Endogenous signalling pathways and caged IP(3) evoke Ca(2+) puffs at the same abundant immobile intracellular sites

The building blocks of intracellular Ca(2+) signals evoked by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are Ca(2+) puffs, transient focal increases in Ca(2+) concentration that reflect the opening of small clusters of IP(3)Rs. We use total internal reflection fluorescence microscopy and automated analyses to detect Ca(2+) puffs evoked by photolysis of caged IP(3) or activation of endogenous muscarinic receptors with carbachol in human embryonic kidney 293 cells. Ca(2+) puffs evoked by carbachol initiated at an estimated 65±7 sites/cell, and the sites remained immobile for many minutes. Photolysis of caged IP(3) evoked Ca(2+) puffs at a similar number of sites (100±35). Increasing the carbachol concentration increased the frequency of Ca(2+) puffs without unmasking additional Ca(2+) release sites. By measuring responses to sequential stimulation with carbachol or photolysed caged IP(3), we established that the two stimuli evoked Ca(2+) puffs at the same sites. We conclude that IP(3)-evoked Ca(2+) puffs initiate at numerous immobile sites and the sites become more likely to fire as the IP(3) concentration increases; there is no evidence that endogenous signalling pathways selectively deliver IP(3) to specific sites.