The Voltage-dependent Anion Channel in Endoplasmic/Sarcoplasmic Reticulum: Characterization,Modulation and Possible Function

In recent years, it has been recognized that there is a metabolic coupling between the cytosol, ER/SR and mitochondria. In this cross-talk, mitochondrial Ca²⁺ homeostasis and ATP production and supply play a major role. The primary transporter of adenine nucleotides, Ca²⁺and other metabolites into and out of mitochondria is the voltage-dependent anion channel (VDAC) located at the outer mitochondrial membrane, at a crucial position in the cell. VDAC has been established as a key player in mitochondrial metabolite and ion signaling and it has also been proposed that VDAC is present in extramitochondrial membranes. Thus, regulation of VDAC, as the main interface between mitochondrial and cellular metabolism, by other molecules is of utmost importance. This article reviews localization and function of VDAC, and focuses on VDAC as a skeletal muscle sarcoplasmic reticulum channel. The regulation of VDAC activity by associated proteins and by inhibitors is also presented. Several aspects of the physiological relevance of VDAC to Ca²⁺ homeostasis and mitochondria-mediated apoptosis will be discussed.     

The SERCA2: A Gatekeeper of Neuronal Calcium Homeostasis in the Brain

Calcium (Ca²⁺) ions are prominent cell signaling regulators that carry information for a variety of cellular processes and are critical for neuronal survival and function. Furthermore, Ca²⁺ acts as a prominent second messenger that modulates divergent intracellular cascades in the nerve cells. Therefore, nerve cells have developed intricate Ca²⁺ signaling pathways to couple the Ca²⁺ signal to their biochemical machinery. Notably, intracellular Ca²⁺ homeostasis greatly relies on the rapid redistribution of Ca²⁺ ions into the diverse subcellular organelles which serve as Ca²⁺ stores, including the endoplasmic reticulum (ER). It is well established that Ca²⁺ released into the neuronal cytoplasm is pumped back into the ER by the sarco-/ER Ca²⁺ ATPase 2 (SERCA2), a P-type ion-motive ATPase that resides on the ER membrane. Even though the SERCA2 is constitutively expressed in nerve cells, its precise role in brain physiology and pathophysiology is not well-characterized. Intriguingly, SERCA2-dependent Ca²⁺ dysregulation has been implicated in several disorders that affect cognitive function, including Darier’s disease, schizophrenia, Alzheimer’s disease, and cerebral ischemia. The current review summarizes knowledge on the expression pattern of the different SERCA2 isoforms in the nervous system, and further discusses evidence of SERCA2 dysregulation in various neuropsychiatric disorders. To the best of our knowledge, this is the first literature review that specifically highlights the critical role of the SERCA2 in the brain. Advancing knowledge on the role of SERCA2 in maintaining neuronal Ca²⁺ homeostasis may ultimately lead to the development of safer and more effective pharmacotherapies to combat debilitating neuropsychiatric disorders.     

Role of SOCE architects STIM and Orai proteins in Cell Death

Calcium (Ca²⁺) signaling plays a critical role in regulating plethora of cellular functions including cell survival, proliferation and migration. The perturbations in cellular Ca²⁺ homeostasis can lead to cell death either by activating autophagic pathways or through induction of apoptosis. Endoplasmic reticulum (ER) is the major storehouse of Ca²⁺ within cells and a number of physiological agonists mediate ER Ca²⁺ release by activating IP₃ receptors (IP₃R). This decrease in ER Ca²⁺ levels is sensed by STIM, which physically interacts and activates plasma membrane Ca²⁺ selective Orai channels. Emerging literature implicates a key role for STIM1, STIM2, Orai1 and Orai3 in regulating both cell survival and death pathways. In this review, we will retrospect the work highlighting the role of STIM and Orai homologs in regulating cell death signaling. We will further discuss the rationales that could explain the dual role of STIM and Orai proteins in regulating cell fate decisions.     

Mitochondria adjust Ca signaling regime to a pattern of stimulation in salivary acinar cells

The salivary acinar cells have unique Ca²⁺ signaling machinery that ensures an extensive secretion. The agonist-induced secretion is governed by Ca²⁺ signals originated from the endoplasmic reticulum (ER) followed by a store-operated Ca²⁺ entry (SOCE). During tasting and chewing food a frequency of parasympathetic stimulation increases up to ten fold, entailing cells to adapt its Ca²⁺ machinery to promote ER refilling and ensure sustained SOCE by yet unknown mechanism. By employing a combination of fluorescent Ca²⁺ imaging in the cytoplasm and inside cellular organelles (ER and mitochondria) we described the role of mitochondria in adjustment of Ca²⁺ signaling regime and ER refilling according to a pattern of agonist stimulation. Under the sustained stimulation, SOCE is increased proportionally to the degree of ER depletion. Cell adapts its Ca²⁺ handling system directing more Ca²⁺ into mitochondria via microdomains of high [Ca²⁺] providing positive feedback on SOCE while intra-mitochondrial tunneling provides adequate ER refilling. In the absence of an agonist, the bulk of ER refilling occurs through Ca²⁺-ATPase-mediated Ca²⁺ uptake within subplasmalemmal space. In conclusion, mitochondria play a key role in the maintenance of sustained SOCE and adequate ER refilling by regulating Ca²⁺ fluxes within the cell that may represent an intrinsic adaptation mechanism to ensure a long-lasting secretion.     

