M3 Receptor Activation Redistribution Assay

• Designed to assay compounds for their ability to modulate activation of the M3 receptor
• 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

The muscarinic acetylcholine receptor 3 belongs to a family of G protein-coupled receptors (GPCRs), which consists of five members (M1-M5). The odd-numbered members (M1, M3, M5) couple to Gαq whereas the even-numbered members (M2, M4) couple to Gαi. Muscarinic acetylcholine receptors mediate the effect of acetylcholine in synapses, and they are involved in several indications related to the central nervous system. In this assay, the muscarinic acetylcholine receptor 3 (M3) has been transfected into the NFATc1 GPCR Reporter Assay for Gq-coupled Receptors (Product ID 017_02), where receptor activation leads to release of cytoplasmic Ca²⁺, which in turn induces NFATc1 translocation. Binding of an agonist to the extracellular parts of M3 causes a conformational change in the receptor. This leads to activation of heterotrimeric Gq proteins, subsequent release of Gαq from the beta-gamma subunit and activation of phospholipase C, which catalyzes the formation of DAG and IP3 from PIP2. Free IP3 diffuses into the cytoplasm and activates IP3 receptors on the endoplasmic reticulum (ER) resulting in Ca²⁺ release from the ER into the cytoplasm. Elevated calcium levels lead to dephosphorylation of NFATc1 and rapid translocation from the cytoplasm to the nucleus.

Figure 1. Nuclear translocation of EGFP-NFATc1. Cells expressing the M3 receptor were treated with 300 nM carbachol for 20 min (right panel) or untreated (DMSO control, left panel). Activation of the receptor leads to nuclear translocation of EGFP-NFATc1, which can be detected by the image analysis algorithm.

Figure 2. Carbachol concentration response in the M3:NFATc1 assay. The EC₅₀ of carbachol is approximately 2.5 nM. Concentration response was measured in 9 point half log dilution series (n=16). Cells were treated with carbachol for 20 min. Cells were then fixed and nuclear translocation was measured using the Cellomics ArrayScan V^TI Reader and the Redistribution V3 BioApplication. % activity was calculated relative to the positive (300 nM carbachol) and negative control (0.25% DMSO).