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A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels.

Chiara Nardin1, Abraham Tettey-Matey1, Viola Donati1

  • 1CNR Institute of Biochemistry and Cell Biology, Monterotondo, 00015 Rome, Italy.

International Journal of Molecular Sciences
|July 9, 2022
PubMed
Summary

Researchers developed a novel all-optical assay to measure calcium uptake through connexin hemichannels. This method enables sensitive, high-throughput screening of potential drug candidates targeting these channels in various cell types.

Keywords:
bicistronic vectorscancerconnexinsdrug discoverygenetically encoded calcium indicatorsgenodermatoseslentivirusmonoclonal antibodies

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Area of Science:

  • Cell Biology
  • Biophysics
  • Pharmacology

Background:

  • Connexin (Cx) hemichannels (HCs) facilitate intercellular communication and metabolite exchange.
  • Dysregulated HC activity is linked to numerous diseases, driving interest in HC inhibitors as drug candidates.
  • Current methods for studying HCs, like electrophysiology and dye uptake, have limitations.

Purpose of the Study:

  • To develop and validate a sensitive, all-optical assay for measuring Cx HC activity.
  • To demonstrate the assay's utility in characterizing Cx HC inhibitors, such as monoclonal antibodies (mAbs).
  • To enable high-throughput screening of HC inhibitors and investigation of HCs in primary cells and microtissues.

Main Methods:

  • Utilized a Ca2+-selective genetically encoded indicator (GCaMP6s) for fluorometric measurement of cytosolic Ca2+ concentration ([Ca2+]cyt) changes.
  • Employed stable HaCaT cell lines overexpressing human Cx26, Cx46, or the Cx26G45E mutant under a Tet-on promoter.
  • Validated the assay using monoclonal antibodies targeting the extracellular domain of HCs on a spinning disk confocal fluorescence microscope.

Main Results:

  • Successfully demonstrated optical tracking of cytosolic Ca2+ concentration changes with high sensitivity.
  • Showcased the assay's effectiveness in characterizing monoclonal antibodies against Cx HCs.
  • Established a versatile platform adaptable for high-throughput screening and analysis of HCs in diverse biological contexts.

Conclusions:

  • The developed all-optical Ca2+ uptake assay offers a sensitive and efficient method for studying Cx HC function.
  • This assay facilitates the discovery and characterization of novel Cx HC inhibitors.
  • The methodology's adaptability supports broad applications in drug discovery and basic research involving HCs.