Calcium signaling around Mitochondria Associated Membranes (MAMs)

Calcium (Ca²⁺) homeostasis is fundamental for cell metabolism, proliferation, differentiation, and cell death. Elevation in intracellular Ca²⁺ concentration is dependent either on Ca²⁺ influx from the extracellular space through the plasma membrane, or on Ca²⁺ release from intracellular Ca²⁺ stores, such as the endoplasmic/sarcoplasmic reticulum (ER/SR). Mitochondria are also major components of calcium signalling, capable of modulating both the amplitude and the spatio-temporal patterns of Ca²⁺ signals. Recent studies revealed zones of close contact between the ER and mitochondria called MAMs (Mitochondria Associated Membranes) crucial for a correct communication between the two organelles, including the selective transmission of physiological and pathological Ca²⁺ signals from the ER to mitochondria. In this review, we summarize the most up-to-date findings on the modulation of intracellular Ca²⁺ release and Ca²⁺ uptake mechanisms. We also explore the tight interplay between ER- and mitochondria-mediated Ca²⁺ signalling, covering the structural and molecular properties of the zones of close contact between these two networks.     

SR/ER-mitochondrial local communication: Calcium and ROS

Mitochondria form junctions with the sarco/endoplasmic reticulum (SR/ER), which support signal transduction and biosynthetic pathways and affect organellar distribution. Recently, these junctions have received attention because of their pivotal role in mediating calcium signal propagation to the mitochondria, which is important for both ATP production and mitochondrial cell death. Many of the SR/ER-mitochondrial calcium transporters and signaling proteins are sensitive to redox regulation and are directly exposed to the reactive oxygen species (ROS) produced in the mitochondria and SR/ER. Although ROS has been emerging as a novel signaling entity, the redox signaling of the SR/ER-mitochondrial interface is yet to be elucidated. We describe here possible mechanisms of the mutual interaction between local Ca²⁺ and ROS signaling in the control of SR/ER-mitochondrial function.     

酿酒酵母细胞中内质网应激与未折叠蛋白反应的研究进展

内质网应激激活的未折叠蛋白反应(Unfolded protein response,UPR)途径在酿酒酵母和哺乳动物细胞中是非常保守的。内质网(Endoplasmic reticulum,ER)是蛋白质合成、折叠和修饰的细胞器,也是贮存钙的主要场所之一。酵母细胞内质网钙平衡与UPR的作用是相互的;两个MAPK途径——HOG途径和CWI途径都是细胞应答内质网应激压力时生存所必需的;重金属镉离子能够激活UPR途径,它通过激活钙离子通道Cch1/Mid1进入细胞影响钙离子的功能。本文结合最新研究进展对酿酒酵母细胞中的两个MAPK途径、镉离子和钙离子稳态与内质网应激激活的UPR途径之间相互关系进行综述。     

Enhanced parkin levels favor ER-mitochondria crosstalk and guarantee Ca2 + transfer to sustain cell bioenergetics

Loss-of-function mutations in PINK1 or parkin genes are associated with juvenile-onset autosomal recessive forms of Parkinson disease. Numerous studies have established that PINK1 and parkin participate in a common mitochondrial-quality control pathway, promoting the selective degradation of dysfunctional mitochondria by mitophagy. Upregulation of parkin mRNA and protein levels has been proposed as protective mechanism against mitochondrial and endoplasmic reticulum (ER) stress. To better understand how parkin could exert protective function we considered the possibility that it could modulate the ER–mitochondria inter-organelles cross talk. To verify this hypothesis we investigated the effects of parkin overexpression on ER–mitochondria crosstalk with respect to the regulation of two key cellular parameters: Ca^2 + homeostasis and ATP production. Our results indicate that parkin overexpression in model cells physically and functionally enhanced ER–mitochondria coupling, favored Ca^2 + transfer from the ER to the mitochondria following cells stimulation with an 1,4,5 inositol trisphosphate (InsP₃) generating agonist and increased the agonist-induced ATP production. The overexpression of a parkin mutant lacking the first 79 residues (ΔUbl) failed to enhance the mitochondrial Ca^2 + transients, thus highlighting the importance of the N-terminal ubiquitin like domain for the observed phenotype. siRNA-mediated parkin silencing caused mitochondrial fragmentation, impaired mitochondrial Ca^2 + handling and reduced the ER–mitochondria tethering. These data support a novel role for parkin in the regulation of mitochondrial homeostasis, Ca^2 + signaling and energy metabolism under physiological conditions.     

