Endoplasmic reticulum Ca2+ dysregulation and endoplasmic reticulum stress following in vitro neuronal ischemia: role of Na+-K+-Cl- cotransporter

We investigated the role of Na(+)-K(+)-Cl(-) cotransporter (NKCC1) in conjunction with Na(+)/Ca(2+) exchanger (NCX) in disruption of endoplasmic reticulum (ER) Ca(2+) homeostasis and ER stress development in primary cortical neurons following in vitro ischemia. Oxygen-glucose deprivation (OGD) and reoxygenation (REOX) caused a rise in [Na(+)](cyt) which was accompanied by an elevation in [Ca(2+)](cyt). Inhibition of NKCC1 with its potent inhibitor bumetanide abolished the OGD/REOX-induced rise in [Na(+)](cyt) and [Ca(2+)](cyt). Moreover, OGD significantly increased Ca(2+)(ER) accumulation. Following REOX, a biphasic change in Ca(2+)(ER) occurred with an initial release of Ca(2+)(ER) which was sensitive to inositol 1,4,5-trisphosphate receptor (IP(3)R) inhibition and a subsequent refilling of Ca(2+)(ER) stores. Inhibition of NKCC1 activity with its inhibitor or genetic ablation prevented the release of Ca(2+)(ER). A similar result was obtained with inhibition of reversed mode operation of NCX (NCX(rev)). OGD/REOX also triggered a transient increase of glucose regulated protein 78 (GRP78), phospho-form of the alpha subunit of eukaryotic initiation factor 2 (p-eIF2alpha), and cleaved caspase 12 proteins. Pre-treatment of neurons with NKCC1 inhibitor bumetanide inhibited upregulation of GRP78 and attenuated the level of cleaved caspase 12 and p-eIF2alpha. Inhibition of NKCC1 reduced cytochrome C release and neuronal death. Taken together, these results suggest that NKCC1 and NCX(rev) may be involved in ischemic cell damage in part via disrupting ER Ca(2+) homeostasis and ER function.     

Sustained ER Ca2+ depletion suppresses protein synthesis and induces activation-enhanced cell death in mast cells

Depletion of Ca(2+) from the endoplasmic reticulum (ER) induces large increases in cytoplasmic Ca(2+), mitochondrial Ca(2+) loading, protein synthesis inhibition, and cell death. To clarify the connections among these events, we have evaluated the effect of Ca(2+) mobilizing agents thapsigargin (Tg), econazole (Ec), and the growth factor Steel Factor (SLF) on bone marrow-derived mast cells (BMMCs). BMMC Ca(2+) stores were found to consist of a Tg-sensitive ER compartment, the Tg-insensitive SIC store, and mitochondrial stores. Low levels of Ec interfered with Tg-stimulated mitochondrial loading while promoting progressive leakage of Ca(2+) from the ER. Low levels of Ec completely reversed Tg toxicity while higher levels blocked store-operated influx and induced cell death in a SLF-enhanced manner. Both Ec and Tg inhibited protein synthesis, however, only SLF plus Tg or very high levels of Ec were able to significantly stimulate EIF-2alpha phosphorylation. Cycloheximide only partially protected BMMCs from Tg toxicity yet strongly synergized with Ec to induce cell death. These results therefore indicate that although both Tg and Ec deplete ER Ca(2+) levels, Ec-induced cell death results from sustained protein synthesis inhibition while Tg toxicity results primarily from mitochondrial Ca(2+) overload and secondarily from ER stress associated with Ca(2+) depletion.     

Low cytoplasmic [Ca(2+)] activates I(CRAC) independently of global Ca(2+) store depletion in RBL-1 cells

