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High‐level expression and purification of Cys‐loop ligand‐gated ion channels in a tetracycline‐inducible stable mammalian cell line: GABA A and serotonin receptors
Author(s) -
Dostalova Zuzana,
Liu Aiping,
Zhou Xiaojuan,
Farmer Sarah L.,
Krenzel Eileen S.,
Arevalo Enrique,
Desai Rooma,
FeinbergZadek Paula L.,
Davies Paul A.,
Yamodo Innocent H.,
Forman Stuart A.,
Miller Keith W.
Publication year - 2010
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.456
Subject(s) - receptor , agonist , ligand gated ion channel , heterologous expression , hek 293 cells , ion channel , gabaa receptor , allosteric regulation , allosteric modulator , chemistry , serotonin , biochemistry , endogenous agonist , ligand (biochemistry) , 5 ht receptor , biology , biophysics , recombinant dna , gene , dopamine receptor d1
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.