Endogenous signalling pathways and caged IP3 evoke Ca2+ puffs at the same abundant immobile intracellular sites.

Abstract

The building blocks of intracellular Ca2+ signals evoked by inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+ puffs, transient focal increases in Ca2+ concentration that reflect the opening of small clusters of IP3Rs. We use total internal reflection fluorescence microscopy and automated analyses to detect Ca2+ puffs evoked by photolysis of caged IP3 or activation of endogenous muscarinic receptors with carbachol in human embryonic kidney 293 cells. Ca2+ puffs evoked by carbachol initiated at an estimated 65±7 sites/cell, and the sites remained immobile for many minutes. Photolysis of caged IP3 evoked Ca2+ puffs at a similar number of sites (100±35). Increasing the carbachol concentration increased the frequency of Ca2+ puffs without unmasking additional Ca2+ release sites. By measuring responses to sequential stimulation with carbachol or photolysed caged IP3, we established that the two stimuli evoked Ca2+ puffs at the same sites. We conclude that IP3-evoked Ca2+ puffs initiate at numerous immobile sites and the sites become more likely to fire as the IP3 concentration increases; there is no evidence that endogenous signalling pathways selectively deliver IP3 to specific sites.

DOI: 10.1242/jcs.208520

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Cite this paper

@article{Keebler2017EndogenousSP, title={Endogenous signalling pathways and caged IP3 evoke Ca2+ puffs at the same abundant immobile intracellular sites.}, author={Michael V Keebler and Colin W Taylor}, journal={Journal of cell science}, year={2017}, volume={130 21}, pages={3728-3739} }