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Measuring IP3 Generation in Real-Time Using a NanoBiT Luminescence Biosensor.

Caroline M Gorvin1,2

  • 1Metabolism and Systems Science and Centre for Diabetes, Endocrinology and Metabolism (CEDAM), University of Birmingham, Birmingham, UK. c.gorvin@bham.ac.uk.

Methods in Molecular Biology (Clifton, N.J.)
|October 12, 2024
PubMed
Summary

This study presents a novel NanoBiT-IP3 luminescent biosensor assay for detecting intracellular calcium release. This method effectively assesses signaling pathways in live cells, crucial for understanding Gq/11-coupled receptor activity.

Keywords:
Calcium-sensing receptorGhrelin receptorGq/11 signalingLuminescencePhospholipase C

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Area of Science:

  • Cellular signaling pathways
  • G protein-coupled receptor (GPCR) research
  • Biochemical assay development

Background:

  • Gq/11-coupled receptors are vital for numerous physiological functions, including neurotransmission and calcium homeostasis.
  • Receptor activation triggers inositol 1,4,5-trisphosphate (IP3) production, leading to intracellular calcium release.
  • Existing methods for IP3 detection can be limited in live-cell applications.

Purpose of the Study:

  • To describe a novel NanoBiT-IP3 luminescent biosensor assay.
  • To enable real-time detection of IP3 generation in live cells.
  • To validate the assay for assessing signaling of specific Gq/11-coupled receptors.

Main Methods:

  • Development and implementation of a NanoBiT-IP3 luminescent biosensor.
  • Utilizing HEK293 cells for assay validation.
  • Measuring IP3 levels in response to receptor activation.

Main Results:

  • The NanoBiT-IP3 assay successfully detects increases in IP3 in live cells.
  • The assay demonstrated efficacy in assessing ghrelin receptor and calcium-sensing receptor signaling.
  • Luminescence signal correlates with IP3 generation, indicating assay sensitivity.

Conclusions:

  • The NanoBiT-IP3 luminescent biosensor provides a sensitive and robust method for studying Gq/11-coupled receptor signaling.
  • This assay facilitates live-cell analysis of IP3 dynamics and intracellular calcium mobilization.
  • The developed assay is applicable to various GPCRs involved in critical physiological processes.