ArticleProtein science : a publication of the Protein Society2010
High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptors.
Article in Protein science : a publication of the Protein Society, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 28 papers.
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Who cites it
28 citing papers in PubMed, 44 citations in OpenAlex.
- Chimeric Approach to Identify Molecular Determinants of Nicotinic Acetylcholine Receptors.International journal of molecular sciences · 2026Review
- Druggable Lipid Binding Sites in Pentameric Ligand-Gated Ion Channels and Transient Receptor Potential Channels.Frontiers in physiology · 2021Review
- Thermostability of a recombinant G protein-coupled receptor expressed at high level in mammalian cell culture.Scientific reports · 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
- Inhibitable photolabeling by neurosteroid diazirine analog in the β3-Subunit of human hetereopentameric type A GABA receptors.European journal of medicinal chemistry · 2019Article
- Cryo-EM structure of the human α1β3γ2 GABANature · 2019Article
- Etomidate and Etomidate Analog Binding and Positive Modulation of γ-Aminobutyric Acid Type A Receptors: Evidence for a State-dependent Cutoff Effect.Anesthesiology · 2018Article
- Identification of binding sites contributing to volatile anesthetic effects on GABA type A receptors.FASEB journal : official publication of the Federation of American Societies for Experimental Biology · 2018Article
- Expression and purification of a functional heteromeric GABAA receptor for structural studies.PloS one · 2018Article
- Article
- Competitive Antagonism of Anesthetic Action at the γ-Aminobutyric Acid Type A Receptor by a Novel Etomidate Analog with Low Intrinsic Efficacy.Anesthesiology · 2017Article
- Mapping General Anesthetic Sites in Heteromeric γ-Aminobutyric Acid Type A Receptors Reveals a Potential For Targeting Receptor Subtypes.Anesthesia and analgesia · 2016Review
- Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β- Interface.The Journal of biological chemistry · 2015Article
- MiRNA mimic screen for improved expression of functional neurotensin receptor from HEK293 cells.Biotechnology and bioengineering · 2015Article
- Multiple propofol-binding sites in a γ-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog.The Journal of biological chemistry · 2014Article
- Engineering cells to improve protein expression.Current opinion in structural biology · 2014Review
- Structural models of ligand-gated ion channels: sites of action for anesthetics and ethanol.Alcoholism, clinical and experimental research · 2014Review
- Human α1β3γ2L gamma-aminobutyric acid type A receptors: High-level production and purification in a functional state.Protein science : a publication of the Protein Society · 2014Article
- Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid type A (GABAA) receptor.The Journal of biological chemistry · 2013Article
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Authors and funding
12 authors at 2 institutions in 1 country.
Funding
Abstract
The human neuronal Cys-loop ligand-gated ion channel superfamily of ion channels are important determinants of human behavior and the target of many drugs. It is essential for their structural characterization to achieve high-level expression in a functional state. The aim of this work was to establish stable mammalian cell lines that enable high-level heterologous production of pure receptors in a state that supports agonist-induced allosteric conformational changes. In a tetracycline-inducible stable human embryonic kidney cells (HEK293S) cell line, GABA(A) receptors containing α1 and β3 subunits could be expressed with specific activities of 29-34 pmol/mg corresponding to 140-170 pmol/plate, the highest expression level reported so far. Comparable figures for serotonin (5-HT(3A)) receptors were 49-63 pmol/mg and 245-315 pmol/plate. The expression of 10 nmol of either receptor in suspension in a bioreactor required 0.3-3.0 L. Both receptor constructs had a FLAG epitope inserted at the N-terminus and could be purified in one step after solubilization using ANTI-FLAG affinity chromatography with yields of 30-40%. Purified receptors were functional. Binding of the agonist [(3)H]muscimol to the purified GABA(A)R was enhanced allosterically by the general anesthetic etomidate, and purified 5-hydroxytryptamine-3A receptor supported serotonin-stimulated cation flux when reconstituted into lipid vesicles.
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