Activation of endoplasmic reticulum calcium leak by 2-APB depends on the luminal calcium concentration

It has been shown that 2-APB is a nonspecific modulator of ion channel activity, while most of the channels are inhibited by this compound, there are few examples of channels that are activated by 2-APB. Additionally, it has been shown that, 2-APB leads to a reduction in the luminal endoplasmic reticulum Ca²⁺ level ([Ca²⁺]_ER) and we have carried out simultaneous recordings of both [Ca²⁺]_i and the [Ca²⁺]_ER in HeLa cell suspensions to assess the mechanism involved in this effect. This approach allowed us to determine that 2-APB induces a reduction in the [Ca²⁺]_ER by activating an ER-resident Ca²⁺ permeable channel more than by inhibiting the activity of SERCA pumps. Interestingly, this effect of 2-APB of reducing the [Ca²⁺]_ER is auto-limited because depends on a replete ER Ca²⁺ store; a condition that thapsigargin does not require to decrease the [Ca²⁺]_ER. Additionally, our data indicate that the ER Ca²⁺ permeable channel activated by 2-APB does not seem to participate in the ER Ca²⁺ leak revealed by inhibiting SERCA pump with thapsigargin. This work suggests that, prolonged incubations with even low concentrations of 2-APB (5 μM) would lead to the reduction in the [Ca²⁺]_ER that might explain the inhibitory effect of this compound on those signals that require Ca²⁺ release from the ER store.     

Exploring the unique features of the ARC channel,a Store-independent Orai channel

The discovery of the Orai proteins, and the identification of STIM1 as the molecule that regulates them, was based on their role in the agonist-activated store-operated entry of calcium via the CRAC channels. However, these same proteins are also essential components of the ARC channels responsible for a similar agonist-activated, but store-independent, arachidonic acid-regulated entry of calcium. The fact that these 2 biophysically similar calcium entry pathways frequently co-exist in the same cells suggests that they must each possess different features that allow them to function in distinct ways to regulate specific cellular activities. This review begins to address this question by describing recent findings characterizing the unique features of the ARC channels—their molecular composition, STIM1-dependent activation, and physiological activities—and the importance of defining such features for the accurate therapeutic targeting of these 2 Orai channel subtypes.     

Potent functional uncoupling between STIM1 and Orai1 by dimeric 2-aminodiphenyl borinate analogs

The coupling of ER Ca²⁺-sensing STIM proteins and PM Orai Ca²⁺ entry channels generates “store-operated” Ca²⁺ signals crucial in controlling responses in many cell types. The dimeric derivative of 2-aminoethoxydiphenyl borinate (2-APB), DPB162-AE, blocks functional coupling between STIM1 and Orai1 with an IC₅₀ (200 nM) 100-fold lower than 2-APB. Unlike 2-APB, DPB162-AE does not affect L-type or TRPC channels or Ca²⁺ pumps at maximal STIM1–Orai1 blocking levels. DPB162-AE blocks STIM1-induced Orai1 or Orai2, but does not block Orai3 or STIM2-mediated effects. We narrowed the DPB162-AE site of action to the STIM–Orai activating region (SOAR) of STIM1. DPB162-AE does not prevent the SOAR–Orai1 interaction but potently blocks SOAR-mediated Orai1 channel activation, yet its action is not as an Orai1 channel pore blocker. Using the SOAR-F394H mutant which prevents both physical and functional coupling to Orai1, we reveal DPB162-AE rapidly restores SOAR–Orai binding but only slowly restores Orai1 channel-mediated Ca²⁺ entry. With the same SOAR mutant, 2-APB induces rapid physical and functional coupling to Orai1, but channel activation is transient. We infer that the actions of both 2-APB and DPB162-AE are directed toward the STIM1–Orai1 coupling interface. Compared to 2-APB, DPB162-AE is a much more potent and specific STIM1/Orai1 functional uncoupler. DPB162-AE provides an important pharmacological tool and a useful mechanistic probe for the function and coupling between STIM1 and Orai1 channels.     

