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Boczek, Tomasz, Marta Sobolczyk, Joanna Mackiewicz, Malwina Lisek, Bozena Ferenc, Feng Guo y Ludmila Zylinska. "Crosstalk among Calcium ATPases: PMCA, SERCA and SPCA in Mental Diseases". International Journal of Molecular Sciences 22, n.º 6 (10 de marzo de 2021): 2785. http://dx.doi.org/10.3390/ijms22062785.
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Calcium in mammalian neurons is essential for developmental processes, neurotransmitter release, apoptosis, and signal transduction. Incorrectly processed Ca2+ signal is well-known to trigger a cascade of events leading to altered response to variety of stimuli and persistent accumulation of pathological changes at the molecular level. To counterbalance potentially detrimental consequences of Ca2+, neurons are equipped with sophisticated mechanisms that function to keep its concentration in a tightly regulated range. Calcium pumps belonging to the P-type family of ATPases: plasma membrane Ca2+-ATPase (PMCA), sarco/endoplasmic Ca2+-ATPase (SERCA) and secretory pathway Ca2+-ATPase (SPCA) are considered efficient line of defense against abnormal Ca2+ rises. However, their role is not limited only to Ca2+ transport, as they present tissue-specific functionality and unique sensitive to the regulation by the main calcium signal decoding protein—calmodulin (CaM). Based on the available literature, in this review we analyze the contribution of these three types of Ca2+-ATPases to neuropathology, with a special emphasis on mental diseases.
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NEWTON, Thomas, John P. J. BLACK, John BUTLER, Anthony G. LEE, John CHAD y J. Malcolm EAST. "Sarco/endoplasmic-reticulum calcium ATPase SERCA1 is maintained in the endoplasmic reticulum by a retrieval signal located between residues 1 and 211". Biochemical Journal 371, n.º 3 (1 de mayo de 2003): 775–82. http://dx.doi.org/10.1042/bj20021477.
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The location of sarco/endoplasmic-reticulum calcium ATPase (SERCA) retention/retrieval motifs in the sequence of the SERCA1 has been investigated by examining the subcellular location in COS-7 cells of enhanced-green-fluorescent-protein-tagged calcium-pump chimaeras. These chimaeras have been constructed from the fast-twitch SERCA1 and the plasma-membrane calcium ATPase PMCA3. The N-terminal, central and C-terminal segments of these calcium pumps were exchanged between SERCA1 and PMCA3. The segments exchanged correspond to residues 1–211, 212–711 and 712–994 of SERCA1, and residues 1–264, 265–788 and 789–1159 of PMCA3 respectively. Only chimaeras containing the N-terminal segment of SERCA1 were located in the endoplasmic reticulum (ER), whereas chimaeras containing the N-terminal segment from PMCA3 were able to escape from the ER and enter the endomembrane pathway en route for the plasma membrane. Co-localization of SERCA1 in COS-7 cells with the ER/Golgi-intermediate compartment marker ERGIC53 indicates that SERCA1 is maintained in the ER by a process of retrieval. These results indicate that the N-terminal region of SERCA1, containing transmembrane helices M1 and M2, contains an ER-retrieval signal.
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Li, Sen, Anant Chopra, Wendy Keung, Camie W. Y. Chan, Kevin D. Costa, Chi-Wing Kong, Roger J. Hajjar, Christopher S. Chen y Ronald A. Li. "Sarco/endoplasmic reticulum Ca2+-ATPase is a more effective calcium remover than sodium-calcium exchanger in human embryonic stem cell-derived cardiomyocytes". American Journal of Physiology-Heart and Circulatory Physiology 317, n.º 5 (1 de noviembre de 2019): H1105—H1115. http://dx.doi.org/10.1152/ajpheart.00540.2018.
