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

Abstract

All three subtypes of inositol 1,4,5-trisphosphate receptor (IP₃R) are intracellular Ca²⁺ channels that are co-regulated by IP₃ and Ca²⁺ This allows IP₃Rs to evoke regenerative Ca²⁺ signals, the smallest of which are Ca²⁺ puffs that reflect the coordinated opening of a few clustered IP₃Rs. We use total internal reflection microscopy (TIRF) microscopy to record Ca²⁺ signals in HEK cells expressing all three IP₃R subtypes or a single native subtype. Ca²⁺ puffs are less frequent in cells expressing one IP₃R subtype, commensurate with them expressing fewer IP₃Rs than wild-type cells. However, all three IP₃R subtypes generate broadly similar Ca²⁺ puffs with similar numbers of IP₃Rs contributing to each. This suggests that IP₃R clusters may be assembled by conserved mechanisms that generate similarly sized clusters across different IP₃R expression levels. The Ca²⁺ puffs evoked by IP₃R2 had slower kinetics and more prolonged durations, which may be due to IP₃ binding with greater affinity to IP₃R2. We conclude that Ca²⁺ puffs are the building blocks for the Ca²⁺ signals evoked by all IP₃Rs.