The endoplasmic reticulum network in the ascidian egg: localization and calcium content

In contrast with most other eggs, where the endoplasmic reticulum is mixed with many other organelles, in ascidians, continuous sheets and tubes of endoplasmic reticulum constitute the only prominent organelle in the immediate layer (0.5-1μm) beneath the plasma membrane, and occupies 16–20% of the cortical volume. We took advantage of this unusual stratification of the organelles in the ascidian egg, to carry X-ray microanalysis. Our measurements provide the first estimate of the calcium content of the endoplasmic reticulum network in an egg, and show it is the main calcium store.     

The protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation and increases sarcoplasmic/endoplasmic reticulum calcium ATPase 2 levels

The ubiquitously expressed protein glycogen synthase kinase-3 (GSK3) is constitutively active, however its activity is markedly diminished following phosphorylation of Ser21 of GSK3alpha and Ser9 of GSK3beta. Although several kinases are known to phosphorylate Ser21/9 of GSK3, for example Akt, relatively much less is known about the mechanisms that cause the dephosphorylation of GSK3 at Ser21/9. In the present study KCl-induced plasma membrane depolarization of SH-SY5Y cells, which increases intracellular calcium concentrations caused a transient decrease in the phosphorylation of Akt at Thr308 and Ser473, and GSK3 at Ser21/9. Overexpression of the selective protein phosphatase-1 inhibitor protein, inhibitor-2, increased basal GSK3 phosphorylation at Ser21/9 and significantly blocked the KCl-induced dephosphorylation of GSK3beta, but not GSK3alpha. The phosphorylation of Akt was not affected by the overexpression of inhibitor-2. GSK3 activity is known to affect sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) levels. Overexpression of inhibitor-2 or treatment of cells with the GSK3 inhibitors lithium and SB216763 increased the levels of SERCA2. These results indicate that the protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation induced by KCl and that GSK3 activity regulates SERCA2 levels.     

Reticulon 3 mediates Bcl-2 accumulation in mitochondria in response to endoplasmic reticulum stress

Reticulon3 (RTN3), firstly isolated from the retina and widely expressed in human tissues with the highest expression in the brain, is presumed to play an important role in the developing axons through the transport of liquids and proteins. We have identified and characterized RTN3 as a RTN4B/ASY interaction protein. Here we demonstrated that ER-stress activated RTN3 expression. CHOP and ATF6 were sufficient to up-regulate the expression of RTN3. The down-regulation of RTN3 would induce apoptosis and attenuate the anti-apoptotic activity of Bcl-2, indicating RTN3 was required for the cellular survival and optimal anti-apoptotic activity of Bcl-2. Our present studies also indicated ER-stress induced RTN3 up-regulation could trigger Bcl-2 translocation from ER to mitochondria. Moreover, the previous studies showed that RTN4B was also a Bcl-2-interacted protein. We found that RTN3 and RTN4B could block the access of Bcl-2 to each other and thereafter determined the Bcl-2 subcellular distribution. Taken together, our findings indicate that RTN3 is directly involved in the ER-constituents trafficking events through dually acting as an essential and important ER-stress sensor, and a trigger for the Bcl-2 translocation. Q. Wan and E. Kuang contributed equally to this work.     

A calcium conducting channel akin to a calcium pump

Summary Calcium conducting channels were studied in blebs of sarcoplasmic reticulum described by Stein & Palade (1988). The calcium channels had at least three conductance states (70 pS, 50 pS and 37 pS) and were weakly selective for calcium ions, with a permeability ratio Ca²⁺ to K⁺ of about 3.4. The open probability of the channel was strongly voltage dependent, decreasing at positive membrane voltages. 10 m ryanodine and 5 m ruthenium red had no effect on this channel; neither did millimolar concentrations of ATP, Mg²⁺, caffeine, and Ca²⁺, implying that the calcium conducting channels are not ryanodine receptors. Several calcium pump inhibitors—namely, vanadate, AlF ₄ ^– , reactive red 120, and cyclopiazonic acid—had obvious effects on the calcium conducting channels, suggesting that the calcium conducting channel of SR membrane blebs is some form of the SR calcium pump.We thank the National Science Foundation for steadfast support.We thank Drs. F. Cohen, A. Fox, R. Levis and E. Rios for much useful help and criticism and Dr. G. Inesi for sending us his paper while in press.     

