Endogenous signalling pathways and caged IP evoke Ca puffs at the same abundant immobile intracellular sites.

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