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

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.