High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA 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 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.

Figure 1

n-Dodecyl-β-d-maltopyranoside (DDM) is the best detergent for solubilizing the FLAG-hGABA_Aα1β3R from HEK293S-TetR cell membranes. The number of [³H]muscimol sites from membrane protein (1 mg/mL) solubilized increased with detergent concentration to a plateau and decreased thereafter. The low-CMC detergents, DDM (filled squares), and C₁₂E₉ (filled circles) solubilized best. Others detergents used were: C₁₂E₉:CHAPS (4:5 molar ratio) (filled triangles); CHAPS (open circles), and CHAPS supplemented with 1.8 mM asolectin (open triangles).

Figure 2

Purification and deglycosylation of FLAG-h5-HT_3A-1D4 (lanes A and B) and FLAG-hGABA_Aα1β3 (lanes D and E) receptors. FLAG-h5-HT_3AR-1D4 (25 pmol/lane): silver stained SDS-PAGE gel (8 %) of the purified, reconstituted receptor (lane A), and after digestion with N-glycosidase F (lane B). The broad band between the 55 and 72 kDa markers is the receptor. After deglycosylation, it becomes a narrow band at 55 kDa. The band at 35 kDa originated from the enzyme. The high M_w bands are aggregated receptors (see text). FLAG-hGABA_Aα1β3R (18 pmol/lane): Coomassie blue stained SDS-PAGE gel (8%) of the purified receptor (lane D) and after digestion with N-glycosidase F (lane E). The presence of both, α1 and β3 subunits (the two diffuse bands at ±55 kDa marker) was confirmed via western blot and mass spectrometry. Deglycosylation produced two sharper, well-separated subunit bands at lower M_w. The gel was run out to increase the separation of the subunits. In regular gels (not shown), there were no contaminants below 40 kDa. Lanes C and F: molecular weight standards.

Figure 3

Serotonin-stimulated efflux from ⁸⁶Rb⁺ loaded reconstituted FLAG-h5-HT_3AR-1D4 lipid vesicles. Vesicles (PC:PA:Chol, molar ratio 67:12:21) were loaded overnight at 4°C with ⁸⁶Rb⁺ before being passed down a Sephadex column containing either buffer (filled circles) or 100 μM 5-HT (open circles) to separate external ⁸⁶Rb⁺ from that retained in vesicles, which eluted in the void volume at 2–4 min. Vesicles in the presence of 5-HT released about half their trapped ⁸⁶Rb⁺ (see Results section). The inset shows the whole chromatogram, which is dominated by the external ⁸⁶Rb⁺ eluting at ≥6 min in equal quantities for both samples.

Figure 4

Purified FLAG-GABA_A-α1β3Rs undergo allosteric conformation changes. Etomidate modulates specific [³H]muscimol binding (2 nM) in membranes (open diamonds with standard deviation (SD) as lines), in solubilized membranes (2.5 mM DDM, open circles with SD not shown for clarity), and after elution from the affinity column (11.5 mM cholate, 0.86 mM asolectin, filled squares with SD shown as capped lines).