AAV ACTOne Gi-GPCR Assay Kit-HTR1A

Serotonin Receptor 1A (HTR1A) is a G protein-coupled receptor (GPCR) that is part of the serotonin receptor family. It is primarily coupled with the Gi/Go class of G proteins, which inhibit adenylate cyclase activity, leading to a reduction in cyclic AMP (cAMP) levels within the cell. This inhibition affects various downstream signaling pathways, ultimately modulating neurotransmitter release and neuronal excitability. HTR1A is widely expressed in the central nervous system (CNS), particularly in regions such as the hippocampus, cortex, and raphe nuclei. It is found both presynaptically and postsynaptically, where it plays different roles. Presynaptically, HTR1A functions as an autoreceptor on serotonin-producing neurons, inhibiting the release of serotonin and thus regulating its levels. Postsynaptically, it modulates the activity of target neurons in response to serotonin. The HTR1A receptor is involved in various physiological processes, including mood regulation, anxiety, cognition, and sleep. Dysregulation of HTR1A signaling is implicated in several psychiatric disorders, including depression, anxiety disorders, and schizophrenia. Due to its critical role in mood regulation, HTR1A is a major target for antidepressant and anxiolytic drugs. Many selective serotonin reuptake inhibitors (SSRIs) and other serotonergic medications indirectly influence HTR1A activity to achieve their therapeutic effects. Research into HTR1A continues to explore its broader role in the brain and its potential as a therapeutic target for treating a range of mental health conditions.
This kit uses AAV vectors with a CMV promoter to co-express the HTR1A and cyclic nucleotide-gated (CNG) channel, allowing researchers to conduct high-throughput screening and functional analysis of potential HTR1A-targeting compounds. The kit provides a sensitive and reliable method for evaluating the pharmacological properties of HTR1A 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.