Development and validation of HTS assay for screening the calcium-activated chloride channel modulators in TMEM16A stably expressed CHO cells.
Calcium-activated chloride channels (CaCCs), for example TMEM16A, are widely expressed in a variety of tissues and are involved in many important physiological functions. We developed and validated an atomic absorption spectroscopy (AAS)-based detection system for high-throughput screening (HTS) of CaCC modulators. With this assay, Cl(-) flux from CHO cells stably transfected with TMEM16A is assayed indirectly, by measuring excess silver ions (Ag(+)) in the supernatant of AgCl precipitates. The screening process involved four steps: (1) TMEM16A CHO cells were incubated in high-K(+) and high-Cl(-) buffer with test compounds, and with ionomycin as Ca(2+) ionophore, for 12 min; (2) cells were washed with a low-K(+), Cl(-)-free and Ca(2+)-free buffer; (3) CaCC/TMEM16A were activated in high-K(+), Cl(-)-free buffer with ionomycin (10 μmol L(-1)) for 12 min; and (4) excess Ag(+) concentration was measured using an ion channel reader (ICR, an AAS system). The assay can be used to screen CaCC activators and inhibitors at the same time. With this assay, positive control drugs, including NPPB, CaCCinh-A01, flufenamic acid (Flu) and Eact, all had good concentration-dependent effects on CaCC/TMEM16A. NPPB and CaCCinh-A01 inhibited the CaCC/TMEM16A currents completely at 300 μmol L(-1), with IC50 values of 39.35 ± 4.72 μmol L(-1) and 6.35 ± 0.27 μmol L(-1), respectively; and Eact, activated CaCC/TMEM16A, with an EC50 value of 3.92 ± 0.87 μmol L(-1).
The Key Laboratory of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, Shijiazhuang, 050017, China.
Jinlong
J
Qi
Yuan
Y
Wang
Yani
Y
Liu
Fan
F
Zhang
Bingcai
B
Guan
Hailin
H
Zhang
D000818:Animals; D000075369:Anoctamin-1; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D065101:Chloride Channel Agonists; D018118:Chloride Channels / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004353:Drug Evaluation, Preclinical / Q000379:methods; D015870:Gene Expression
C531181:ANO1 protein, mouse; D000075369:Anoctamin-1; D065101:Chloride Channel Agonists; D018118:Chloride Channels; D002118:Calcium