Establishment of a nanobit-based cytosolic Ca2+ sensor by optimizing calmodulin-binding motif and protein expression levels

Lan Phuong Nguyen, Huong Thi Nguyen, Hyo Jeong Yong, Arfaxad Reyes-Alcaraz, Yoo Na Lee, Hee Kyung Park, Yun Hee Na, Cheol Soon Lee, Byung Joo Ham, Jae Young Seong, Jong Ik Hwang

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

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.

Original languageEnglish
Pages (from-to)1-12
Number of pages12
JournalMolecules and cells
Volume43
Issue number10
DOIs
Publication statusPublished - 2020

Keywords

  • Calmodulin
  • Cytosolic Ca sensor
  • Internal ribosome entry site
  • Myosin light chainC kinase 1/2
  • NanoBiT assay

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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