Oxidant stress induces constitutive calcium entry by tacking glutathiones onto the Orai CRAC channel activator STIM1.
Inositol 1,4,5-trisphosphate receptors (IP 3 R) 2 are a family of intracellular release channels that play an essential role in evoking Ca 2ϩ signals triggered by the occupation of numerous types of cell-surface receptors that are coupled to enhanced inositol-lipid turnover (1). Three different IP 3 R isoforms have been identified, and most cells appear to express multiple isoforms (2, 3). IP 3 receptors have been shown to be substrates for several different protein kinases in vivo and/or in vitro. These include protein kinase A (4, 5), protein kinase G (6, 7), protein kinase C (8), CaM kinase-II (8), Fyn tyrosine kinase (9, 10) and cdk1/CyB (11). By far the best-characterized of these effects are those of protein kinases A and G. Two serine residues have been identified as potential sites of phosphorylation in the type I IP 3 R isoform (12). Mutagenesis studies in a DT40 IP 3 R expression system suggest that both protein kinases A and G act to enhance IP 3 -mediated Ca 2ϩ release (13). However, the preferred sites of phosphorylation and functional effects on IP 3 R channel function may differ in different cell types and between alternatively spliced variants (6, 13-15). The regulatory role of the other protein kinases phosphorylating IP 3 Rs are poorly understood.The serine/threonine protein kinase Akt/protein kinase B is the cellular homologue of the viral oncogene v-Akt and is activated by various growth factors and cytokines. The membrane translocation and initial activation of the enzyme is dependent on the production of 3-phosphorylated inositol lipids catalyzed by PI 3-kinase. The activated Akt kinase then phosphorylates a number of key substrates involved in the stimulation of intermediary metabolism and promotion of cell survival, proliferation, and growth (reviewed in ). An examination of the sequence of all three IP 3 R isoforms indicates the presence of a consensus RXRXX(S/T) sequence for phosphorylation by Akt kinase (19). In the present study, we have shown for the first time that IP 3 Rs are substrates for activated Akt kinase in vivo and have investigated several possible functional consequences of this phosphorylation on Ca 2ϩ signaling.
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.
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.Inositol 1,4,5-trisphosphate receptors (IP 3 Rs) 3 are tetrameric ligand-gated ion channels located in the endoplasmic reticulum membrane that are co-activated by IP 3 and Ca 2ϩ (reviewed in Ref. 1). The Ca 2ϩ released through these channels can activate a diverse array of physiological processes, depending on the amplitude and frequency of Ca 2ϩ release (2). The IP 3 R contains a ligand-binding domain in the N-terminal region (amino acids 226 -576) and six transmembrane segments (S1-S6; amino acids 2276 -2590) in the C-terminal region of the receptor (3, 4). Several key aspects of IP 3 R function remain to be addressed, including the identification of critical residues that line the channel conduction pore and the exact mechanism of the IP 3 -activated gating process.In common with many voltage-gated ion channels, the IP 3 R and its close relative the ryanodine receptor (RyR) contain a short luminal pore helix and a selectivity filter (1). In the IP 3 R, these are located between the S5 and S6 helices. Experimental evidence suggests that the minimal channel domain of the IP 3 R lies in the portion of the receptor encoded by the sequences distal to the S5 helix (5). Previous mutagenesis studies of this region of the IP 3 R have focused on the selectivity filter sequence 2547 GVGD 2550 (numbering according to rat type-I IP 3 R). The results showed that the V2548I mutation increased conductance through the channel (6, 7), that the D2550A mutation inactivated channel function (6), and that the D2550E mutation decreased the divalent cation selectivity of the channel (6). Examination of the sequences of all three IP 3 Rs and RyR isoforms from several species in the region of the putative pore helix and selectivity filter indicates the presence of 8 highly conserved residues. In the present study, we have examined the effect of mutating these conserved amino acids on IP 3 R function.The N-terminal ligand-binding domain and the channel domain of the receptor show a direct intersubunit interaction in the tetrameric channel (8). A model for the gating mechanism of the IP 3 R channel has been proposed in which the N-terminal region of the receptor interacts with the channel domain ...
Inter-␣-trypsin inhibitor-4 (Itih-4) is a liver-restricted member of the serine protease inhibitor family with diverse functions as an anti-apoptotic and matrix stabilizing molecule that are important throughout development. We investigate the functional role of Itih-4 in liver formation, regeneration (LR) and examine its role in calcium and hyaluronic acid binding. Itih-4 expression is prominent in early liver development at E9 and later at E16, being restricted to hepatoblasts, immature hepatocytes, and differentiated hepatocytes. We note a marked and differential increase in Itih-4 labeling in proliferating hepatocytes, compared with bile duct cells in liver explant cultures treated with interleukin-6 (IL-6). After partial hepatectomy, maximal Itih-4 expression occurs in a bimodal manner at 30 min and at 12 hr, with a predominant centrizonal distribution. There is no detectable binding of glutathione transferase-fusion Itih-4 protein to calcium and hyaluronic acid, indicating a possible requirement for posttranslational modifications for these functions. These results suggest that in LR, Itih-4 expression corresponds to that of immediate early genes and may contribute to the entry of normally quiescent hepatocytes into the early stages of the cell cycle. The markedly high expression of Itih-4 in early liver development and in explants treated with IL-6 suggests a prominent role for Itih-4 at key points in liver formation.
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