title: Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) has InsP(3)-independent gating and disrupts intracellular Ca(2+) homeostasis.
abstract: The type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) is a ubiquitous intracellular Ca(2+) release channel that is vital to intracellular Ca(2+) signaling. InsP(3)R1 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(3)R1 and utilized a recombinant truncated form of the channel (capn-InsP(3)R1) corresponding to the stable, carboxyl-terminal fragment to examine the functional consequences of channel proteolysis. Single-channel recordings of capn-InsP(3)R1 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(3)R1 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(3)R1. Using a cleavage-specific antibody, we identified calpain-cleaved InsP(3)R1 in selectively vulnerable cerebellar Purkinje neurons after in vivo cardiac arrest. These findings indicate that calpain proteolysis of InsP(3)R1 generates a dysregulated channel that disrupts cellular Ca(2+) homeostasis. Furthermore, our results demonstrate that calpain cleaves InsP(3)R1 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.
authors.affiliation: Department of Emergency Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Kopil
authors.affiliation: Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Horia
authors.initials: H
authors.lastname: Vais
authors.affiliation: Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104; Department of Physiology, University of Hong Kong, Pok Fu Lam, Hong Kong, China.
authors.forename: King-Ho
authors.initials: KH
authors.lastname: Cheung
authors.affiliation: Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Siebert
authors.affiliation: Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Don-On Daniel
authors.initials: DD
authors.lastname: Mak
authors.affiliation: Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104; Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: J Kevin
authors.initials: JK
authors.lastname: Foskett
authors.affiliation: Department of Emergency Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Electronic address: neumarr@uphs.upenn.edu.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Neumar
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D061207:Calcium Ionophores / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects*; D002154:Calpain / Q000378:metabolism / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006706:Homeostasis / Q000187:drug effects*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects*; D015759:Ionomycin / Q000494:pharmacology; D008297:Male; D059748:Proteolysis / Q000187:drug effects; D011689:Purkinje Cells / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans
chemical_list: D015220:Calcium Channels; D061207:Calcium Ionophores; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015759:Ionomycin; D002154:Calpain; D002118:Calcium