Human α1β3γ2L gamma-aminobutyric acid type A receptors: High-level production and purification in a functional state

Abstract

Gamma-aminobutyric acid type A receptors (GABA(A)Rs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA(A)Rs determine their function and pharmacological profile. GABAA Rs are heteropentamers of subunits, and (α1)2 (β3)2 (γ2L)1 is a common subtype. Biochemical and biophysical studies of GABA(A)Rs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high-level production of active human α1β3 GABA(A)R using tetracycline-inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline-inducible HEK293-TetR cell line expressing human (N)-FLAG-α1β3γ2L-(C)-(GGS)3 GK-1D4 GABA(A)R. These cells achieved expression levels of 70-90 pmol [(3)H]muscimol binding sites/15-cm plate at a specific activity of 15-30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [(3)H]flunitrazepam to [(3)H]muscimol binding sites and sensitivity of GABA-induced currents to benzodiazepines and zinc. The α1β3γ2L GABA(A)Rs were solubilized in dodecyl-D-maltoside, purified by anti-FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ∼ 30%. Typical purifications yielded 1.0-1.5 nmoles of [(3)H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [(3)H]muscimol binding were maintained in the purified state.

Keywords: HEK293 TetR cells; human α1β3γ2 GABAAR; purification; reconstitution.

Figure 1

FLAG–α1β3γ2L–L3–1D4 GABA_ARs in plasma membranes contain γ–subunits. Whole-cell patch-clamp recordings of GABA–induced chloride currents after induction of GABA_AR expression. (A) Resistance to inhibition by Zn²⁺ demonstrated in paired pulses with and without Zn²⁺. Right panel, statistics of n determinations compared to control when Zn²⁺ was omitted from the second pulse. (B) Enhancement of GABA currents. Upper panel shows a representative trace; lower panel, the statistics relative to control without diazepam in the second pulse. (C) GABA concentration–response curve. Peak currents elicited with varying GABA concentrations were normalized to the second pulse peak elicited with 10 mM GABA.

Figure 2

FLAG–α1β3γ2L–L3–1D4 GABA_ARs in cell membranes contain γ–subunits. Binding curves of [³H]muscimol and [³H]flunitrazepam determined by filtration assays using cell membranes. Binding curves were fitted to the Hill equation by nonlinear least squares (see Table1 and text for parameters).

Figure 3

Purification and subunit composition of FLAG–α1β3γ2L–L3–1D4 GABA_ARs. Receptors were purified by anti-FLAG Chromatography. (A) Coomassie blue stain on a 14×15 cm SDS–PAGE gel of solubilized (30 mM DDM; lane 1) and purified reconstituted samples (5 mM CHAPS + 25 μM Asolectin; lane 2, 4, 5, loaded with 4, 25, 45 pmoles respectively). Lane 3 shows purified receptor deglycosylated with PNGase F. (B) Western blots from 8 × 8 cm SDS–PAGE gels of purified reconstituted receptors before (–) and after (+) deglycosylation with PNGase F: Antibodies used for detection where: α1, anti-FLAG; β3, anti-β3; γ2, anti-1D4.

Figure 4

Purified FLAG–α1β3γ2L–L3–1D4 GABA_ARs reconstituted in 5 mM CHAPS plus 25 µM asolectin contain γ–subunits (other details as in Figure 2).

Figure 5

Etomidate modulates muscimol binding to FLAG–α1β3γ2L–L3–1D4 GABA_ARs in membranes and purified reconstituted (5 mM CHAPS plus 25 µM asolectin) preparations. The [³H]muscimol concentration was 2 nM. The data were fitted to the Hill equation with fixed slope of 1.25 and normalized to the maximum for display purpose (see text for results).