All three IP 3 receptor isoforms generate Ca 2+ puffs that display similar characteristics

Abstract

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