High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptors |
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Authors: | Zuzana Dostalova Aiping Liu Xiaojuan Zhou Sarah L Farmer Eileen S Krenzel Enrique Arevalo Rooma Desai Paula L Feinberg-Zadek Paul A Davies Innocent H Yamodo Stuart A Forman Keith W Miller |
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Affiliation: | 1Department of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114;2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 |
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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, GABAA 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‐HT3A) 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 [3H]muscimol to the purified GABAAR 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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Keywords: | GABAA α1β3 receptor 5‐HT3A receptor inducible expression mammalian cell lines purification functional reconstitution |
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