Ca transfer from the ER to mitochondria: When, how and why

The heterogenous subcellular distribution of a wide array of channels, pumps and exchangers allows extracellular stimuli to induce increases in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) with highly defined spatial and temporal patterns, that in turn induce specific cellular responses (e.g. contraction, secretion, proliferation or cell death). In this extreme complexity, the role of mitochondria was considered marginal, till the direct measurement with targeted indicators allowed to appreciate that rapid and large increases of the [Ca²⁺] in the mitochondrial matrix ([Ca²⁺]_m) invariably follow the cytosolic rises. Given the low affinity of the mitochondrial Ca²⁺ transporters, the close proximity to the endoplasmic reticulum (ER) Ca²⁺-releasing channels was shown to be responsible for the prompt responsiveness of mitochondria. In this review, we will summarize the current knowledge of: i) the mitochondrial and ER Ca²⁺ channels mediating the ion transfer, ii) the structural and molecular foundations of the signaling contacts between the two organelles, iii) the functional consequences of the [Ca²⁺]_m increases, and iv) the effects of oncogene-mediated signals on mitochondrial Ca²⁺ homeostasis. Despite the rapid progress carried out in the latest years, a deeper molecular understanding is still needed to unlock the secrets of Ca²⁺ signaling machinery.     

Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca²⁺ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP₃Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca²⁺ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP₃Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP₃Rs were located in the SR. [Ca²⁺]i, [Ca²⁺]_m and [Ca²⁺]_SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca²⁺]_SR, increased [Ca²⁺]_i and [Ca²⁺]_m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca²⁺ release from the SR into the mitochondria through IP₃Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.     

The mystery of mitochondria-ER contact sites in physiology and pathology: A cancer perspective

Mitochondria-associated membranes (MAM), physical platforms that enable communication between mitochondria and the endoplasmic reticulum (ER), are enriched with many proteins and enzymes involved in several crucial cellular processes, such as calcium (Ca²⁺) homeostasis, lipid synthesis and trafficking, autophagy and reactive oxygen species (ROS) production. Accumulating studies indicate that tumor suppressors and oncogenes are present at these intimate contacts between mitochondria and the ER, where they influence Ca²⁺ flux between mitochondria and the ER or affect lipid homeostasis at MAM, consequently impacting cell metabolism and cell fate. Understanding these fundamental roles of mitochondria-ER contact sites as important domains for tumor suppressors and oncogenes can support the search for new and more precise anticancer therapies. In the present review, we summarize the current understanding of basic MAM biology, composition and function and discuss the possible role of MAM-resident oncogenes and tumor suppressors.     

Oxidative Protein Folding in the Secretory Pathway and Redox Signaling Across Compartments and Cells

The endoplasmic reticulum (ER) is central for many essential cellular activities, such as folding, assembly and quality control of secretory and membrane proteins, disulfide bond formation, glycosylation, lipid biosynthesis, Ca²⁺ storage and signaling. In addition, this multifunctional organelle integrates many adaptive and/or maladaptive signaling cues reporting on metabolism, proteostasis, Ca²⁺ and redox homeostasis. We are beginning to understand how these functions and pathways are integrated with one another to regulate homeostasis at cell, tissue and organism levels. The mechanisms underlying the introduction of the proper set of disulfide bonds into secretory proteins (oxidative folding) are strictly related to redox homeostasis, ER stress sensing and signaling and provide a good example of the integration systems operative in the early secretory compartment.     

Molecular architecture of Ca2+ signaling control in muscle and heart cells

Ca²⁺ signaling in skeletal and cardiac muscles is a bi-directional process that involves cross-talk between signaling molecules in the sarcolemmal membrane and Ca²⁺ release machinery in the intracellular organelles. Maintenance of a junctional membrane structure between the sarcolemmal membrane and the sarcoplasmic reticulum (SR) provides a framework for the conversion of action potential arrived at the sarcolemma into release of Ca²⁺ from the SR, leading to activation of a variety of physiological processes. Activity-dependent changes in Ca²⁺ storage inside the SR provides a retrograde signal for the activation of store-operated Ca²⁺ channel (SOC) on the sarcolemmal membrane, which plays important roles in the maintenance of Ca²⁺ homeostasis in physiology and pathophysiology. Research progress during the last 30 years had advanced our understanding of the cellular and molecular mechanisms for the control of Ca²⁺ signaling in muscle and cardiovascular physiology. Here we summarize the functions of three key molecules that are located in the junctional membrane complex of skeletal and cardiac muscle cells: junctophilin as a “glue” that physiologically links the SR membrane to the sarcolemmal membrane for formation of the junctional membrane framework, mitsugumin29 as a muscle-specific synaptophysin family protein that contributes to maintain the coordinated Ca²⁺ signaling in skeletal muscle, and TRIC as a novel cation-selective channel located on the SR membrane that provides counter-ion current during the rapid process of Ca²⁺ release from the SR.     

