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Establishment of a Cell Model for Dynamic Monitoring of Intracellular Calcium Concentration and High-Throughput Screening of P2Y2 Regulators

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. The P2Y2 receptor subtype is expressed in a variety of cell types and plays an important role in physiological and pathophysiological processes such as inflammatory responses and neuropathic pain. Based on this, the...

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Autores principales: Wu, Mingda, Hu, Cheng, Liu, Jingsong, Wu, Chuannan, Liu, Xueying, Hao, Feng, Li, Wenliang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9103248/
https://www.ncbi.nlm.nih.gov/pubmed/35566353
http://dx.doi.org/10.3390/molecules27093003
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author Wu, Mingda
Hu, Cheng
Liu, Jingsong
Wu, Chuannan
Liu, Xueying
Hao, Feng
Li, Wenliang
author_facet Wu, Mingda
Hu, Cheng
Liu, Jingsong
Wu, Chuannan
Liu, Xueying
Hao, Feng
Li, Wenliang
author_sort Wu, Mingda
collection PubMed
description P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. The P2Y2 receptor subtype is expressed in a variety of cell types and plays an important role in physiological and pathophysiological processes such as inflammatory responses and neuropathic pain. Based on this, the P2Y2 has been identified as an important drug target. The specificity of current P2Y2 receptor modulators is relatively poor, and currently, specific and efficient P2Y2 receptor modulators and efficient screening strategies are lacking. In this study, a cell model based on calcium-activated chloride channels (CaCCs) was established that can detect changes in intracellular calcium concentrations and can be used to high-throughput screen for P2Y2 receptor-specific regulators. This screening strategy is suitable for screening of most G-protein-coupled receptor regulators that mediate increases in intracellular calcium signals. The cell model consists of three components that include the endogenously expressed P2Y2 receptor protein, the exogenously expressed calcium-activated chloride channel Anoctamin-1 (Ano1), and a yellow fluorescent protein mutant expressed within the cell that is highly sensitive to iodine ions. This model will allow for high-throughput screening of GPCR regulators that mediate increased intracellular calcium signaling using the calcium-activated transport of iodide ions by Ano1. We verified the ability of the model to detect intracellular calcium ion concentration using fluorescence quenching kinetic experiments by applying existing P2Y2 agonists and inhibitors to validate the screening function of the model, and we also evaluated the performance of the model in the context of high-throughput screening studies. The experimental results revealed that the model could sensitively detect intracellular calcium ion concentration changes and that the model was accurate in regard to detecting P2Y2 modulators. The resultant value of the Z-factor was 0.69, thus indicating that the model possesses good sensitivity and specificity.
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spelling pubmed-91032482022-05-14 Establishment of a Cell Model for Dynamic Monitoring of Intracellular Calcium Concentration and High-Throughput Screening of P2Y2 Regulators Wu, Mingda Hu, Cheng Liu, Jingsong Wu, Chuannan Liu, Xueying Hao, Feng Li, Wenliang Molecules Article P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. The P2Y2 receptor subtype is expressed in a variety of cell types and plays an important role in physiological and pathophysiological processes such as inflammatory responses and neuropathic pain. Based on this, the P2Y2 has been identified as an important drug target. The specificity of current P2Y2 receptor modulators is relatively poor, and currently, specific and efficient P2Y2 receptor modulators and efficient screening strategies are lacking. In this study, a cell model based on calcium-activated chloride channels (CaCCs) was established that can detect changes in intracellular calcium concentrations and can be used to high-throughput screen for P2Y2 receptor-specific regulators. This screening strategy is suitable for screening of most G-protein-coupled receptor regulators that mediate increases in intracellular calcium signals. The cell model consists of three components that include the endogenously expressed P2Y2 receptor protein, the exogenously expressed calcium-activated chloride channel Anoctamin-1 (Ano1), and a yellow fluorescent protein mutant expressed within the cell that is highly sensitive to iodine ions. This model will allow for high-throughput screening of GPCR regulators that mediate increased intracellular calcium signaling using the calcium-activated transport of iodide ions by Ano1. We verified the ability of the model to detect intracellular calcium ion concentration using fluorescence quenching kinetic experiments by applying existing P2Y2 agonists and inhibitors to validate the screening function of the model, and we also evaluated the performance of the model in the context of high-throughput screening studies. The experimental results revealed that the model could sensitively detect intracellular calcium ion concentration changes and that the model was accurate in regard to detecting P2Y2 modulators. The resultant value of the Z-factor was 0.69, thus indicating that the model possesses good sensitivity and specificity. MDPI 2022-05-07 /pmc/articles/PMC9103248/ /pubmed/35566353 http://dx.doi.org/10.3390/molecules27093003 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Wu, Mingda
Hu, Cheng
Liu, Jingsong
Wu, Chuannan
Liu, Xueying
Hao, Feng
Li, Wenliang
Establishment of a Cell Model for Dynamic Monitoring of Intracellular Calcium Concentration and High-Throughput Screening of P2Y2 Regulators
title Establishment of a Cell Model for Dynamic Monitoring of Intracellular Calcium Concentration and High-Throughput Screening of P2Y2 Regulators
title_full Establishment of a Cell Model for Dynamic Monitoring of Intracellular Calcium Concentration and High-Throughput Screening of P2Y2 Regulators
title_fullStr Establishment of a Cell Model for Dynamic Monitoring of Intracellular Calcium Concentration and High-Throughput Screening of P2Y2 Regulators
title_full_unstemmed Establishment of a Cell Model for Dynamic Monitoring of Intracellular Calcium Concentration and High-Throughput Screening of P2Y2 Regulators
title_short Establishment of a Cell Model for Dynamic Monitoring of Intracellular Calcium Concentration and High-Throughput Screening of P2Y2 Regulators
title_sort establishment of a cell model for dynamic monitoring of intracellular calcium concentration and high-throughput screening of p2y2 regulators
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9103248/
https://www.ncbi.nlm.nih.gov/pubmed/35566353
http://dx.doi.org/10.3390/molecules27093003
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