Release of Ca(2+) from inositol (1,4,5)-trisphosphate-sensitive Ca(2+) stores causes "capacitative calcium entry," which is mediated by the so-called "Ca(2+) release-activated Ca(2+) current" (I(CRAC)) in RBL-1 cells. Refilling of the Ca(2+) stores or high cytoplasmic [Ca(2+)] ([Ca(2+)](cyt)) inactivate I(CRAC). Here we address the question if also [Ca(2+)](cyt) lower than the resting [Ca(2+)](cyt) influences store-operated channels. We therefore combined patch clamp and mag fura-2 fluorescence methods to determine simultaneously both I(CRAC) and [Ca(2+)] within Ca(2+) stores of RBL-1 cells ([Ca(2+)](store)). We found that low [Ca(2+)](cyt) in the range of 30-50 nM activates I(CRAC) and Ca(2+) influx spontaneously and independently of global Ca(2+) store depletion, while elevation of [Ca(2+)](cyt) to the resting [Ca(2+)](cyt) (100 nM) resulted in store dependence of I(CRAC) activation. We conclude that spontaneous activation of I(CRAC) by low [Ca(2+)](cyt) could serve as a feedback mechanism keeping the resting [Ca(2+)](cyt) constant.     

ERK1/2-p90RSK-mediated phosphorylation of Na+/H+ exchanger isoform 1. A role in ischemic neuronal death

The function and regulation of Na(+)/H(+) exchanger isoform 1 (NHE1) following cerebral ischemia are not well understood. In this study, we demonstrate that extracellular signal-related kinases (ERK1/2) play a role in stimulation of neuronal NHE1 following in vitro ischemia. NHE1 activity was significantly increased during 10-60 min reoxygenation (REOX) after 2-h oxygen and glucose deprivation (OGD). OGD/REOX not only increased the V(max) for NHE1 but also shifted the K(m) toward decreased [H(+)](i). These changes in NHE1 kinetics were absent when MAPK/ERK kinase (MEK) was inhibited by the MEK inhibitor U0126. There were no changes in the levels of phosphorylated ERK1/2 (p-ERK1/2) after 2 h OGD. The p-ERK1/2 level was significantly increased during 10-60 min REOX, which was accompanied by nuclear translocation. U0126 abolished REOX-induced elevation and translocation of p-ERK1/2. We further examined the ERK/90-kDa ribosomal S6 kinase (p90(RSK)) signaling pathways. At 10 min REOX, phosphorylated NHE1 was increased with a concurrent elevation of phosphorylation of p90(RSK), a known NHE1 kinase. Inhibition of MEK activity with U0126 abolished phosphorylation of both NHE1 and p90(RSK). Moreover, neuroprotection was observed with U0126 or genetic ablation or pharmacological inhibition of NHE1 following OGD/REOX. Taken together, these results suggest that activation of ERK1/2-p90(RSK) pathways following in vitro ischemia phosphorylates NHE1 and increases its activity, which subsequently contributes to neuronal damage.     

Local cytosolic Ca2+ elevations are required for stromal interaction molecule 1 (STIM1) de-oligomerization and termination of store-operated Ca2+ entry

The Ca(2+) depletion of the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca(2+) entry (SOCE) pathway that sustains long-term Ca(2+) signals critical for cellular functions. ER Ca(2+) depletion initiates the oligomerization of stromal interaction molecules (STIM) that control SOCE activation, but whether ER Ca(2+) refilling controls STIM de-oligomerization and SOCE termination is not known. Here, we correlate the changes in free luminal ER Ca(2+) concentrations ([Ca(2+)](ER)) and in STIM1 oligomerization, using fluorescence resonance energy transfer (FRET) between CFP-STIM1 and YFP-STIM1. We observed that STIM1 de-oligomerized at much lower [Ca(2+)](ER) levels during store refilling than it oligomerized during store depletion. We then refilled ER stores without adding exogenous Ca(2+) using a membrane-permeable Ca(2+) chelator to provide a large reservoir of buffered Ca(2+). This procedure rapidly restored pre-stimulatory [Ca(2+)](ER) levels but did not trigger STIM1 de-oligomerization, the FRET signals remaining elevated as long as the external [Ca(2+)] remained low. STIM1 dissociation evoked by Ca(2+) readmission was prevented by SOC channel inhibition and was associated with cytosolic Ca(2+) elevations restricted to STIM1 puncta, indicating that Ca(2+) acts on a cytosolic target close to STIM1 clusters. These data indicate that the refilling of ER Ca(2+) stores is not sufficient to induce STIM1 de-oligomerization and that localized Ca(2+) elevations in the vicinity of assembled SOCE complexes are required for the termination of SOCE.     

