As one of the ubiquitous second messengers, the intracellular Ca, has been revealed to be a pivotal regulator of various cellular functions. Two major sources are involved in the initiation of Ca-dependent signals: influx from the extracellular space and release from the intracellular Ca stores such as the endoplasmic/sarcoplasmic reticulum (ER/SR). To manipulate the Ca release from the stores under high spatiotemporal precision, we established a new method termed "organelle optogenetics." That is, one of the light-sensitive cation channels (channelrhodopsin-green receiver, ChRGR), which is Ca-permeable, was specifically targeted to the ER/SR. The expression specificity as well as the functional operation of the ER/SR-targeted ChRGR (ChRGR) was evaluated using mouse skeletal myoblasts (C2C12): (1) the ChRGR co-localized with the ER-marker KDEL; (2) no membrane current was generated by light under whole-cell clamp of cells expressing ChRGR; (3) an increase of fluorometric Ca was evoked by the optical stimulation (OS) in the cells expressing ChRGR in a manner independent on the extracellular Ca concentration ([Ca]); (4) the Δ/ was sensitive to the inhibitor of sarco/endoplasmic reticulum Ca-ATPase (SERCA) and (5) the store-operated Ca entry (SOCE) was induced by the OS in the ChRGR-expressing cells. Our organelle optogenetics effectively manipulated the ER/SR to release Ca from intracellular stores. The use of organelle optogenetics would reveal the neuroscientific significance of intracellular Ca dynamics under spatiotemporal precision.
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| http://dx.doi.org/10.3389/fnins.2018.00561 | DOI Listing |
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