of distinct receptor properties has been hindered by the absence of high resolution structural information for heteromeric assemblies. Robust heterologous expression and purification protocols of high expressing receptor constructs are vital for structural studies. Here, we describe a unique approach to screen for well-behaving and functional GABAA receptor subunit assemblies by using the Xenopus oocyte as an expression host in combination with fluorescence detection size exclusion chromatography (FSEC). To detect receptor expression, GFP fusions were introduced into the α1 subunit isoform. In contrast to expression of α1 alone, co-expression with the β subunit promoted formation of monodisperse assemblies. Mutagenesis experiments suggest that the α and β subunits can tolerate large truncations in the non-conserved M3/M4 cytoplasmic loop without compromising oligomeric assembly or GABA-gated channel activity, although removal of N-linked glycosylation sites is negatively correlated with expression level. Additionally, we report methods to improve GABAA receptor expression in mammalian cell culture that employ recombinant baculovirus transduction. From these methods we have identified a well-behaving minimal functional construct for the α1/β1 GABAA receptor subtype that can be purified in milligram quantities while retaining high affinity agonist binding activity.",
author = "Claxton, {Derek P.} and Eric Gouaux",
year = "2018",
month = "7",
day = "1",
doi = "10.1371/journal.pone.0201210",
language = "English (US)",
volume = "13",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "7",

}

TY - JOUR

T1 - Expression and purification of a functional heteromeric GABAA receptor for structural studies

AU - Claxton, Derek P.

AU - Gouaux, Eric

PY - 2018/7/1

Y1 - 2018/7/1

N2 - The GABA-gated chloride channels of the Cys-loop receptor family, known as GABAA receptors, function as the primary gatekeepers of fast inhibitory neurotransmission in the central nervous system. Formed by the pentameric arrangement of five identical or homologous subunits, GABAA receptor subtypes are defined by the subunit composition that shape ion channel properties. An understanding of the structural basis of distinct receptor properties has been hindered by the absence of high resolution structural information for heteromeric assemblies. Robust heterologous expression and purification protocols of high expressing receptor constructs are vital for structural studies. Here, we describe a unique approach to screen for well-behaving and functional GABAA receptor subunit assemblies by using the Xenopus oocyte as an expression host in combination with fluorescence detection size exclusion chromatography (FSEC). To detect receptor expression, GFP fusions were introduced into the α1 subunit isoform. In contrast to expression of α1 alone, co-expression with the β subunit promoted formation of monodisperse assemblies. Mutagenesis experiments suggest that the α and β subunits can tolerate large truncations in the non-conserved M3/M4 cytoplasmic loop without compromising oligomeric assembly or GABA-gated channel activity, although removal of N-linked glycosylation sites is negatively correlated with expression level. Additionally, we report methods to improve GABAA receptor expression in mammalian cell culture that employ recombinant baculovirus transduction. From these methods we have identified a well-behaving minimal functional construct for the α1/β1 GABAA receptor subtype that can be purified in milligram quantities while retaining high affinity agonist binding activity.

AB - The GABA-gated chloride channels of the Cys-loop receptor family, known as GABAA receptors, function as the primary gatekeepers of fast inhibitory neurotransmission in the central nervous system. Formed by the pentameric arrangement of five identical or homologous subunits, GABAA receptor subtypes are defined by the subunit composition that shape ion channel properties. An understanding of the structural basis of distinct receptor properties has been hindered by the absence of high resolution structural information for heteromeric assemblies. Robust heterologous expression and purification protocols of high expressing receptor constructs are vital for structural studies. Here, we describe a unique approach to screen for well-behaving and functional GABAA receptor subunit assemblies by using the Xenopus oocyte as an expression host in combination with fluorescence detection size exclusion chromatography (FSEC). To detect receptor expression, GFP fusions were introduced into the α1 subunit isoform. In contrast to expression of α1 alone, co-expression with the β subunit promoted formation of monodisperse assemblies. Mutagenesis experiments suggest that the α and β subunits can tolerate large truncations in the non-conserved M3/M4 cytoplasmic loop without compromising oligomeric assembly or GABA-gated channel activity, although removal of N-linked glycosylation sites is negatively correlated with expression level. Additionally, we report methods to improve GABAA receptor expression in mammalian cell culture that employ recombinant baculovirus transduction. From these methods we have identified a well-behaving minimal functional construct for the α1/β1 GABAA receptor subtype that can be purified in milligram quantities while retaining high affinity agonist binding activity.

UR - http://www.scopus.com/inward/record.url?scp=85051444522&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85051444522&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0201210

DO - 10.1371/journal.pone.0201210

M3 - Article

VL - 13

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 7

M1 - e0201210

ER -