ChemiScreen™ M5 Calcium-Optimized FLIPR Cell Lines

ChemiScreen™ M5 Calcium-Optimized FLIPR Cell Lines

• ChemiScreen
• Chemicon (Millipore)

2 vials

Full-length human CHRM5 cDNA, encoding M5

The muscarinic acetylcholine receptor family consists of five GPCRs that mediate some of the neurotransmission functions of acetylcholine in the CNS and the periphery. The M5 receptor, along with the M1 and M3 receptors, signal through Gq/11 and subsequent release of Ca++ from the ER (Caulfield and Birdsall, 1998). Although M5 is expressed in the CNS at relatively low levels, it appears to be the only muscarinic acetylcholine receptor expressed in midbrain dopaminergic neurons, and it has been shown to mediate dopamine release from these neurons. The M5 receptor is also expressed in blood vessels in the brain and the periphery, and studies with M5 knockout mice demonstrate that M5 mediates the vasodilatory action of acetylcholine on cerebral microvessels. Further studies with M5 knockout mice also indicate a role for M5 in the rewarding effects of cocaine and morphine (Wess, 2004). Chemicon's cloned human M5-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant M5 expression on the cell surface and contains high levels of the promiscuous G protein Galpha15 to enhance coupling of the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between M5 and its ligands.

Calcium flux assay, ligand binding assays

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The muscarinic cholinergic receptors belong to a larger family of G protein-coupled receptors. The functional diversity of these receptors is defined by the binding of acetylcholine and includes cellular responses such as adenylate cyclase inhibition, phosphoinositide degeneration, and potassium channel mediation. Muscarinic receptors influence many effects of acetylcholine in the central and peripheral nervous system. The clinical implications of this receptor are unknown; however, stimulation of this receptor is known to increase cyclic AMP levels.

FUNCTION: SwissProt: P08912 # The muscarinic acetylcholine receptor mediates various cellular responses, including inhibition of adenylate cyclase, breakdown of phosphoinositides and modulation of potassium channels through the action of G proteins. Primary transducing effect is Pi turnover.
SIZE: 532 amino acids; 60074 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
SIMILARITY: SwissProt: P08912 ## Belongs to the G-protein coupled receptor 1 family.

Human

SPECIFICATIONS: EC50 for calcium mobilization by carbamoyl choline: ~ 19.5 nM

Chem-1

GPCR Cell Lines

Chemicon

Chem-1

Cells are frozen at 2 x 10e6 cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.

M5

P08912

non-peptide

Acetylcholine (muscarinic)

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.

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A

1 mL per vial

• CHRM5
• MGC41838
• HM5

GPCRs

ChemiScreen™ M5 Calcium-Optimized FLIPR Cell Lines

NM_012125.2

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)/100 U/ml each penicillin and streptomycin

2 x 10e6 cells/mL