High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptors.
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.
Dostalova
Zuzana
Z
Department of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
Liu
Aiping
A
Zhou
Xiaojuan
X
Farmer
Sarah L
SL
Krenzel
Eileen S
ES
Arevalo
Enrique
E
Desai
Rooma
R
Feinberg-Zadek
Paula L
PL
Davies
Paul A
PA
Yamodo
Innocent H
IH
Forman
Stuart A
SA
Miller
Keith W
KW
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D004594:Electrophysiology; D015870:Gene Expression / Q000187:drug effects; D057809:HEK293 Cells / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D011963:Receptors, GABA-A / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D044406:Receptors, Serotonin, 5-HT3 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection / Q000379:methods
D000900:Anti-Bacterial Agents; D008024:Ligands; D011963:Receptors, GABA-A; D044406:Receptors, Serotonin, 5-HT3; D013752:Tetracycline