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

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 a1 and b3 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 Abbreviations: 5-HT 3 AR, 5-hydroxytryptamine-3A receptor; CMC, critical micelle concentration; DDM, n-dodecyl-b-D-maltopyranoside; EPR, electron paramagnetic resonance; GABA A a1b3R, gamma-aminobutyric acid type A receptor with a1 and b3 subunits; GPCR, G protein-coupled receptor; GlyR, glycine receptor; HEK293, human embryonic kidney cells; LGIC, ligand-gated ion channel; nAChR, nicotinic acetylcholine receptor; NMR, nuclear magnetic resonance; PEG, poly(ethylene glycol).Additional Supporting Information may be found in the online version of this article. Published by Wiley-Blackwell. V C 2010 The Protein Society 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.

Specificity of Intersubunit General Anesthetic-binding Sites in the Transmembrane Domain of the Human α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor*

“…Saccharomyces aureus endoproteinase Glu-C (EndoGlu-C) and Lysobacter enzymogenes endoproteinase Lys-C (EndoLys-C) were from Worthington and Roche Applied Science, respectively. Purification of Human ␣1␤3␥2 GABA A R-␣1␤3␥2 GABA A Rs with a FLAG epitope at the N terminus of the ␣1 subunit were expressed in a tetracycline-inducible, stably transfected HEK293S cell line and purified on an anti-FLAG affinity resin with modifications of procedures used to purify a previously characterized tetracycline-inducible FLAG-␣1␤3 GABA A R (15,20 (14). The total concentration of sites was determined at 500 nM [ 3 H]muscimol and with 1 mM GABA to determine nonspecific binding.…”

“…The total concentration of sites was determined at 500 nM [ 3 H]muscimol and with 1 mM GABA to determine nonspecific binding. Anesthetic modulation of 2-3 nM [ 3 H]muscimol binding was measured as described (15,20), except that samples were incubated for 60 min at room temperature before addition of polyethylene glycol and ␥-globulins and then filtered after a 30-min incubation at room temperature. The modulation results are presented as the percentage of the specifically bound [ 3 H]muscimol over that without modulators.…”

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

“…Electrophysiology-Whole-cell patch clamp recordings were obtained from induced HEK293-TetR cells expressing either ␣1␤3 or ␣1␤3␥2L GABA A receptors using methods described previously (28,29). Briefly, cells were seeded on a glass coverslip, and protein expression was induced with tetracycline (2 g/ml) for 5-26 h before recordings.…”

“…Purification of Expressed Human ␣1␤3␥2 GABA A Rs-␣1␤3␥2 L and ␣1␤3 GABA A Rs containing a FLAG epitope at the N terminus of the mature ␣1 subunit (MRK…SYGDYKDDDDKQPS…) were purified from tetracycline-inducible, stably transfected HEK293S cell lines using an anti-FLAG affinity resin as described previously (23,24,28,29). GABA A R was solubilized in 30 mM n-dodecyl ␤-D-maltopyranoside, and column wash and elution buffers contained 5 mM CHAPS and 0.2 mM asolectin.…”

Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog

“…Purification of Expressed ␣1␤3 GABA A Rs-␣1␤3 GABA A Rs containing the FLAG epitope at the N terminus of the ␣1 subunit were purified from a tetracycline-inducible, stably transfected HEK293S cell line (27). Briefly, membrane fractions containing 6 -10 nmol of [ 3 H]muscimol-binding sites, collected from cells growing on 40 -60 tissue culture dishes (15 cm), were resuspended at 1 mg of protein/ml and solubilized overnight in 300 ml of a purification buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM CaCl 2 , 5 mM KCl, 5 mM MgCl 2 , 4 mM EDTA, 20% glycerol, pepstatin, chymostatin, and leupeptin (10 g/ml each), 2 g/ml aprotinin, and 1 mM phenylmethanesulfonyl fluoride) supplemented with 2.5 mM n-dodecyl-␤-D-maltopyranoside.…”