Rapid turnover of calcium in the endoplasmic reticulum during signaling. Studies with cameleon calcium indicators

HEK293 cells expressing the thyrotropin-releasing hormone (TRH) receptor were transfected with cameleon Ca(2+) indicators designed to measure the free Ca(2+) concentration in the cytoplasm, [Ca(2+)](cyt), and the endoplasmic reticulum (ER), [Ca(2+)](er). Basal [Ca(2+)](cyt) was about 50 nm; thyrotropin-releasing hormone (TRH) or other agonists increased [Ca(2+)](cyt) to 1 micrometer or higher. Basal [Ca(2+)](er) averaged 500 micrometer and fell to 50-100 micrometer over 10 min in the presence of thapsigargin. TRH consistently decreased [Ca(2+)](er) to 100 micrometer, independent of extracellular Ca(2+), whereas agonists for endogenous receptors generally caused a smaller decline. When added with thapsigargin, all agonists rapidly decreased [Ca(2+)](er) to 5-10 micrometer, indicating that there is substantial store refilling during signaling. TRH increased [Ca(2+)](cyt) and decreased [Ca(2+)](er) if applied after other agonists, whereas other agonists did not alter [Ca(2+)](cyt) or [Ca(2+)](er) if added after TRH. When Ca(2+) was added back to cells that had been incubated with TRH in Ca(2+)-free medium, [Ca(2+)](cyt) and [Ca(2+)](er) increased rapidly. The increase in [Ca(2+)](er) was only partially blocked by thapsigargin but was completely blocked if cells were loaded with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In conclusion, these new Ca(2+) indicators showed that basal [Ca(2+)](er) is approximately 500 micrometer, that [Ca(2+)](er) has to be >100 micrometer to support an increase in [Ca(2+)](cyt) by agonists, and that during signaling, intracellular Ca(2+) stores are continuously refilled with cytoplasmic Ca(2+) by the sarcoendoplasmic reticulum Ca(2+)-ATPase pump.     

ER Ca2+ depletion triggers apoptotic signals for endoplasmic reticulum (ER) overload response induced by overexpressed reticulon 3 (RTN3/HAP)

Perturbance of endoplasmic reticulum (ER) function, either by the mutant proteins not folding correctly, or by an excessive accumulation of proteins in the organelle, will lead to the unfolded protein response (UPR) or ER overload response (EOR). The signal-transducing pathways for UPR have been identified, whereas the pathway for EOR remains to be elucidated. Our previous study demonstrated that the overexpression of reticulon 3 (RTN3, also named HAP, homologue of ASY protein) caused apoptosis with the depletion of ER Ca(2+) stores. In present research, we characterized RTN3 as a novel EOR-induced protein, triggering the apoptotic signals through the release of ER Ca(2+) and the elevation of cytosolic Ca(2+). Our studies showed that overexpressed RTN3 induced EOR, eliciting ER-specific apoptosis with activation of caspase-12 and mitochondrial dysfunction through ER Ca(2+) depletion and the sustained elevation of cytosolic Ca(2+). Furthermore, we demonstrated that overexpressed RTN3 and stimuli that activate both EOR and UPR, not UPR only, were able to induce up-regulation of inducible nitric oxide synthase (iNOS) in HeLa cells through ER Ca(2+) release and reactive oxygen intermediates (ROIs), resulting in endogenous calcium-dependent nitric oxide protecting cells against ER specific apoptosis, which suggested that the nitric oxide and iNOS represented a likely protective response to EOR, not the UPR. These results supported that the release of ER Ca(2+) stores triggered the initial signal-transducing pathways for EOR induced by overexpressed RTN3.     

Store-operated Ca2+ entry in astrocytes: different spatial arrangement of endoplasmic reticulum explains functional diversity in vitro and in situ

