ChemiSCREEN Human M₃ Muscarinic Acetylcholine Receptor Membrane Preparation

Full-length human CHRM3 cDNA encoding M₃

The muscarinic acetylcholine receptor (mAChR) family consists of five GPCRs that mediate some of the neurotransmission functions of acetylcholine in the CNS and the periphery. The M₁, M₃ and M₅ receptors couple to G_q to mobilize intracellular calcium, whereas the M₂ and M₄ receptors couple to G_i/o to inhibit cAMP production (Caulfield and Birdsall, 1998). M₃ is expressed prominently in smooth muscle, and plays a primary role in mediating mAChR agonist-induced contractility. Mice lacking M₃ have dilated pupils, which indicates a role for M₃ in regulating tone of the pupillary sphincter muscle. In addition, Msub3 plays a role in feeding, as indicated by the lean and hypophagic phenotype of M₃-null mice (Wess, 2004). Chemicon′s M₃ membrane preparations are crude membrane preparations made from our proprietary stable recombinant cell lines to ensure high-level of GPCR surface expression; thus, they are ideal HTS tools for screening of antagonists of M₃ interactions with 4-DAMP. The membrane preparations exhibit a Kd of 0.72-1 nM for [³H]-4-DAMP. With 10 µg/well M₃ Membrane Prep and 0.75 nM [³H]-4-DAMP, a greater than 4-fold signal-to-background ratio was obtained.

Human M3 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Radioligand binding assay and GTPγS binding.

GPCR Class: A

Protein Target: M3

Target Sub-Family: Acetylcholine (muscarinic)

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of M₃ membrane prep:

	20 µg/well	10 µg/well
Signal:Background	5.17	5.10
Specific Binding (cpm)	1224	1030

SPECIFICATIONS: 1 unit = 10 µg
Bmax: 3.75 pmol/mg
Kd: 0.86 nM

Inucbation Conditions
Membranes are mixed with radioactive ligand and unlabeled competitor (see Figures 1 and 2 for concentrations tested) in binding buffer in a nonbinding 96-well plate, and incubated for 1-2 h. Prior to filtration, a GF/C 96-well filter plate is coated with 0.33% polyethyleneimine for 30 min, then washed with 50mM HEPES, pH 7.4, 0.5% BSA. Binding reaction is transferred to the filter plate, and washed 3 times (1 mL per well per wash) with Wash Buffer. The plate is dried and counted.
Binding buffer: 50 mM Hepes, pH 7.4, 5 mM MgCl₂, 1 mM CaCl₂, 0.2% BSA, filtered and stored at 4°C
Radioligand: [³H]-4-DAMP (Perkin Elmer#:NET1040 )
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, filtered and stored at 4°C.
One package contains enough membranes for at least 200 assays (units), where a unit is the amount of membrane that will yield greater than 4-fold signal:background with ³H labeled 4-DAMP at 0.75 nM

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein were adjusted to 0.5 mg/ml in 1 mL packaging buffer, rapidly frozen, and stored at -80°C.

Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.

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