Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor (InsP₃R1) has InsP₃-independent gating and disrupts intracellular Ca²⁺ homeostasis

The type 1 inositol 1,4,5-trisphosphate receptor (InsP₃R1) is a ubiquitous intracellular Ca²⁺ release channel that is vital to intracellular Ca²⁺ signaling. InsP₃R1 is a proteolytic target of calpain, which cleaves the channel to form a 95-kDa carboxyl-terminal fragment that includes the transmembrane domains, which contain the ion pore. However, the functional consequences of calpain proteolysis on channel behavior and Ca ²⁺homeostasis are unknown. In the present study we have identified a unique calpain cleavage site in InsP₃R1 and utilized a recombinant truncated form of the channel (capn-InsP₃R1) corresponding to the stable, carboxyl-terminal fragment to examine the functional consequences of channel proteolysis. Single-channel recordings of capn-InsP₃R1 revealed InsP₃-independent gating and high open probability (P_o) under optimal cytoplasmic Ca²⁺ concentration ([Ca²⁺] _i) conditions. However, some [Ca²⁺] _i regulation of the cleaved channel remained, with a lower P_o in suboptimal and inhibitory [Ca²⁺] _i. Expression of capn-InsP₃R1 in N2a cells reduced the Ca²⁺ content of ionomycin-releasable intracellular stores and decreased endoplasmic reticulum Ca²⁺ loading compared with control cells expressing full-length InsP₃R1. Using a cleavage-specific antibody, we identified calpain-cleaved InsP₃R1 in selectively vulnerable cerebellar Purkinje neurons after in vivo cardiac arrest. These findings indicate that calpain proteolysis of InsP₃R1 generates a dysregulated channel that disrupts cellular Ca²⁺ homeostasis. Furthermore, our results demonstrate that calpain cleaves InsP₃R1 in a clinically relevant injury model, suggesting that Ca²⁺ leak through the proteolyzed channel may act as a feed-forward mechanism to enhance cell death.