GT-repeat polymorphism in the heme oxygenase-1 gene promoter and the risk of carotid atherosclerosis related to arsenic exposure

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca²⁺ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP₃Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca²⁺ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP₃Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP₃Rs were located in the SR. [Ca²⁺]i, [Ca²⁺]_m and [Ca²⁺]_SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca²⁺]_SR, increased [Ca²⁺]_i and [Ca²⁺]_m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca²⁺ release from the SR into the mitochondria through IP₃Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.     

Mitochondria Are Linked to Calcium Stores in Striated Muscle by Developmentally Regulated Tethering Structures

Bi-directional calcium (Ca²⁺) signaling between mitochondria and intracellular stores (endoplasmic/sarcoplasmic reticulum) underlies important cellular functions, including oxidative ATP production. In striated muscle, this coupling is achieved by mitochondria being located adjacent to Ca²⁺ stores (sarcoplasmic reticulum [SR]) and in proximity of release sites (Ca²⁺ release units [CRUs]). However, limited information is available with regard to the mechanisms of mitochondrial-SR coupling. Using electron microscopy and electron tomography, we identified small bridges, or tethers, that link the outer mitochondrial membrane to the intracellular Ca²⁺ stores of muscle. This association is sufficiently strong that treatment with hypotonic solution results in stretching of the SR membrane in correspondence of tethers. We also show that the association of mitochondria to the SR is 1) developmentally regulated, 2) involves a progressive shift from a longitudinal clustering at birth to a specific CRU-coupled transversal orientation in adult, and 3) results in a change in the mitochondrial polarization state, as shown by confocal imaging after JC1 staining. Our results suggest that tethers 1) establish and maintain SR–mitochondrial association during postnatal maturation and in adult muscle and 2) likely provide a structural framework for bi-directional signaling between the two organelles in striated muscle.     

Proteome-wide study of endoplasmic reticulum stress induced by thapsigargin in N2a neuroblastoma cells

Disturbances in intraluminal endoplasmic reticulum (ER) Ca²⁺ concentration leads to the accumulation of unfolded proteins and perturbation of intracellular Ca²⁺ homeostasis, which has a huge impact on mitochondrial functioning under normal and stress conditions and can trigger cell death. Thapsigargin (TG) is widely used to model cellular ER stress as it is a selective and powerful inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPases. Here we provide a representative proteome-wide picture of ER stress induced by TG in N2a neuroblastoma cells. Our proteomics study revealed numerous significant protein expression changes in TG-treated N2a cell lysates analysed by two-dimensional electrophoresis followed by mass spectrometric protein identification. The proteomic signature supports the evidence of increased bioenergetic activity of mitochondria as several mitochondrial enzymes with roles in ATP-production, tricarboxylic acid cycle and other mitochondrial metabolic processes were upregulated. In addition, the upregulation of the main ER resident proteins confirmed the onset of ER stress during TG treatment. It has become widely accepted that metabolic activity of mitochondria is induced in the early phases in ER stress, which can trigger mitochondrial collapse and subsequent cell death. Further investigations of this cellular stress response in different neuronal model systems like N2a cells could help to elucidate several neurodegenerative disorders in which ER stress is implicated.     

Bcl-2 Modulates Endoplasmic Reticulum and Mitochondrial Calcium Stores in PC12 Cells

Endoplasmic reticulum (ER) and mitochondria are intracellular organelles and their interactions are directly involved in different processes such as Ca²⁺ signaling in cell survival and death mechanisms. Bcl-2 is an anti-apoptotic protein intrinsically related to ER and mitochondria, modulating Ca²⁺ content in these organelles. We investigated the effects of Bcl-2 overexpression on ER and mitochondrial Ca²⁺ dynamics in PC12 cells. Bcl-2 overexpressing and control cells were loaded with Fura 2/AM and stimulated with different drugs. Results showed that in Bcl-2 cells, ACh induced a lower Ca²⁺ response compared to control. Ca²⁺ release induced by TG was decreased in Bcl-2 cells, however, it was greater in Caff induced Ca²⁺ rise. In addition, FCCP induced a higher Ca²⁺ release in Bcl-2 cells. These results suggest that Bcl-2 overexpression modulate the ER Ca²⁺ pools differently and the release of ER Ca²⁺ may increase mitochondrial Ca²⁺ accumulation. These alterations of intracellular Ca²⁺ stores are important mechanisms for the control of Ca²⁺ signaling.