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Human pluripotent stem cell (hPSCs)-derived ventricular (V) cardiomyocytes (CMs) display immature Ca2+–handing properties with smaller transient amplitudes and slower kinetics due to such differences in crucial Ca2+-handling proteins as the poor sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump but robust Na+-Ca2+ exchanger (NCX) activities in human embryonic stem cell (ESC)-derived VCMs compared with adult. Despite their fundamental importance in excitation-contraction coupling, the relative contribution of SERCA and NCX to Ca2+-handling of hPSC-VCMs remains unexplored. We systematically altered the activities of SERCA and NCX in human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) and their engineered microtissues, followed by examining the resultant phenotypic consequences. SERCA overexpression in hESC-VCMs shortened the decay of Ca2+ transient at low frequencies (0.5 Hz) without affecting the amplitude, SR Ca2+ content and Ca2+ baseline. Interestingly, short hairpin RNA-based NCX suppression did not prolong the transient decay, indicating a compensatory response for Ca2+ removal. Although hESC-VCMs and their derived microtissues exhibited negative frequency-transient/force responses, SERCA overexpression rendered them less negative at high frequencies (>2 Hz) by accelerating Ca2+ sequestration. We conclude that for hESC-VCMs and their microtissues, SERCA, rather than NCX, is the main Ca2+ remover during diastole; poor SERCA expression is the leading cause for immature negative-frequency/force responses, which can be partially reverted by forced expression. Combinatorial approach to mature calcium handling in hESC-VCMs may help shed further mechanistic insights. NEW & NOTEWORTHY In this study of human pluripotent stem cell-derived cardiomyocytes, we studied the role of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) in Ca2+ handling. Our data support the notion that SERCA is more effective in cytosolic calcium removal than the NCX.
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Rathod, Nishadh, Jessi J. Bak, Joseph O. Primeau, M’Lynn E. Fisher, Lennane Michel Espinoza-Fonseca, Mary Joanne Lemieux y Howard S. Young. "Nothing Regular about the Regulins: Distinct Functional Properties of SERCA Transmembrane Peptide Regulatory Subunits". International Journal of Molecular Sciences 22, n.º 16 (18 de agosto de 2021): 8891. http://dx.doi.org/10.3390/ijms22168891.
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The sarco-endoplasmic reticulum calcium ATPase (SERCA) is responsible for maintaining calcium homeostasis in all eukaryotic cells by actively transporting calcium from the cytosol into the sarco-endoplasmic reticulum (SR/ER) lumen. Calcium is an important signaling ion, and the activity of SERCA is critical for a variety of cellular processes such as muscle contraction, neuronal activity, and energy metabolism. SERCA is regulated by several small transmembrane peptide subunits that are collectively known as the “regulins”. Phospholamban (PLN) and sarcolipin (SLN) are the original and most extensively studied members of the regulin family. PLN and SLN inhibit the calcium transport properties of SERCA and they are required for the proper functioning of cardiac and skeletal muscles, respectively. Myoregulin (MLN), dwarf open reading frame (DWORF), endoregulin (ELN), and another-regulin (ALN) are newly discovered tissue-specific regulators of SERCA. Herein, we compare the functional properties of the regulin family of SERCA transmembrane peptide subunits and consider their regulatory mechanisms in the context of the physiological and pathophysiological roles of these peptides. We present new functional data for human MLN, ELN, and ALN, demonstrating that they are inhibitors of SERCA with distinct functional consequences. Molecular modeling and molecular dynamics simulations of SERCA in complex with the transmembrane domains of MLN and ALN provide insights into how differential binding to the so-called inhibitory groove of SERCA—formed by transmembrane helices M2, M6, and M9—can result in distinct functional outcomes.
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Kempton, Robert J., Taylor A. Kidd-Kautz, Soizic Laurenceau y Stefan Paula. "Heck- and Suzuki-coupling approaches to novel hydroquinone inhibitors of calcium ATPase". Beilstein Journal of Organic Chemistry 15 (24 de abril de 2019): 971–75. http://dx.doi.org/10.3762/bjoc.15.94.
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In this study, we explored Heck- and Suzuki-coupling methodology to modify the template 2,5-di-tert-butylhydroquinone (BHQ, 2), an inhibitor of the enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). We found that by utilizing Suzuki coupling, we could successfully attach a six-carbon tether to BHQ that terminated in a leucine moiety to obtain target 14. Similar to related compounds based on the structure of the natural product thapsigargin, 14 displayed inhibitory potency against SERCA activity. This makes 14 a suitable candidate for the future attachment of a deactivating peptide to convey specificity for prostate cancer cells.
