Journal
PROTEIN SCIENCE
Volume 23, Issue 2, Pages 157-166Publisher
WILEY-BLACKWELL
DOI: 10.1002/pro.2401
Keywords
human 132 GABA(A)R; HEK293 TetR cells; purification; reconstitution
Categories
Funding
- National Institute of General Medical Sciences [GM 58448]
- Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital
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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 (1)(2)(3)(2)(2L)(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 13 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-132L-(C)-(GGS)(3)GK-1D4 GABA(A)R. These cells achieved expression levels of 70-90 pmol [H-3]muscimol binding sites/15-cm plate at a specific activity of 15-30 pmol/mg of membrane protein. Incorporation of the 2 subunit was confirmed by the ratio of [H-3]flunitrazepam to [H-3]muscimol binding sites and sensitivity of GABA-induced currents to benzodiazepines and zinc. The 132L 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 approximate to 30%. Typical purifications yielded 1.0-1.5 nmoles of [H-3]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 [H-3]muscimol binding were maintained in the purified state.
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