Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Establishment of a NanoBiT-Based Cytosolic Ca2+ Sensor by Optimizing Calmodulin-Binding Motif and Protein Expression Levels

  • Nguyen, Lan Phuong (Department of Biomedical Sciences, Korea University College of Medicine) ;
  • Nguyen, Huong Thi (Department of Biomedical Sciences, Korea University College of Medicine) ;
  • Yong, Hyo Jeong (Department of Biomedical Sciences, Korea University College of Medicine) ;
  • Reyes-Alcaraz, Arfaxad (College of Pharmacy, University of Houston) ;
  • Lee, Yoo-Na (Department of Biomedical Sciences, Korea University College of Medicine) ;
  • Park, Hee-Kyung (Department of Biomedical Sciences, Korea University College of Medicine) ;
  • Na, Yun Hee (Department of Biomedical Sciences, Korea University College of Medicine) ;
  • Lee, Cheol Soon (Department of Biomedical Sciences, Korea University College of Medicine) ;
  • Ham, Byung-Joo (Department of Psychiatry, Korea University College of Medicine) ;
  • Seong, Jae Young (Department of Biomedical Sciences, Korea University College of Medicine) ;
  • Hwang, Jong-Ik (Department of Biomedical Sciences, Korea University College of Medicine)
  • Received : 2020.07.04
  • Accepted : 2020.09.17
  • Published : 2020.11.30
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Abstract

Cytosolic Ca2+ levels ([Ca2+]c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca2+]c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca2+]c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca2+]c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca2+-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca2+]c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca2+]c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca2+]c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca2+]c in single cells and animal models.

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References

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