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.
N1 - This work was supported, in whole or in part, by National Institutes of Health Grant Grants NS039481 (to R. W. N.), NS069951 (to R. W. N.), NS071828 (to C. M. K.), MH059937 (to J. K. F.), and GM065830 (D.-O. D. M. and J. K. F.).
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 -