Ca(2+) signaling is the astrocyte form of excitability and the endoplasmic reticulum (ER) plays an important role as an intracellular Ca(2+) store. Since the subcellular distribution of the ER influences Ca(2+) signaling, we compared the arrangement of ER in astrocytes of hippocampus tissue and astrocytes in cell culture by electron microscopy. While the ER was usually located in close apposition to the plasma membrane in astrocytes in situ, the ER in cultured astrocytes was close to the nuclear membrane. Activation of metabotropic receptors linked to release of Ca(2+) from ER stores triggered distinct responses in cultured and in situ astrocytes. In culture, Ca(2+) signals were commonly first recorded close to the nucleus and with a delay at peripheral regions of the cells. Store-operated Ca(2+) entry (SOC) as a route to refill the Ca(2+) stores could be easily identified in cultured astrocytes as the Zn(2+)-sensitive component of the Ca(2+) signal. In contrast, such a Zn(2+)-sensitive component was not recorded in astrocytes from hippocampal slices despite of evidence for SOC. Our data indicate that both, astrocytes in situ and in vitro express SOC necessary to refill stores, but that a SOC-related signal is not recorded in the cytoplasm of astrocytes in situ since the stores are close to the plasma membrane and the refill does not affect cytoplasmic Ca(2+) levels.     

Upregulated TRP and enhanced capacitative Ca(2+) entry in human pulmonary artery myocytes during proliferation

A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) influx through plasmalemmal Ca(2+) channels plays a critical role in mitogen-mediated cell growth. Depletion of intracellular Ca(2+) stores triggers capacitative Ca(2+) entry (CCE), a mechanism involved in maintaining Ca(2+) influx and refilling intracellular Ca(2+) stores. Transient receptor potential (TRP) genes have been demonstrated to encode the store-operated Ca(2+) channels that are activated by Ca(2+) store depletion. In this study, we examined whether CCE, activity of store-operated Ca(2+) channels, and human TRP1 (hTRP1) expression are essential in human pulmonary arterial smooth muscle cell (PASMC) proliferation. Chelation of extracellular Ca(2+) and depletion of intracellularly stored Ca(2+) inhibited PASMC growth in media containing serum and growth factors. Resting [Ca(2+)](cyt) as well as the increases in [Ca(2+)](cyt) due to Ca(2+) release and CCE were all significantly greater in proliferating PASMC than in growth-arrested cells. Consistently, whole cell inward currents activated by depletion of intracellular Ca(2+) stores and the mRNA level of hTRP1 were much greater in proliferating PASMC than in growth-arrested cells. These results suggest that elevated [Ca(2+)](cyt) and intracellularly stored [Ca(2+)] play an important role in pulmonary vascular smooth muscle cell growth. CCE, potentially via hTRP1-encoded Ca(2+)-permeable channels, may be an important mechanism required to maintain the elevated [Ca(2+)](cyt) and stored [Ca(2+)] in human PASMC during proliferation.     

Evaluation, using targeted aequorins, of the roles of the endoplasmic reticulum and its (Ca2++Mg2+)ATP-ases in the activation of store-operated Ca2+ channels in liver cells

The process by which store-operated Ca2+ channels (SOCs) deliver Ca2+ to the endoplasmic reticulum (ER) and the role of (Ca2++Mg2+)ATP-ases of the ER in the activation of SOCs in H4-IIE liver cells were investigated using cell lines stably transfected with apo-aequorin targeted to the cytoplasmic space or the ER. In order to measure the concentration of Ca2+ in the ER ([Ca2+]er), cells were pre-treated with 2,5-di-tert-butylhydroquinone (DBHQ) to deplete Ca2+ in the ER before reconstitution of holo-aequorin. The addition of extracellular Ca2+ (Cao2+) to Ca2+-depleted cells induced refilling of the ER, which was complete within 5 min. This was associated with a sharp transient increase in the cytoplasmic Ca2+ concentration ([Ca2+]cyt) of about 15 s duration (a Cao2+-induced [Ca2+]cyt spike) after which [Ca2+]cyt remained elevated slightly above the basal value for a period of about 2 min (low [Ca2+]cyt plateau). The Cao2+-induced [Ca2+]cyt spike was inhibited by Gd3+, not affected by tetrakis-(2-pyridymethyl) ethylenediamine (TPEN), and broadened by ionomycin and the intracellular Ca2+ chelators BAPTA and EGTA. Refilling of the ER was inhibited by caffeine. Neither thapsigargin nor DBHQ caused a detectable inhibition or change in shape of the Cao2+-induced [Ca2+]cyt spike or the low [Ca2+]cyt plateau whereas each inhibited the inflow of Ca2+ to the ER by about 80%. Experiments conducted with carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) indicated that thapsigargin did not alter the amount of Ca2+ accumulated in mitochondria. The changes in [Ca2+]cyt reported by aequorin were compared with those reported by fura-2. It is concluded that (i) there are significant quantitative differences between the manner in which aequorin and fura-2 sense changes in [Ca2+]cyt and (ii) thapsigargin and DBHQ inhibit the uptake of Ca2+ to the bulk of the ER but this is not associated with inhibition of the activation of SOCs. The possible involvement of a small sub-region of the ER (or another intracellular Ca2+ store), which contains thapsigargin-insensitive (Ca2++Mg2+)ATP-ases, in the activation of SOCs is briefly discussed.     

