All three IP(3) receptor subtypes generate Ca(2+) puffs, the universal building blocks of IP(3)-evoked Ca(2+) signals

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.