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

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.