All three IP₃ receptor isoforms generate Ca²⁺ puffs that display similar characteristics

Inositol 1,4,5-trisphosphate (IP₃) evokes Ca²⁺ release through IP₃ receptors (IP₃Rs) to generate both local Ca²⁺ puffs arising from concerted openings of clustered IP₃Rs and cell-wide Ca²⁺ waves. Imaging Ca²⁺ puffs with single-channel resolution yields information on the localization and properties of native IP₃Rs in intact cells, but interpretation has been complicated because cells express varying proportions of three structurally and functionally distinct isoforms of IP₃Rs. Here, we used TIRF and light-sheet microscopy to image Ca²⁺ puffs in HEK-293 cell lines generated by CRISPR-Cas9 technology to express exclusively IP₃R type 1, 2, or 3. Photorelease of the IP₃ analog i-IP₃ in all three cell lines evoked puffs with largely similar mean amplitudes, temporal characteristics, and spatial extents. Moreover, the single-channel Ca²⁺ flux was similar among isoforms, indicating that clusters of different IP₃R isoforms contain comparable numbers of active channels. Our results show that all three IP₃R isoforms cluster to generate local Ca²⁺ puffs and, contrary to findings of divergent properties from in vitro electrophysiological studies, display similar conductances and gating kinetics in intact cells.