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Related Experiment Video

Updated: Jun 23, 2025

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Using Fluorescent GAP Indicators to Monitor ER Ca2.

Jonathan Rojo-Ruiz1, Cinthia Sánchez-Rabadán1, Belen Calvo1

  • 1Unidad de Excelencia, Instituto de Biomedicina y Genética Molecular de Valladolid (IBGM), Universidad de Valladolid y Consejo Superior de Investigaciones Científicas (CSIC), Valladolid, Spain.

Current Protocols
|June 26, 2024
PubMed
Summary
This summary is machine-generated.

Accurate measurement of endoplasmic reticulum (ER) calcium (Ca2+) dynamics is now possible using the novel erGAP3 indicator. This genetically encoded tool allows for precise imaging of ER Ca2+, alone or with cytosolic Ca2+.

Keywords:
Ca2+ calibrationGFPaequorinfluorescenceimaging

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Area of Science:

  • Cell Biology
  • Biochemistry
  • Genetics

Background:

  • The endoplasmic reticulum (ER) is a critical calcium (Ca2+) reservoir.
  • Accurate measurement of ER Ca2+ dynamics has been a significant challenge.
  • Genetically encoded Ca2+ indicators offer potential solutions for live-cell imaging.

Purpose of the Study:

  • To develop and validate a novel genetically encoded Ca2+ indicator for ER lumen.
  • To enable quantitative and dynamic imaging of Ca2+ within the ER.
  • To provide methods for simultaneous ER and cytosolic Ca2+ monitoring.

Main Methods:

  • Engineering of a low Ca2+ affinity GFP-Aequorin Protein (GAP) variant (GAP3).
  • Targeting GAP3 to the ER lumen to create erGAP3.
  • Utilizing ratiometric fluorescence measurements for Ca2+ quantification.
  • Immunofluorescence for erGAP3 detection and Ca2+ imaging protocols.

Main Results:

  • erGAP3 enables accurate, quantitative measurement of ER Ca2+ concentrations ([Ca2+]ER).
  • The indicator is ratiometric, insensitive to Mg2+ and pH variations, and has a Hill coefficient of 1.
  • Simultaneous imaging of ER and cytosolic Ca2+ is achievable using erGAP3 with other indicators.

Conclusions:

  • erGAP3 is a robust tool for studying ER Ca2+ homeostasis.
  • The developed methods facilitate precise assessment of ER Ca2+ dynamics.
  • This technology opens new avenues for understanding Ca2+ signaling in various cellular processes.