Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor (InsP 3R1) has InsP 3-independent gating and disrupts intracellular Ca 2+ homeostasis

Abstract

The type 1 inositol 1,4,5-trisphosphate receptor (InsP 3R1) is a ubiquitous intracellular Ca 2+ release channel that is vital to intracellular Ca 2+ signaling. InsP 3R1 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 InsP 3R1 and utilized a recombinant truncated form of the channel (capn-InsP 3R1) corresponding to the stable, carboxyl-terminal fragment to examine the functional consequences of channel proteolysis. Single-channel recordings of capn-InsP 3R1 revealed InsP 3-independent gating and high open probability (P o) under optimal cytoplasmic Ca 2+ concentration ([Ca 2+] i) conditions. However, some [Ca 2+] i regulation of the cleaved channel remained, with a lower P o in suboptimal and inhibitory [Ca 2+] i. Expression of capn-InsP 3R1 in N2a cells reduced the Ca 2+ content of ionomycin-releasable intracellular stores and decreased endoplasmic reticulum Ca 2+ loading compared with control cells expressing full-length InsP 3R1. Using a cleavage-specific antibody, we identified calpain-cleaved InsP 3R1 in selectively vulnerable cerebellar Purkinje neurons after in vivo cardiac arrest. These findings indicate that calpain proteolysis of InsP 3R1 generates a dysregulated channel that disrupts cellular Ca 2+ homeostasis. Furthermore, our results demonstrate that calpain cleaves InsP 3R1 in a clinically relevant injury model, suggesting that Ca 2+ leak through the proteolyzed channel may act as a feed-forward mechanism to enhance cell death. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.