ChemiScreen™ Human recombinant GABA_B1b/ GABA_B2Receptor, Calcium-Optimized Stable Cell Line

ChemiScreen™ Human recombinant GABA_B1b/ GABA_B2Receptor, Calcium-Optimized Stable Cell Line

ChemiScreen

TRANSFECTION: Full-length human GABBR1b and GPR51 cDNAs encoding GABAB1b/GABAB2

The neurotransmitter -aminobutyric acid (GABA) exerts its effects through an ion channel, GABA_A, and a GPCR, GABA_B. Functional GABA_B is a heterodimer composed of the GABA_B1 and GABA_B2 subunits, which share 35% sequence identity and belong to the class 3 family of GPCRs. The GABA_B1 subunit, which exists as splice variants GABA_B1a and GABA_B1b, binds directly to GABA and is required for agonist activation. The GABA_B2 and GABA_B1 subunits associate by formation of a coiled coil by their C-terminal tails; this association masks an ER retention sequence in GABA_B1 to permit export from the ER and trafficking to the cell surface. In addition to its chaperone function, GABA_B2 is the component that couples to G_i to reduce intracellular cAMP. Agonists of GABA_B, such as baclofen, are used clinically for treatment of muscle spasticity, migraine headache and musculoskeletal pain (Bowery et al., 2002). Chemicon's cloned human GABA_B1b/GABA_B2-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant GABA_B1b/GABA_B2 expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between GABA_B and its ligands.

Calcium flux assay, ligand binding assays

Human

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GPCR Cell Lines

2 x 10⁶ cells/mL

Chem-1, an adherent cell line expressing the promiscuous G protein, Gα15.

2 x 10⁶ cells/mL

GROWTH MEDIA: DMEM containing 4.5 g/L glucose/10% heat inactivated fetal bovine serum/1x nonessential amino acids/10 mM HEPES/0.25 mg/ml Geneticin (G418)/0.5 mg/mL Hygromycin/100 U/ml each penicillin and streptomycin.

Chem-1

• GABBR1
• dJ271M21.1.1
• GABBR1-3
• GABAB(1e)
• hGB1a
• GABABR1
• dJ271M21.1.2
• Gb1
• GABA-B-R1
• GPRC3A

Cell Line

Cells are frozen at 2 x 10⁶ cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.

SPECIFICATIONS: EC50 for calcium mobilization by GABA: ~ 280 nM

GPCRs

Human

Place cells in liquid nitrogen immediately upon receipt. Maintain frozen in liquid nitrogen for up to 5 years. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37ºC water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing 20 mL growth media, and place in a humidified 37°C incubator with 5% CO2.. After 8-24 h, cells will adhere to the plate, at which time the media should be replaced to remove residual DMSO. Cells are passaged by washing with Ca⁺⁺ and Mg⁺⁺-free HBSS (10 mL/T75), incubating with 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) for 5-10 minutes at 37°C, and rapping the side of the flask to dislodge the cells. Neutralize the trypsin by addition of 4 volumes growth media. Cells are typically passaged 1:10 with every 3-4 days, and should be passaged at least once after thawing prior to use in calcium flux assays.