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. GABA A Rs are heteropentamers of subunits, and (a1) 2 (b3) 2 (c2L) 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 a1b3 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-a1b3c2L-(C)-(GGS) 3 GK-1D4 GABA A R. These cells achieved expression levels of 70-90 pmol Keywords: human a1b3c2 GABA A R; HEK293 TetR cells; purification; reconstitution Abbreviations: a1b3 GABA A R, GABA A R with a1 and b3 subunits; a1b3g2 GABA A R, GABA A R with a1, b3, and g2 subunits; 5HT 3A R, 5-hydroxytryptamine-3A receptor; DDM, ndodecyl-b-D-maltopyranoside; GABA A R, gamma-aminobutyric acid type A receptor; K d , equilibrium dissociation constant of the ligand.

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.…”

Dodecyl Maltopyranoside Enabled Purification of Active Human GABA Type A Receptors for Deep and Direct Proteomic Sequencing*

“…These results paved the way for streamlined high-performance proteomics of GABA A R, and more importantly allowed directly coupling proteomics with crystallography to map both static and dynamic structures of membrane proteins in crystallization pipelines. Cell Growth, Membrane Isolation and GABA A R Purification-HEK293-TetR cells stably expressing human (N)-FLAG-␣1␤3␥2L-(GGS) 3 GK-1D4 GABA A R were obtained as recently described (56). Briefly, the genes coding for GABA A R subunits were individually cloned using independent antibiotic selections and introduced to HEK293-TetR at ␣1 (bovine): ␤3 (human): ␥2L (human) subunit plasmid copy ratio of 2:2:1.…”

Instant Integrated Ultradeep Quantitative-structural Membrane Proteomics Discovered Post-translational Modification Signatures for Human Cys-loop Receptor Subunit Bias

“…GABA A R Protein Purification from HEK293 Cells-HEK293-TetR cells stably over-expressing full-length human (N)-FLAG-␣1␤3␥2L-(GGS) 3 GK-1D4 GABA A R at ␣1 (bovine): ␤3 isoform 2 (human): ␥2L (human) subunit plasmid copy ratio of 2:2:1 were provided by Dr. Keith W. Miller (35), and final active GABA A R (at typical concentration of 25 M in the 0.05% DDM/0.0125% CHS/10% glycerol protein buffer) was obtained by affinity purification on a low-flow-rate anti-FLAG column, as described earlier (34). Briefly, following 72-hour growth and 24-hour tetracycline-induced GABA A R expression in 5% CO 2 at 37°C, cells at 90% confluence on 15-cm plates were PBSwashed, harvested into hypotonic lysis buffer (10 mM HEPES pH 7.5, protease inhibitors LACP and PMSF), and frozen slowly at Ϫ80°C to promote lysis.…”