ArticleProtein science : a publication of the Protein Society2014
Human α1β3γ2L gamma-aminobutyric acid type A receptors: High-level production and purification in a functional state.
Article in Protein science : a publication of the Protein Society, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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Who cites it
30 citing papers in PubMed, 37 citations in OpenAlex.
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- Mechanism of NACHO-mediated assembly of pentameric ligand-gated ion channels.bioRxiv : the preprint server for biology · 2024Article
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- EMC rectifies the topology of multipass membrane proteins.Nature structural & molecular biology · 2024Article
- Differential assembly diversifies GABANature · 2022Article
- Electrophysiology of ionotropic GABA receptors.Cellular and molecular life sciences : CMLS · 2021Review
- Druggable Lipid Binding Sites in Pentameric Ligand-Gated Ion Channels and Transient Receptor Potential Channels.Frontiers in physiology · 2021Review
- Enhancement of Muscimol Binding and Gating by Allosteric Modulators of the GABAMolecular pharmacology · 2020Article
- Site-specific effects of neurosteroids on GABAeLife · 2020Article
- Competitive Antagonism of Etomidate Action by Diazepam: In Vitro GABAA Receptor and In Vivo Zebrafish Studies.Anesthesiology · 2020Article
- Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABAThe Journal of biological chemistry · 2020Article
- A potent photoreactive general anesthetic with novel binding site selectivity for GABAEuropean journal of medicinal chemistry · 2020Article
- Cryo-electron microscopy reveals informative details of GABAAnnals of translational medicine · 2019Article
- Identifying Drugs that Bind Selectively to Intersubunit General Anesthetic Sites in theMolecular pharmacology · 2019Article
- Boundary lipids of the nicotinic acetylcholine receptor: Spontaneous partitioning via coarse-grained molecular dynamics simulation.Biochimica et biophysica acta. Biomembranes · 2019Article
- Inhibitable photolabeling by neurosteroid diazirine analog in the β3-Subunit of human hetereopentameric type A GABA receptors.European journal of medicinal chemistry · 2019Article
- GABANature · 2019Article
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Authors and funding
8 authors at 2 institutions in 1 country.
Funding
Abstract
Gamma-aminobutyric acid type A receptors (GABA(A)Rs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA(A)Rs determine their function and pharmacological profile. GABAA Rs are heteropentamers of subunits, and (α1)2 (β3)2 (γ2L)1 is a common subtype. Biochemical and biophysical studies of GABA(A)Rs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high-level production of active human α1β3 GABA(A)R using tetracycline-inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline-inducible HEK293-TetR cell line expressing human (N)-FLAG-α1β3γ2L-(C)-(GGS)3 GK-1D4 GABA(A)R. These cells achieved expression levels of 70-90 pmol [(3)H]muscimol binding sites/15-cm plate at a specific activity of 15-30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [(3)H]flunitrazepam to [(3)H]muscimol binding sites and sensitivity of GABA-induced currents to benzodiazepines and zinc. The α1β3γ2L GABA(A)Rs were solubilized in dodecyl-D-maltoside, purified by anti-FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ∼ 30%. Typical purifications yielded 1.0-1.5 nmoles of [(3)H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [(3)H]muscimol binding were maintained in the purified state.
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