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Development and validation of HTS assay for screening the calcium-activated chloride channel modulators in TMEM16A stably expressed CHO cells

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Abstract

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 Ca2+ ionophore, for 12 min; (2) cells were washed with a low-K+, Cl−-free and Ca2+-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.

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Acknowledgments

This work supported by the National Natural Science Foundation of China (31270882 to HZ), the National Basic Research Program of China (2013CB531302 to HZ) and the Science Foundation of Hebei Province, China (H2013206048, 20130462 and YQ2013033 to J.Q).

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Authors and Affiliations

  1. The Key Laboratory of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, Shijiazhuang, 050017, China

    Jinlong Qi, Yuan Wang, Yani Liu, Bingcai Guan & Hailin Zhang

  2. The Key Laboratory of New Drug Pharmacology and Toxicology, Hebei Province, Hebei Medical University, Shijiazhuang, 050017, China

    Jinlong Qi, Yuan Wang, Yani Liu, Bingcai Guan & Hailin Zhang

  3. Department of Pharmacology, Hebei Medical University, Shijiazhuang, 050017, China

    Jinlong Qi, Yuan Wang, Yani Liu, Bingcai Guan & Hailin Zhang

  4. Department of Biochemistry, Hebei Medical University, Shijiazhuang, 050017, China

    Fan Zhang

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  1. Jinlong Qi
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Correspondence to Hailin Zhang.

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Qi, J., Wang, Y., Liu, Y. et al. Development and validation of HTS assay for screening the calcium-activated chloride channel modulators in TMEM16A stably expressed CHO cells. Anal Bioanal Chem 406, 1713–1721 (2014). https://doi.org/10.1007/s00216-013-7550-5

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