Increased calcium leak associated with reduced calsequestrin expression in hyperthyroid cardiomyocytes

IntroductionCalcium (Ca²⁺) leak during cardiac diastole is chiefly mediated by intracellular Ca²⁺ channel/Ryanodine Receptors. Increased diastolic Ca²⁺ leak has been proposed as the mechanism underlying the appearance of hereditary arrhythmias. However, little is known about alterations in diastolic Ca²⁺ leak and the specific roles played by key intracellular Ca²⁺-handling proteins in hyperthyroidism, a known arrhythmogenic condition.AimWe sought to determine whether there were modifications in diastolic Ca²⁺ leak, based on the recording of Ca²⁺ sparks and Ca²⁺ waves; we also investigated changes in the expression and activity of key Ca²⁺ handling proteins, including ryanodine receptors, Sarco-Endoplasmic Reticulum Ca²⁺ ATPase pump and calsequestrin in isolated left-ventricular cardiomyocytes isolated from hyperthyroid rats.Materials and methodsElectrocardiography (ECG) recordings were performed in control and hyperthyroid rats. Ca²⁺ sparks, Ca²⁺ waves, and electrically-stimulated Ca²⁺ transients were recorded in Fluo-3-loaded cardiomyocytes from both experimental groups using confocal microscopy. In addition, left-ventricular homogenates and Ryanodine Receptor-enriched membrane fractions were prepared for assessing [³H]-ryanodine binding, hydrolytic ATPase activity of SERCA pump and expression levels of key proteins by Western blot, and cDNA for real-time qPCR.Results and conclusionsExtrasystoles were observed in hearts of hyperthyroid rats by ECG recordings. Arrhythmogenic activity, high incidence of Ca²⁺ waves, and de novo Ca²⁺ wavelets −in the absence of sarcoplasmic reticulum Ca²⁺ overload- were recorded in these cardiomyocytes. The exacerbated diastolic Ca²⁺ leak and arrhythmogenic activities were related to a diminished expression of calsequestrin along with increased SERCA pump activity, which, in effect, promoted a gain-of-function in RyRs without alterations in SR Ca²⁺ load, RyR expression or its Ca²⁺ sensitivity.     

A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels,an arachidonate-regulated store-independent Orai channel

The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.     

A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels,an arachidonate-regulated store-independent Orai channel

The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.     

ATP regulation of a large conductance voltage-gated cation channel in rough endoplasmic reticulum of rat hepatocytes

ATP-sensitive K⁺ channels play an important role in regulating membrane potential during metabolic stress. In this work we report the effect of ATP and ADP-Mg on a K⁺ channel present in the membrane of rough endoplasmic reticulum (RER) from rat hepatocytes incorporated into lipid bilayers. Channel activity was found to decrease in presence of ATP 100 μM on the cytoplasmic side and was totaly inhibited at ATP concentrations greater than 0.25 mM. The effect appeared voltage dependent, suggesting that the ATP binding site was becoming available upon channel opening. Channel activity was suppressed by the nonhydrolyzable ATP analog (ATPγS), ruling out a phosphorylation-based mechanism. Notably addition of 2.5 mM ADP-Mg to the cytosolic side increased the channel open probability at negative potentials. We conclude that the large conductance voltage-gated cation channel in RER of rat hepatocytes is an ATP and ADP sensitive channel likely to be involved in cellular processes such as Ca²⁺ signaling or control of membrane potential across the endoplasmic reticulum membrane.     

Presynaptic store-operated Ca2+ entry drives excitatory spontaneous neurotransmission and augments endoplasmic reticulum stress

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Remodelling of the endoplasmic reticulum during store-operated calcium entry

