Calpain-cleaved Type 1 Inositol 1,4,5-Trisphosphate Receptor (InsP3R1) Has InsP3-independent Gating and Disrupts Intracellular Ca2+ Homeostasis

Abstract

(8), and ischemia (9). Observations in brain ischemia models also suggest altered InsP 3 R function, specifically, decreased InsP 3 binding (10, 11), decreased InsP 3 -induced Ca 2ϩ release (12), and depletion of releasable Ca 2ϩ stores (13,14). Proteolytic cleavage of InsP 3 R could explain these observations.The type 1 InsP 3 R (InsP 3 R1), the predominant neuronal isoform, is a substrate for both the caspase and calpain families of cysteine proteases (15). These proteases are indirectly (caspase-3) and directly (calpain) activated by Ca 2ϩ and are known to play a central role in apoptotic and necrotic cell death pathways (16). Proteolytic activity of these enzymes is limited and site-specific, typically altering rather than eliminating substrate function. In the case of Ca 2ϩ regulatory proteins, this may initiate a positive feedback loop that further increases protease activation via increases in [Ca 2ϩ ] i . Caspase-3 or calpain cleavage of InsP 3 R1 generates carboxylterminal fragments of ϳ95 kDa (17). Caspase-3 cleaves InsP 3 R1 at a highly conserved DEVD consensus sequence within the coupling domain (17) (Fig. 1A). Caspase-mediated proteolysis of the channel has been observed in several models of apoptosis (17)(18)(19). Previous studies have demonstrated altered ER Ca

Fragmented Inositol 1,4,5-Trisphosphate Receptors Retain Tetrameric Architecture and Form Functional Ca2+ Release Channels

“…7E. Previous studies showed that transient expression of the C-terminal 95-kDa-fragment resulted in a modest depletion of ER stores (22,27,30). However, examination of resting Ca 2ϩ levels and the content of intracellular Ca 2ϩ stores, as judged by the rate and magnitude of cyclopiazonic acid -induced Ca 2ϩ release, showed no significant difference between these cell lines, expressing channel-only fragments and IP 3 R1 cells or FIGURE 7.…”

“…Fig. 1C shows the predicted N-terminal and C-terminal fragments resulting from calpain cleavage of IP 3 R1 after glutamic acid residue 1917 (30). Finally, the membrane and soluble fragments of IP 3 R2, generated at a site corresponding to calpain cleavage of IP 3 R1, together with the epitope locations of ␣-IP 3 R2 antibodies used in this study, are shown in Fig.…”

“…There was a lower band detected with the C-terminal antibody UCD but not with SC (an epitope located within residues aa 1894 -1973), and, therefore, we concluded that this lower band could result from an alternative translation or a degradation product. IP 3 R1 is also a known substrate for calpain, and cleavage has been reported to result in fragments, including an ϳ95-kDa membrane retained fragment analogous to those produced by caspase activity on IP 3 R1 (30,37,38). Further, recent reports have suggested that calpain-mediated IP 3 R1 proteolysis is implicated in the disruption of Ca 2ϩ homeostasis during ischemic brain injury (30,39).…”

“…The NotI-NotI fragment coding for R1 casp mem was inserted into the TPV that was digested with NotI. IP 3 R1 fragments corresponding to cytosolic (R1 calp sol) and membrane (R1 calp mem) fragments, predicted to result from calpain cleavage rIP 3 R1 after glutamic acid 1917 (30) (Fig. 1C), were constructed in an identical manner using 5Ј-CCGGGATCAGCTCTTGGAATAGAATTCG-CGGCCGCGCTAGCATGGCATCTGCTGCCACCAGAAA-AGCC-3Ј, which introduced a stop codon after glutamic acid 1917 and a Kozak sequence and initiation methionine in-frame with the membrane fragment to ensure efficient expression.…”

“…IP 3 R1 is also a known substrate for calpain, and cleavage has been reported to result in fragments, including an ϳ95-kDa membrane retained fragment analogous to those produced by caspase activity on IP 3 R1 (30,37,38). Further, recent reports have suggested that calpain-mediated IP 3 R1 proteolysis is implicated in the disruption of Ca 2ϩ homeostasis during ischemic brain injury (30,39). Therefore, to extend these studies, IP 3 R1 constructs encoding fragments on the basis of the putative calpain cleavage site after aa residue 1917 were generated.…”