S-glutathionylation activates STIM1 and alters mitochondrial homeostasis.

Oxidant stress influences many cellular processes, including cell growth, differentiation, and cell death. A well-recognized link between these processes and oxidant stress is via alterations in Ca(2+) signaling. However, precisely how oxidants influence Ca(2+) signaling remains unclear. Oxidant stress led to a phenotypic shift in Ca(2+) mobilization from an oscillatory to a sustained elevated pattern via calcium release-activated calcium (CRAC)-mediated capacitive Ca(2+) entry, and stromal interaction molecule 1 (STIM1)- and Orai1-deficient cells are resistant to oxidant stress. Functionally, oxidant-induced Ca(2+) entry alters mitochondrial Ca(2+) handling and bioenergetics and triggers cell death. STIM1 is S-glutathionylated at cysteine 56 in response to oxidant stress and evokes constitutive Ca(2+) entry independent of intracellular Ca(2+) stores. These experiments reveal that cysteine 56 is a sensor for oxidant-dependent activation of STIM1 and demonstrate a molecular link between oxidant stress and Ca(2+) signaling via the CRAC channel.

Molecular characterization of the inositol 1,4,5-trisphosphate receptor pore-forming segment.

Specific residues in the putative pore helix, selectivity filter, and S6 transmembrane helix of the inositol 1,4,5-trisphosphate receptor were mutated in order to examine their effects on channel function. Mutation of 5 of 8 highly conserved residues in the pore helix/selectivity filter region inactivated the channel (C2533A, G2541A, G2545A, G2546A, and G2547A). Of the remaining three mutants, C2527A and R2543A were partially active and G2549A behaved like wild type receptor. Mutation of a putative glycine hinge residue in the S6 helix (G2586A) or a putative gating residue at the cytosolic end of S6 helix (F2592A) had minimal effects on function, although channel function was inactivated by G2586P and F2592D mutations. The mutagenesis data are interpreted in the context of a structural homology model of the inositol 1,4,5-trisphosphate receptor.

Role of inositol 1,4,5-trisphosphate receptors in apoptosis in DT40 lymphocytes.

The role of inositol 1,4,5-trisphosphate receptors (IP(3)R) in caspase-3 activation and cell death was investigated in DT40 chicken B-lymphocytes stably expressing various IP(3)R constructs. Both full-length type-I IP(3)R and a truncated construct corresponding to the caspase-3 cleaved "channel-only" fragment were able to support staurosporine (STS)-induced caspase-3 activation and cell death even when the IP(3)R construct harbored a mutation that inactivates the pore of the Ca(2+) channel (D2550A). However, a full-length wild-type IP(3)R did not promote caspase-3 activation when the 159-amino acid cytosol-exposed C-terminal tail was deleted. STS caused an increase in cytosolic free Ca(2+) in DT40 cells expressing wild-type or pore-dead IP(3)R mutants. However, in the latter case all the Ca(2+) increase originated from Ca(2+) entry across the plasma membrane. Caspase-3 activation of pore-dead DT40 cells was also more sensitive to extracellular Ca(2+) chelation when compared with wild-type cells. STS-mediated release of cytochrome c into the cytosol and mitochondrial membrane potential depolarization could also be observed in DT40 cells lacking IP(3)Rs or containing the pore-dead mutant. We conclude that nonfunctional IP(3)Rs can sustain apoptosis in DT40 lymphocytes, because they facilitate Ca(2+) entry mechanisms across the plasma membrane. Although the intrinsic ion-channel function of IP(3)Rs is dispensable for apoptosis induced by STS, the C-terminal tail of IP(3)Rs appears to be essential, possibly reflecting key protein-protein interactions with this domain.

Mechanism of proteasomal degradation of inositol trisphosphate receptors in CHO-K1 cells.

myo-Inositol 1,4,5-trisphosphate receptor (IP3R) degradation occurs in response to carbachol (Cch) stimulation of CHO-K1 cells. The response was mediated by endogenous muscarinic receptors and was blocked by atropine or proteasomal inhibitors. We have used these cells to identify the sites of ubiquitination on IP3Rs and study the role of Ca2+ and substrate recognition properties of the degradation system using exogenously expressed IP3R constructs. Employing caspase-3 for IP3R cleavage, we show that Cch promotes polyubiquitination in the N-terminal domain and monoubiquitination in the C-terminal domain. The addition of extracellular Ca2+ to Ca2+-depleted Chinese hamster ovary (CHO) cells initiates IP3R degradation provided Cch is present. This effect is inhibited by thapsigargin. The data suggest that both a sustained elevation of IP3 and a minimal content of Ca2+ in the endoplasmic reticulum lumen is required to initiate IP3R degradation. Transient transfection of IP3R constructs into CHO cells indicated the selective degradation of only the SI+ splice variant of the type I IP3R. This was also the splice form present endogenously in these cells. A pore-defective, nonfunctional SI+ IP3R mutant (D2550A) was also degraded in Cch-stimulated cells. The Cch-mediated response in CHO cells provides a convenient model system to further analyze the Ca2+ dependence and structural requirements of the IP3R proteasomal degradation pathway.

Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis.

Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2*- that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2*--triggered Ca2+ mobilization preceded a loss in mitochondrial membrane potential that was independent of other oxidants and mitochondrially derived ROS. Activation of apoptosis occurred selectively in response to O2*- and could be prevented by [Ca2+]i buffering. This study provides evidence that O2*- facilitates an InsP3R-linked apoptotic cascade and may serve a critical function in I/R injury and inflammation.