Apoaequorin monitors degradation of endoplasmic reticulum (ER) proteins initiated by loss of ER Ca(2+)

Apoaequorin was targeted to the cytosol, nucleus, and endoplasmic reticulum of HeLa cells in order to determine the effect of Ca(2+) release from the ER on protein degradation. In resting cells apoaequorin had a rapid half-life (ca. 20-30 min) in the cytosol or nucleus, but was relatively stable for up to 24 h in the ER (t(1/2) > 24 h). However, release of Ca(2+) from the ER, initiated by the addition of inhibitors of the ER Ca(2+)/Mg(2+) ATPase such as 2 microM thapsigargin or 1 microM ionomycin, initiated rapid loss of apoaequorin in the ER, but had no detectable effect on apoaequorin turnover in the cytosol nor the nucleus. This loss of apoprotein was not the result of secretion into the external fluid, and could not be inhibited by inhibitors of protein degradation by proteosomes. Proteolysis of apoaequorin in cell extracts (t(1/2) < 20 min) was completely inhibited in the presence of 1 mM Ca(2+), and this effect was independent of the ER retention signal KDEL at the C-terminus. Proteolysis was unaffected by the presence of selected serine protease inhibitors, or 10 microM Zn(2+), a known caspase-3 inhibitor. The results show that apoaequorin can monitor proteolysis of ER proteins activated by loss of ER Ca(2+). Several Ca(2+)-binding proteins exist in the ER, acting as the Ca(2+) store and chaperones. Our results have important implications both for the role of ER Ca(2+) in cell activation and stress and when using aequorin for monitoring free ER Ca(2+) over long time periods.     

Epstein-Barr virus latent membrane protein 1 increases calcium influx through store-operated channels in B lymphoid cells

Ca(2+) signaling plays an important role in B cell survival and activation and is dependent on Ca(2+) trapped in the endoplasmic reticulum (ER) and on extracellular Ca(2+). Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis. Previously, we showed that the ER Ca(2+) content of Burkitt lymphoma cell lines was increased following infection with immortalization-competent virus expressing the full set of EBV latency genes (B95-8). In contrast, infection with an immortalization-deficient virus (P3HR-1) not expressing LMP-1 is without effect. LMP-1 protein expression was sufficient to increase the ER Ca(2+) content and to increase the cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this follow-up study, we showed that the resting [Ca(2+)](cyt) of P3HR-1-infected cells was decreased, implying that EBV not only modified the ER homeostasis but also affected the cytosolic Ca(2+) homeostasis. Furthermore, even if the store-operated calcium entry (SOCE) of these cells was normal, the [Ca(2+)](cyt) increase after thapsigargin + CaCl(2) stimulation was blunted. In contrast, the resting [Ca(2+)](cyt) of B95-8 infected cells was not changed, even if their SOCE was increased significantly. When expressed alone, LMP-1 induced an increase of the SOCE amplitude and the expression of the protein allowing this influx, Orai1, showing the effect of EBV on SOCE of B cells are mediated by LMP-1. However, other hitherto unidentified EBV processes, unmasked in P3HR-1 infected cells, counteract this LMP-1-dependent increase of SOCE amplitude to impair a general and potentially toxic increase of [Ca(2+)](i). Thus, EBV infection modifies the cellular Ca(2+) homeostasis by acting on the ER and plasma membrane transporters.     

