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Development of a functional cell-based HTS assay for identification of NKCC1-negative modulators

  • Article
  • Genomics
  • Published:
Chinese Science Bulletin

Abstract

Na–K–Cl cotransporter 1 (NKCC1) cotransports Na+, K+, and Cl− ions across the plasma membrane into cells. Accumulation of Cl− ions in dorsal root ganglion neurons induces depolarizing GABAA receptors, which mediate presynaptic inhibition and filtration of sensory noise. The activity of the Na–K–Cl cotransporter is modulated by high-dose loop diuretics, such as furosemide and bumetanide. To identify NKCC1 modulators, we developed a functional cell-based assay feasible for high-throughput screening (HTS), in which the activity of NKCC1 was detected by a BTC-AM dye-based thallium transportation assay. We demonstrated that the influx of Tl+ was mediated by NKCC1, which required the existence of Cl− ions and could be inhibited by bumetanide and furosemide. Our results demonstrated that the assay was stable, reproducible, and suitable for HTS of negative modulators for NKCC1.

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Acknowledgments

This work was supported by “Significant new drugs creation” science and technology major special “Twelfth Five” plan, and Key Technology Research of GPCR targeting Drug Screening and Mechanism of Action Studies Project (2012ZX09301003-003). We thank HD Biosciences for providing the instruments and compounds used in this study.

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Authors and Affiliations

  1. Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China

    Yanqiu Bai, Lianghui Ma & Shiyou Li

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Yanqiu Bai, Lianghui Ma & Shiyou Li

  3. Laboratory of Disease Genomics and Individualized Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China

    Shiyou Li

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  1. Yanqiu Bai
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  2. Lianghui Ma
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  3. Shiyou Li
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Correspondence to Shiyou Li.

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Bai, Y., Ma, L. & Li, S. Development of a functional cell-based HTS assay for identification of NKCC1-negative modulators. Chin. Sci. Bull. 59, 630–638 (2014). https://doi.org/10.1007/s11434-013-0083-7

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  • DOI: https://doi.org/10.1007/s11434-013-0083-7

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