ChemiSCREEN Membrane Preparation Recombinant Human M₂ Muscarinic Acetylcholine Receptor

Table 1. Signal:background and specific binding values obtained in a competition binding assay with M₂ membrane prep and unlabeled Atropine.

	10 µg/well
Signal:Background	3.2
Specific Binding (cpm)	606.8

SPECIFICATIONS: 1 unit = 10 µg
Bmax for [³H]-NMS binding: 0.5 pmol/mg protein
K_d for [³H]-NMS binding: ~0.24 nM

Protein Target: M2

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, an FC 96-well harvest plate (Millipore cat. # MAHF C1H) is coated with 0.33% polyethyleneimine for 30 min, then washed with 4mM Na₂HPO₄, 1mM KH₂PO₄, pH7.4. 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 and Wash buffer: 4mM Na₂HPO₄, 1mM KH₂PO₄, pH7.4, filtered and stored at 4&degC Radioligand: [³H] NMS (Perkin Elmer#:NEX-636 ) One package contains enough membranes for at least 200 assays (units), where a unit is the amount of membrane that will yield greater than 3-fold signal:background with ³H-labeled NMS at 0.5 nM

Full-length human CHRM2 cDNA encoding M₂

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). In urinary bladder trachea and stomach, M₂ augments the function of M₃ in promoting contractility, and activation of M₂ serves to counteract relaxation induced by increased cAMP levels (Ehlert et al., 2005; Wess, 2004). In addition, the ability of mAChR agonists to decrease heart rate appears to be mediated primarily by M₂. Agonists of mAChRs induce tremor, hypothermia, corticosterone release, and analgesia; each of these functions is mediated at least in part by M₂ (Wess, 2004). Chemicon′s M₂ 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 M₂ interactions with its ligands. The membrane preparations exhibit a Kd of 0.24 nM for [³H]-Scopolamine methyl chloride (NMS). With 10 µg/well M₂ Membrane Prep and 0.5 nM [³H]-NMS, greater than 3-fold signal-to-background ratio was obtained.

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80oC.

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