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

ABSTRACT

The building blocks of intracellular Ca ²⁺ signals evoked by inositol 1,4,5-trisphosphate receptors (IP ₃Rs) are Ca ²⁺ puffs, transient focal increases in Ca ²⁺ concentration that reflect the opening of small clusters of IP ₃Rs. 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.

Abstract

Summary: Ca ²⁺ puffs are the building blocks for IP ₃-evoked Ca ²⁺ signals. Ca ²⁺ puffs evoked by caged IP ₃ or via endogenous signalling pathways initiate at the same fixed intracellular sites.