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http://dx.doi.org/10.14348/molcells.2020.0144

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)
Publication Information
Molecules and Cells / v.43, no.11, 2020 , pp. 909-920 More about this Journal
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.
Keywords
calmodulin; cytosolic $Ca^{2+}$ sensor; internal ribosome entry site; myosin light chainC kinase 1/2; NanoBiT assay;
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