SOCE (store-operated calcium entry) is a ubiquitous cellular mechanism linking the calcium depletion of the ER (endoplasmic reticulum) to the activation of PM (plasma membrane) Ca2+-permeable channels. The activation of SOCE channels favours the entry of extracellular Ca2+ into the cytosol, thereby promoting the refilling of the depleted ER Ca2+ stores as well as the generation of long-lasting calcium signals. The molecules that govern SOCE activation comprise ER Ca2+ sensors [STIM1 (stromal interaction molecule 1) and STIM2], PM Ca2+-permeable channels {Orai and TRPC [TRP (transient receptor potential) canonical]} and regulatory Ca2+-sensitive cytosolic proteins {CRACR2 [CRAC (Ca2+ release-activated Ca2+ current) regulator 2]}. Upon Ca2+ depletion of the ER, STIM molecules move towards the PM to bind and activate Orai or TRPC channels, initiating calcium entry and store refilling. This molecular rearrangement is accompanied by the formation of specialized compartments derived from the ER, the pre-cER (cortical ER) and cER. The pre-cER appears on the electron microscope as thin ER tubules enriched in STIM1 that extend along microtubules and that are devoid of contacts with the PM. The cER is located in immediate proximity to the PM and comprises thinner sections enriched in STIM1 and devoid of chaperones that might be dedicated to calcium signalling. Here, we review the molecular interactions and the morphological changes in ER structure that occur during the SOCE process.     

New insights in the role of Bcl-2 Bcl-2 and the endoplasmic reticulum

The oncogenic protein Bcl-2 which is expressed in membranes of different subcellular organelles protects cells from apoptosis induced by endogenic stimuli. Most of the results published so far emphasise the importance of Bcl-2 at the mitochondria. Several recent observations suggest a role of Bcl-2 at the endoplasmic reticulum (ER). Bcl-2 located at the ER was shown to interfere with apoptosis induction by Bax, ceramides, ionising radiation, serum withdrawal and c-myc expression. Although the detailed functions of Bcl-2 at the ER remain elusive, several speculative mechanisms may be supposed. For instance, Bcl-2 at the ER may regulate calcium fluxes between the ER and the mitochondria. In addition, Bcl-2 is able to interact with the endoplasmic protein Bap31 thus avoiding caspase activation at the ER. Bcl-2 may also abrogate the function of ER located pro-apoptotic Bcl-2 like proteins by heterodimerization. Current data on the function of Bcl-2 at the ER, its role for the modulation of calcium fluxes and its influence on caspase activation at the ER are reviewed.     

SRO9基因参与调控酿酒酵母内质网应激反应

【目的】研究酵母SRO9基因在内质网应激(Endoplasmic reticulum stress,ERS)中的作用。【方法】利用PCR介导的同源重组方法构建SRO9基因缺失菌株,检测其在内质网应激诱导剂衣霉素处理条件下的克隆形成能力;通过比色法检测细胞内的H2O2含量,超氧化物歧化酶SOD活性和细胞增殖能力;通过实时荧光定量PCR检测内质网应激靶基因和超氧化物歧化酶编码基因SOD1及SOD2的转录水平。【结果】相对于野生型酵母菌株,SRO9基因缺失酵母菌株对内质网应激诱导剂衣霉素的抗性增强,参与内质网应激反应的靶基因转录上调;细胞内H2O2含量下降,SOD1、SOD2转录水平降低,总SOD活性降低;对氧化剂CHP和VK3的抵抗性减弱,复制寿命明显缩短。【结论】SRO9基因缺失酵母细胞对内质网应激诱导剂衣霉素的抗性增强,原因可能是由于SRO9基因缺失激活了细胞的内质网应激反应。     

SERP1 prevents hypoxia-reoxygenation-induced H9c2 apoptosis through activating JAK2/STAT3 pathway-dependent attenuation of endoplasmic reticulum stress

The endoplasmic reticulum (ER) stress plays an important role in myocardial ischemia/reperfusion (MI/R) injury. SERP1, the stress-associated endoplasmic reticulum protein 1, is involved in regulating ER stress response. However, whether it associates with MI/R injury is not identified. Here, we show that SERP1 is induced in the mouse heart after MI/R injury as well as in H9c2 cells under hypoxia/reoxygenation (H/R) treatment. Additionally, SERP1 overexpression reduces H/R-induced H9c2 apoptosis. Moreover, SERP1 overexpression suppresses H/R-induced ER stress and activates JAK2/STAT3 pathway. Furthermore, JAK2/STAT3 pathway inhibition by the specific inhibitor JSI-124 minimizes the suppressive effect of SERP1 overexpression on H/R-induced ER stress and H9c2 apoptosis. Together, these results uncover the protection of SERP1 against H/R-induced H9c2 apoptosis and further relate it to JAK2/STAT3 pathway-dependent attenuation of ER stress. This study suggests SERP1 as a potential regulator invovled in the pathophysiology of MI/R injury.     

