Ca2+ Mobilization Assay

As G-protein-coupled receptors (GPCRs) can activate several signaling pathways dependent on G-protein subunits they bind to, there have been extensive efforts to develop new, sensitive, and easy-to-use assays to measure second messengers as readout for GPCR activation. Ca2+, serving as a second messenger or charge carrier, mediates the functional responses of a variety of membrane receptors and ion channels. Functional assays measuring changes in intracellular Ca2+ levels have been commonly used to screen for compounds that modulate the activities of target receptors or ion channels.

Ca2+ mobilization is the result of activation of GPCRs binding to specific Gα subunits Gq and Gi. Gq-coupled receptors activate the classical phospholipase C pathway, leading to an increase in inositol triphosphate (IP3) level. IP3 binds to IP3-sensitive calcium channels on the endoplasmic reticulum resulting in the release of calcium from the endoplasmic reticulum into the cytoplasm. Changes in intracellular calcium that reflect GPCR activity can be measured conveniently and accurately using calcium-sensitive dyes.

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