2. Academic Validation
  3. Establishment of a Cell Model for Dynamic Monitoring of Intracellular Calcium Concentration and High-Throughput Screening of P2Y2 Regulators

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

  • Molecules. 2022 May 7;27(9):3003. doi: 10.3390/molecules27093003.
Mingda Wu 1 2,  Cheng Hu 1,  Jingsong Liu 3,  Chuannan Wu 4,  Xueying Liu 5,  Feng Hao 1,  Wenliang Li 6
Affiliations

Affiliations

  • 1 College of Laboratory Medicine, Jilin Medical University, Jilin 132000, China.
  • 2 Medical College, Yanbian University, Yanji 136200, China.
  • 3 Department of Thoracic Surgery, The Affiliated Hospital of Xuzhou Medical University, Xuzhou 221000, China.
  • 4 The Municipal Hospital Affiliated to Xuzhou Medical University, Xuzhou 221000, China.
  • 5 School of Medical Technology, Beihua University, Jilin 132000, China.
  • 6 Jilin Collaborative Innovation Center for Antibody Engineering, Jilin Medical University, Jilin 132000, China.
PMID: 35566353 DOI: 10.3390/molecules27093003
Abstract

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.

Keywords

GPCRs; P2Y2; calcium concentration; high-throughput screening; regulators.

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