TY - JOUR
T1 - Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) has InsP3-independent gating and disrupts intracellular Ca2+ homeostasis
AU - Kopil, Catherine M.
AU - Vais, Horia
AU - Cheung, King Ho
AU - Siebert, Adam P.
AU - Mak, Don On Daniel
AU - Foskett, J. Kevin
AU - Neumar, Robert W.
PY - 2011/10/14
Y1 - 2011/10/14
N2 - 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 (Po) under optimal cytoplasmic Ca2+ concentration ([Ca2+] i) conditions. However, some [Ca2+] i regulation of the cleaved channel remained, with a lower Po 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.
AB - 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 (Po) under optimal cytoplasmic Ca2+ concentration ([Ca2+] i) conditions. However, some [Ca2+] i regulation of the cleaved channel remained, with a lower Po 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.
UR - http://www.scopus.com/inward/record.url?scp=80053925001&partnerID=8YFLogxK
U2 - 10.1074/jbc.M111.254177
DO - 10.1074/jbc.M111.254177
M3 - Journal article
C2 - 21859719
SN - 0021-9258
VL - 286
SP - 35998
EP - 36010
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 41
ER -