Abstract：OBJECTIVE To establish an efficient and sensitive drug cell screening model and method targeting Chrm3 based on calcium-activated chloride channel(CaCC). METHODS RT-PCR, immunofluorescence and Western blot were applied to detect the expression of Chrm3 in FRT cells. The FRT cell model co-expressing ANO1 and YFP-H148Q/I152L was constructed, and the CaCC cell model was identified by an inverted fluorescence microscope and fluorescence quenching kinetics test. The Fura-2 fluorescent probe was used to detect the calcium concentration in cytoplasm after adding Chrm3 activator. The validation of the cell model which could screen Chrm3 modulators was verified by the fluorescence quenching kinetics experiments. Z' factor was calculated to evaluate the sensitivity and specificity of the cell model. RESULTS FRT cells endogenously expressed Chrm3. The FRT cell model stably co-expressing ANO1 and YFP-H148Q/I152L was successfully constructed. The cell model could sensitively detect intracellular calcium concentration. The value of relative fluorescence intensity changed with the concentration of Chrm3 modulators in a dose-dependent manner, and the model could screen Chrm3 modulators. The Z' factor was 0.678, which met the requirement for high-throughput screening. CONCLUSION The cell model can be applied for high-throughput screening Chrm3 modulators, and it can sensitively detect calcium signals. The cell model can also screen other GPCR regulators-associated calcium signaling.
BERNDT-PAETZ M, HERBST L, WEIMANN A, et al. Highly specific detection of muscarinic M3 receptor, G protein interaction and intracellular trafficking in human detrusor using proximity ligation assay (PLA)[J]. Acta Histochem, 2018, 120(4):329-339.
[2]
KRUSE A C, HU J, PAN A C, et al. Structure and dynamics of the M3 muscarinic acetylcholine receptor[J]. Nature, 2012, 482(7386):552-556.
[3]
KATAYAMA I. Abberant sudomotor functions in Sjögren's syndrome: comparable study with atopic dermatitis on dry skin manifestation[J]. Curr Probl Dermatol, 2016, 51:62-74.
[4]
LI D L, LIU Y, WANG L K, et al. Protective effects of M3 receptor agonist pilocarpine on myocardial ischemia-reperfusion injury in isolated rat hearts[J]. Chin Pharm J (中国药学杂志), 2007, 42(19):1473-1476.
[5]
ALCÁNTARA MONTERO A. Developments in the medical treatment of overactive bladder[J]. Semergen, 2016, 42(8):557-565.
[6]
PATEL K R, BAI Y, TRIEU K G, et al. Targeting acetylcholine receptor M3 prevents the progression of airway hyperreactivity in a mouse model of childhood asthma[J]. FASEB J, 2017, 31(10):4335-4346.
[7]
WAIN L V, SHRINE N, ARTIGAS M S, et al. Genome-wide association analyses for lung function and chronic obstructive pulmonary disease identify new loci and potential druggable targets[J]. Nat Genet, 2017, 49(3):416-425.
[8]
BHARUCHA A E, RAVI K, ZINSMEISTER A R. Comparison of selective M3 and nonselective muscarinic receptor antagonists on gastrointestinal transit and bowel habits in humans[J]. Am J Physiol Gastrointest Liver Physiol, 2010, 299(1):G215-G219.
[9]
FELTON J, HU S, RAUFMAN J P. Targeting M3 muscarinic receptors for colon cancer therapy[J]. Curr Mol Pharmacol, 2018, 11(3):184-190.
[10]
FENG Y, HU X, LIU G, et al. M3 muscarinic acetylcholine receptors regulate epithelial-mesenchymal transition, perineural invasion, and migration/metastasis in cholangiocarcinoma through the AKT pathway[J]. Cancer Cell Int, 2018, 18:173.
[11]
LIU H, HOFMANN J, FISH I, et al. Structure-guided development of selective M3 muscarinic acetylcholine receptor antagonists[J]. Proc Natl Acad Sci USA, 2018, 115(47):12046-12050.
[12]
JING L I, XIN X. High-throughput screening assays for G-protein-coupled receptors-targeted drug discovery[J]. J Int Pharm Res, 2012, 39(5):353-357.
[13]
JIMÉNEZ E, PAVÍA J, MORELL V, et al. Muscarinic receptor subtypes and calcium signaling in Fischer rat thyroid cells[J]. Biochem Pharmacol, 2001, 61(3):337-342.
[14]
HAO F, BAI X S, JU X H, et al. Expression of TMEM16A as a calcium activated chloride channelin Fischer ratthyroid follicular epithelial cells and its electrophysiologic properties [J]. Chin J Pathophysiol (中国病理生理杂志), 2014, 30(9):1633-1639.
[15]
REBBECK R T, SINGH D P, JANICEK K A, et al. RyR1-targeted drug discovery pipeline integrating FRET-based high-throughput screening and human myofiber dynamic Ca2+ assays[J]. Sci Rep, 2020, 10(1):1791.
[16]
WU F, SONG G, DE GRAAF C, et al. Structure and function of peptide-binding G protein-coupled receptors[J]. J Mol Biol, 2017, 429(17):2726-2745.
[17]
KRUSE A C, LI J, HU J, et al. Novel insights into M3 muscarinic acetylcholine receptor physiology and structure[J]. J Mol Neurosci, 2014, 53(3):316-323.
2021, Vol. 56 
