Subcellular Ca 2+ Puffs Mediated By Different Inositol Trisphosphate Receptor Isoforms

The second messenger inositol trisphosphate (IP 3 ) evokes Ca 2+ liberation through IP 3 receptor/channels (IP 3 Rs) in the ER membrane to generate a hierarchy of cytosolic signals, including local Ca 2+ puffs that arise from concerted openings of clustered IP 3 Rs, and global Ca 2+ waves that propagate through the cell. Mammalian cells express three isoforms of IP 3 Rs that are encoded by different genes, have ~ 60–80% sequence homology and are reported to display differing functional characteristics. Imaging Ca 2+ puffs with single‐channel resolution provides important information on the localization and properties of IP 3 Rs in intact cells. However, interpretation has been complicated because studies have been limited to native cell types that typically express varying proportions of two or three IP 3 R isoforms, with additional complexity arising from hetero‐multimerization within the tetrameric IP 3 R. Here, we applied high‐resolution TIRF microscopy to image Ca 2+ puffs in HEK‐293 cell lines in which IP 3 R isoforms were knocked‐out in pairs by CRISPR/Cas9 technology to generate cells expressing exclusively IP 3 R types 1, 2 or 3. Puffs were evoked by photoreleased i‐IP 3 in all three cell lines, indicating that IP 3 Rs of each isoform are able to assemble into the clusters that underlie these local Ca 2+ signals. Moreover, Ca 2+ puffs mediated by all three isoforms displayed largely similar mean amplitudes, temporal characteristics and spatial extents, except that type 3 IP 3 R‐mediated puffs showed appreciably faster kinetics. A quantal analysis of fluorescence signals revealed the single‐channel Ca 2+ flux to be equivalent between isoforms, further suggesting that clusters of different IP 3 R isoforms contain similar numbers of active channels. Finally, mapping the distances between puff centroid localizations suggested that clusters of type 1 IP 3 Rs are more dispersed than clusters of type 2 or 3 IP 3 Rs. Overall, our results indicate that, although there is substantial functional redundancy among the IP 3 R isoforms, each exhibit distinct properties and spatial arrangements that likely shape the spatial‐temporal patterning of cellular Ca 2+ signals observed in native cells. Support or Funding Information This work was supported by NIH grants R01 DE019245 (D.I.Y) and R37 GM048071 (I.P) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .