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All three subtypes of inositol 1,4,5-trisphosphate receptor (IP 3 R) are intracellular Ca 2+ channels that are co-regulated by IP 3 and Ca 2+ This allows IP 3 Rs to evoke regenerative Ca 2+ signals, the smallest of which are Ca 2+ puffs that reflect the coordinated opening of a few clustered IP 3 Rs. We use total internal reflection microscopy (TIRF) microscopy to record Ca 2+ signals in HEK cells expressing all three IP 3 R subtypes or a single native subtype. Ca 2+ puffs are less frequent in cells expressing one IP 3 R subtype, commensurate with them expressing fewer IP 3 Rs than wild-type cells. However, all three IP 3 R subtypes generate broadly similar Ca 2+ puffs with similar numbers of IP 3 Rs contributing to each. This suggests that IP 3 R clusters may be assembled by conserved mechanisms that generate similarly sized clusters across different IP 3 R expression levels. The Ca 2+ puffs evoked by IP 3 R2 had slower kinetics and more prolonged durations, which may be due to IP 3 binding with greater affinity to IP 3 R2. We conclude that Ca 2+ puffs are the building blocks for the Ca 2+ signals evoked by all IP 3 Rs.

All three IP3 receptor subtypes generate Ca2+ puffs, the universal building blocks of IP3-evoked Ca2+ signals