AAV ACTOne Gi-GPCR Assay Kit-CHRM2

The Cholinergic Receptor Muscarinic 2 (CHRM2), also known as the Muscarinic Acetylcholine Receptor M2, is a subtype of the muscarinic acetylcholine receptors, which are G protein-coupled receptors (GPCRs) that respond to the neurotransmitter acetylcholine. The M2 receptor is primarily coupled with the Gi/Go class of G proteins, leading to inhibition of adenylate cyclase and a subsequent decrease in cyclic AMP (cAMP) levels. This action results in several physiological effects, including slowing the heart rate, reducing the force of cardiac contraction, and decreasing the speed of electrical conduction in the heart. The M2 receptor is predominantly found in the heart, where it mediates the parasympathetic nervous system's control over heart rate, making it crucial for regulating cardiovascular function. Additionally, M2 receptors are also present in the central nervous system, where they play a role in modulating neuronal activity and cognitive functions. Given its role in heart rate regulation and neuronal signaling, the M2 receptor is involved in various physiological processes and is a therapeutic target for conditions such as bradycardia, arrhythmias, and certain neurological disorders.
This kit uses AAV vectors with a CMV promoter to co-express the CHRM2 and cyclic nucleotide-gated (CNG) channel, allowing researchers to conduct high-throughput screening and functional analysis of potential CHRM2-targeting compounds. The kit provides a sensitive and reliable method for evaluating the pharmacological properties of CHRM2 drugs, such as agonists and antagonists, in a live-cell environment.

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Background

ACTOne™ is the only high-throughput GPCR screening technology that can directly measure the intracellular changes of the secondary messenger cyclic AMP (cAMP) in living cells, in real-time. It uses a proprietary modified cyclic nucleotide-gated (CNG) channel, which is co-localized with adenylate cyclase at the plasma membrane, as a biosensor of cAMP activity. The CNG channel opens when the cAMP level near the plasma membrane increases, resulting in ion flux and cell membrane depolarization. The influx of cations through the CNG channel can be quantified using fluorescent ion indicators or membrane potential (MP) dyes. It provides information on real time intracellular cAMP changes and is highly sensitive. By combining kinetic and endpoint readouts, we are able to capture and analyze transient responses from endogenous GPCRs and weak responses caused by weak Gs or Gi coupled GPCR activities. Using ACTOne, we are able to detect the subcellular cAMP concentration changes directly caused by GPCR activation. Real-time kinetic readouts minimize artifacts, and provide greater content and more statistically relevant data. The intensity of signal increase caused by GPCR activation is directly related to the receptor number on cell surface. Using ACTOne assay, we were able to detect activities of some endogenous Gs coupled receptors in HEK293 cells that have not been reported in literature. In addition, we have also detected weak Gs coupled activity of a GPCR that was widely considered to be only linked to Gq coupled pathway. The ACTOne assay also provides a useful tool for GPCR de-orphanization.