Establishment of a NanoBiT-Based Cytosolic Ca2+ Sensor by Optimizing Calmodulin-Binding Motif and Protein Expression Levels.
Mol Cells
; 43(11): 909-920, 2020 Nov 30.
Article
in En
| MEDLINE
| ID: mdl-33162399
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, LgBiTSmBiT) 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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Calmodulin
/
Proteins
/
Cytosol
Type of study:
Prognostic_studies
Limits:
Humans
Language:
En
Journal:
Mol Cells
Journal subject:
BIOLOGIA MOLECULAR
Year:
2020
Document type:
Article
Country of publication:
COREA DEL SUR
/
CORÉIA DO SUL
/
KR
/
SOUTH KOREA