The calcium binding protein ALG-2 binds and stabilizes Scotin, a p53-inducible gene product localized at the endoplasmic reticulum membrane

ALG-2 (apoptosis linked gene 2 product) is a calcium binding protein for which no clear cellular function has been established. In this study we identified Scotin as a novel ALG-2 target protein containing 6 PXY and 4 PYP repeats, earlier identified in the ALG-2 binding regions of AIP1/ALIX and TSG101, respectively. An in vitro synthesized C-terminal fragment of Scotin bound specifically to immobilized recombinant ALG-2 and tagged ALG-2 and Scotin were shown by immunoprecipitation to interact in MCF7 and U2OS cell lines. Furthermore ALG-2 bound to endogenous Scotin in extracts from mouse NIH3T3 cells. Overexpression of ALG-2 led to accumulation of Scotin in MCF7 and H1299 cells. In vitro and in vivo binding of ALG-2 to Scotin was demonstrated to be strictly calcium dependent indicating a role of this interaction in calcium signaling pathways.     

2-脱氧葡萄糖诱导大鼠内质网应激预处理模型的最佳剂量

目的探讨2-脱氧葡萄糖(2-deoxy-glucose,2-DG)诱导大鼠内质网应激模型的最佳剂量。方法选择Wistar雄性大鼠108只,体质量240～260 g,采用不同剂量2-DG建立大鼠内质网应激的模型,随机分为6组:2-DG 50、100、150、200 mg/kg组,假手术组,缺血再灌注组。2-DG组按工作浓度为50 mg/mL溶于双蒸水腹腔注射7d,假手术组和缺血再灌注组腹腔注射双蒸水7 d,于脑缺血再灌注后12 h处死,对各组大鼠进行神经行为学评分,采用HE染色观察脑组织的病理形态,免疫组化法和Westernblot法测定GRP78蛋白的表达,用PCR法检测GRP78mRNA的表达。结果与脑缺血再灌注组相比,2-DG各剂量组大鼠的神经行为学评分明显减低(P<0.01),而以100 mg/kg组评分减低最为明显(P<0.05);2-DG各剂量组能不同程度的改善大鼠脑海马CA1区神经细胞的核深染、核固缩程度,减少细胞及间质的水肿,使胞膜趋于清楚、形态接近正常、核仁清晰可见的神经细胞数目增多,而以2-DG100 mg/kg组的效果最为明显。2-DG各剂量组GRP78蛋白表达明显增加,与脑缺血再灌注组比较,差异有显著性(P<0.01),而以100 mg/kg剂量组的GRP78蛋白表达最高,与其他2-DG剂量组比较差异有显著性(P<0.05)。结论 2-DG对脑缺血再灌注所致的神经细胞损伤具有保护作用,其最佳剂量为100 mg/kg。2-DG具有诱导大鼠内质网应激的作用,其最佳剂量是100 mg/kg。     

Disruption of the endoplasmic reticulum and increases in cytoplasmic calcium are early events in cell death induced by the natural triterpenoid Asiatic acid

Triterpenoids are a novel class of compounds being investigated as potential therapeutic agents for the treatment of prostate cancer and other malignancies. Asiatic acid (AA) is a member of the ursane family of triterpenoids and has anticancer activity, but its mechanism of action is not completely understood. To investigate its mechanism of action, PPC-1 prostate cancer cells were treated with AA at increasing concentrations and times. AA induced rapid caspase-dependent and independent cell death that peaked within 8 h of treatment. AA-induced death was associated with early activation of caspases 2, 3, and 8, but not caspase 9. Within 2.5 h of treatment, release of calcium from intracellular stores and dilatation of the endoplasmic reticulum was observed. Thus, disruption of the endoplasmic reticulum and alterations in calcium homeostasis are early events in AA-induced death.     

Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation

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