M2 Receptor Internalization Redistribution Assay

• Designed to assay compounds for their ability to modulate Muscarinic acetylcholine receptor 2 (M2) internalization
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

Muscarinic acetylcholine receptors mediate their effect by exerting metabotropic actions of acetylcholine in the nervous system. The receptors belong to the G-protein-coupled receptor (GPCR) superfamily that transduces signals through multiple intracellular signalling cascades to regulate a wide variety of physiological responses. At present, five receptor subtypes have been identified, where the odd-numbered receptors (M1, M3, M5) preferentially couple to Gαq and activate phospholipase C, thereby initiating the phosphatidylinositol trisphosphate (PIP3) cascade leading to intracellular Ca²⁺ mobilization and activation of protein kinase C. The even-numbered receptors M2 and M4 couple to Gαi/o and inhibit adenylate cyclase activity.

Drugs targeting muscarinic acetylcholine receptors are currently used in the treatment of chronic obstructive pulmonary disorder and overactive bladder, but these receptors also represent potential therapeutic targets for cognitive disorders such as Alzheimer’s disease and schizophrenia. Selective M1 agonists or mixed M1 agonists/M2 antagonists may provide an approach to the treatment of cognitive disorders, while M3 antagonists or mixed M2/M3 antagonists are approved for the treatment of contractility disorders including overactive bladder and chronic obstructive pulmonary disease.

Figure 1. Internalization of M2-EGFP stimulated with carbachol. Cells were treated with 1 mM carbachol for 1 hr (right panel). Arrows indicate the M2-EGFP internalization that is detected by the image analysis algorithm.

Figure 2. Carbachol concentration response in the M2 assay. Concentration response was measured in 9 point half log dilution series (n=16). The EC₅₀ of carbachol is ~40 μM. Cells were incubated with carbachol for 1 hr. Cells were then fixed and internalization was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (1 mM carbachol) and negative control (0.25% DMSO).