Abstract:

Calcium ion (Ca2+) has several physiological and pathophysiological functions such as communication, cell death and development in neurons. Normally, Ca2+ concentration is too high in out of the neurons (1-3 mM) as compared to the inside of the neurons (50-100 nM). Ca2+ passes the cell membranes through passive and active channels. Passive channels are leak channels. Well known active channels are including several channels such as voltage gated channels, chemical channels, store operated channels and mechanical channels (Kumar et al. 2014).  In addition, Ca2+ is released from intracellular organelles to cytosol by activation IP3 and ryanodine receptors. Apart from the well-known cell membrane Ca2+ channels, transient receptor potential (TRP) channels were discovered within the last decades. The TRP channels have 28 members within the 6 subgroups in mammalian. Activation and inhibition mechanisms of the TRP channels are very different from the well-known Ca2+ channels. For example, TRP vanilloid 1 (TRPV1) channel is activated by hot chili pepper component (capsaicin), acidic pH, high temperature and the vanilloids (Caterina et al. 1997).  TRP melastatin 2 (TRPM2) channel is activated by ADP-Ribose and NAD+. TRPM2 and TRPV1 channels are also activated by oxidative stress (Nazıroğlu and Braidy, 2017). In several neuronal diseases such as epilepsy and Alzheimer’s disease, intracellular free Ca2+ concentration is increased by the oxidative stress. Hence, measurement of intracellular free Ca2+ concentration is very important for discovering new calcium channel blocker drugs. In the cytosol of neurons, intracellular free Ca2+ concentration was measured by using Ca2+ indicators.       There are two main classes of calcium indicators namely chemical indicators and genetically encoded calcium indicators. Chemical indicators of free intracellular Ca2+ are Fura-2, Fluo-3, Fluo-4 and Rhod2. These dyes are often used with acetoxymethyl esters, in order to render the molecule lyphophlilic and to allow easy entrance into the cell. Genetically encoded indicators do not need to be loaded into cells, instead the genes encoding for these proteins can be easily transfected to cells. These indicators are fluorescent proteins derived from green fluorescent protein (GFP). In this presentation, I will summarize Ca2+ signaling and using the fluorescent dyes for Ca2+ imaging. In conclusion, intracellular free Ca2+ concentration can be measured by using the indicators. In the measurement techniques, laser confocal microscopy seems best technique.

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