Journal
MOLECULES AND CELLS
Volume 43, Issue 11, Pages 909-920Publisher
KOREAN SOC MOLECULAR & CELLULAR BIOLOGY
DOI: 10.14348/molcells.2020.0144
Keywords
calmodulin; cytosolic Ca2+ sensor; internal ribosome entry site; myosin light chainC kinase 1/2; NanoBiT assay
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Funding
- Korea Research Foundation - Ministry of Science and ICT [NRF-2019R1A2C1090051, NRF-2020M3E5D 9080792]
- Korea University Grant
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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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