The novel endoplasmic reticulum (ER)-targeted protein HAP induces cell apoptosis by the depletion of the ER Ca(2+) stores

HAP, a novel human apoptosis-inducing protein, was identified to localize exclusively to the endoplasmic reticulum (ER) in our previous work. In the present work, we reported that ectopic overexpression of HAP proteins caused the rapid and sustained elevation of the intracellular cytosolic Ca(2+), which originated from the reversible ER Ca(2+) stores release and the extracellular Ca(2+) influx. The HeLa cells apoptosis induced by HAP proteins was not prevented by establishing the clamped cytosolic Ca(2+) condition, or by buffering of the extracellular Ca(2+) with EGTA, suggesting that the depletion of ER Ca(2+) stores rather than the elevation of cytosolic Ca(2+) or the extracellular Ca(2+) entry contributed to HAP-induced HeLa cells apoptosis. Caspase-3 was also activated in the process of HAP-triggered apoptotic cell death.     

Function of a STIM1 homologue in C. elegans: evidence that store-operated Ca2+ entry is not essential for oscillatory Ca2+ signaling and ER Ca2+ homeostasis

1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca(2+) signaling processes and for maintenance of store Ca(2+) levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.     

Capturing ER calcium dynamics

The lumen of the endoplasmic reticulum (ER) contributes to the dynamics of Ca(2+) signaling by acting as a source or sink of signal Ca(2+). Despite its relevance for the understanding of the cell biology and pathophysiology of the luminal calcium store, the direct measurement of luminal Ca(2+) release and uptake is still critical when Ca(2+) homeostasis is analyzed in neural cells. For the analysis of Ca(2+)-dependent signaling, synthetic Ca(2+) indicators have become popular. The properties of these indicators allow only limited targeting to subcellular structures such as the ER. Recently, we introduced a new strategy for the targeting of synthetic Ca(2+) indicators to the lumen of the ER. The method, termed Targeted-Esterase-induced Dye loading (TED) is based on the targeted recombinant expression of a high carboxylesterase (CES) activity in the lumen of the ER, which is needed to trap synthetic indicators. The method combines the selectivity of protein targeting with the biochemical advantages of low-affinity synthetic Ca(2+) indicators. TED permits direct and non-disruptive measurement and imaging of Ca(2+)-store dynamics. Here, we summarize major topics in the cell biology of ER Ca(2+) signaling and discuss the perspectives of the TED method for the morphological and physiological analysis of temporal and spatial Ca(2+)-dynamics in neural cells.     

BiP-mediated closing of the Sec61 channel limits Ca(2+) leakage from the ER

In mammalian cells, signal peptide-dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic protein-conducting channel, the Sec61 complex. Previous work has characterized the Sec61 channel as a potential ER Ca(2+) leak channel and identified calmodulin as limiting Ca(2+) leakage in a Ca(2+)-dependent manner by binding to an IQ motif in the cytosolic aminoterminus of Sec61α. Here, we manipulated the concentration of the ER lumenal chaperone BiP in cells in different ways and used live cell Ca(2+) imaging to monitor the effects of reduced levels of BiP on ER Ca(2+) leakage. Regardless of how the BiP concentration was lowered, the absence of available BiP led to increased Ca(2+) leakage via the Sec61 complex. When we replaced wild-type Sec61α with mutant Sec61αY344H in the same model cell, however, Ca(2+) leakage from the ER increased and was no longer affected by manipulation of the BiP concentration. Thus, BiP limits ER Ca(2+) leakage through the Sec61 complex by binding to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344.     

Calcium-Dependent Protein Kinase C Activation in Acutely Isolated Neurons During Oxygen and Glucose Deprivation

Glutamate excitotoxicity and necrotic cell death are characteristic features of ischemic neuronal injury. In the penumbral area, glutamate exposure is less pronounced and neuronal death is delayed. Recent studies suggest that delayed neuronal death is propagated by intracellular signalling pathways. Protein kinase C (PKC) activation may initiate apoptosis, but its role in ischemia is still not clear. In this study the PKC activity was investigated during non-excitotoxic ischemia in acutely dissociated rat CA1 neurons. During oxygen and glucose deprivation (OGD) the PKC activity measured with the fluorescent dye Fim-1 increased rapidly reaching a maximum of 31+/-8% (P < 0.05) after 5 min. When extracellular Ca2+ was depleted, the fluorescence intensity increased by 20+/-8% (P<0.05), but with a slower onset. In neurons treated with thapsigargin in a Ca2+ depleted solution, however, OGD did not trigger PKC activation. The results suggest that the PKC activation is mainly triggered by Ca2+ release from endogenous stores.