Opening of the MPTP collapses the mitochondrial membrane potential, which results in release of the accumulated calcium from the matrix, causing a sudden and sustained increase in ﬂuorescence signal 7. Each addition causes a spike in signal that dissipates due to the uptake of calcium into the mitochondrial matrix. This assay is commonly performed by adding a ﬁxed volume of calcium chloride to a mitochondrial sample at known intervals. The calcium retention capacity of mitochondria can be measured using Calcium Green™-5N – a low-affinity membrane-impermeable dye that increases its fluorescence emission upon calcium binding – to analyse the opening and closing of the MPTP and study the efficacy of MPTP inhibitors. This leads to an increase in inner mitochondrial membrane permeability, which then permits unregulated movement of ions, including calcium 7 (Fig. Abnormal mitochondrial function is linked to a whole host of diseases and is the consequence of the heightened opening of the mitochondrial permeability transition pore (MPTP). Measuring calcium retention is a good indicator of mitochondrial function.

This leads to a release of Ca 2+ from the sarcoplasmic reticulum, which also initiates a contraction by activating Ca 2+-dependent contractile proteins. When studying events such as synaptic transmission at the neuromuscular junction, an action potential arrives at the cell membrane, the membrane potential is depolarised, causing voltage-gated L-type calcium channels to open to allow an influx of Ca 2+ into the cell.

Although these processes may display different mechanisms and mobilisation of calcium, there are common approaches to monitoring calcium levels.Ĭalcium flux assays detect intracellular calcium mobilisation in cells and follow the release of Ca 2+ into the cytoplasm 6. GPCRs, in particular, are common targets for a variety of drugs, as they are implicated in a diverse range of physiological processes and diseases. Such tests can assist in predicting potential unwanted drug side effects, and it is critical to have in vitro assays that mimic the in vivo environment as accurately as possible to ensure reliable predictive data.Ĭell-based calcium assays allow the study of intracellular calcium levels, which are important indicators for the activation state of ion channels and G-protein coupled receptors (GPCRs), as well as for following the cell damage and apoptosis pathways 5. In this way, studying calcium signaling can help in determining the mechanism of action (MOA) of drugs 4 prior to clinical trials. Even small calcium imbalances can therefore have pathogenic consequences, with links to neurological, endocrine, cardiovascular, pulmonary, digestive, and metabolic diseases, as well as cancers 4.Ĭalcium signaling can be studied in different cells and model organisms to gain a deeper understanding of the mechanisms of a range of physiological processes, including the pathogenesis of diseases such as cancers and neurodegenerative disorders. This allows the intracellular concentration of Ca 2+ to be maintained at the very low levels required for signaling. The unique chemistry of calcium ions allows complex molecules to easily bind, even in the presence of large amounts of other ions. It even acts as a cofactor for certain enzymes 3. For instance, calcium is important in the transmission of nerve impulses via neurotransmitter release but also aids in triggering muscle contraction by communicating with regulatory proteins that allow the interaction of actin and myosin. Calcium ions contribute to signal transduction by affecting local electrostatic fields and interacting with proteins to alter their conformations, and many receptors, channels, pumps, transporters, enzymes, and transcription factors play a role in the generation and translation of calcium signals 2. Calcium is a ubiquitous second messenger, involved in everything from the contractility of the cardiac and skeletal muscles to blood clotting, bone mineralisation, and the functioning of the nervous system.

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