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Launay, Sophie, Maurizio Giannı̀, Tünde Kovàcs, Raymonde Bredoux, Arlette Bruel, Pascal Gélébart, Fabien Zassadowski, Christine Chomienne, Jocelyne Enouf y Béla Papp. "Lineage-Specific Modulation of Calcium Pump Expression During Myeloid Differentiation". Blood 93, n.º 12 (15 de junio de 1999): 4395–405. http://dx.doi.org/10.1182/blood.v93.12.4395.
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Abstract Calcium is accumulated from the cytosol into the endoplasmic reticulum by sarco-endoplasmic reticulum calcium transport ATPase (SERCA) enzymes. Because calcium stored in the endoplasmic reticulum is essential for cell growth, differentiation, calcium signaling, and apoptosis and because different SERCA enzymes possess distinct functional characteristics, in the present report we explored SERCA expression during in vitro differentiation of the human myeloid/promyelocytic cell lines HL-60 and NB4 and of freshly isolated acute promyelocytic leukemia cells. Two SERCA species have been found to be coexpressed in these cells: SERCA 2b and another isoform, SERCAPLIM, which is recognized by the PLIM430 monoclonal antibody. Induction of differentiation along the neutrophil granulocytic lineage by all-trans retinoic acid or cyclic AMP analogs led to an increased expression of SERCAPLIM, whereas the expression of the SERCA 2b isoform was decreased. The modulation of SERCA expression was manifest also on the mRNA level. Experiments with retinoic acid receptor isoform-specific retinoids indicated that SERCA expression is modulated by retinoic acid receptor -dependent signaling. SERCA expression of retinoic acid-resistant cell variants was refractory to treatment. Differentiation along the monocyte/macrophage lineage by phorbol ester resulted in an increased expression of both SERCA isoforms. In addition, when cells were treated by phorbol ester in the presence of the glucocorticoid dexamethasone, a known inhibitor of monocyte differentiation, a selective blockage of the induction of SERCAPLIM was observed. Altered SERCA expression modified the functional characteristics of calcium transport into the endoplasmic reticulum. These observations show for the first time that the modulation of calcium pump expression is an integral component of the differentiation program of myeloid precursors and indicate that a lineage-specific remodelling of the endoplasmic reticulum occurs during cell maturation. In addition, these data show that SERCA isoforms may serve as useful markers for the study of myeloid differentiation.
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Launay, Sophie, Maurizio Giannı̀, Tünde Kovàcs, Raymonde Bredoux, Arlette Bruel, Pascal Gélébart, Fabien Zassadowski, Christine Chomienne, Jocelyne Enouf y Béla Papp. "Lineage-Specific Modulation of Calcium Pump Expression During Myeloid Differentiation". Blood 93, n.º 12 (15 de junio de 1999): 4395–405. http://dx.doi.org/10.1182/blood.v93.12.4395.412k06_4395_4405.
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Calcium is accumulated from the cytosol into the endoplasmic reticulum by sarco-endoplasmic reticulum calcium transport ATPase (SERCA) enzymes. Because calcium stored in the endoplasmic reticulum is essential for cell growth, differentiation, calcium signaling, and apoptosis and because different SERCA enzymes possess distinct functional characteristics, in the present report we explored SERCA expression during in vitro differentiation of the human myeloid/promyelocytic cell lines HL-60 and NB4 and of freshly isolated acute promyelocytic leukemia cells. Two SERCA species have been found to be coexpressed in these cells: SERCA 2b and another isoform, SERCAPLIM, which is recognized by the PLIM430 monoclonal antibody. Induction of differentiation along the neutrophil granulocytic lineage by all-trans retinoic acid or cyclic AMP analogs led to an increased expression of SERCAPLIM, whereas the expression of the SERCA 2b isoform was decreased. The modulation of SERCA expression was manifest also on the mRNA level. Experiments with retinoic acid receptor isoform-specific retinoids indicated that SERCA expression is modulated by retinoic acid receptor -dependent signaling. SERCA expression of retinoic acid-resistant cell variants was refractory to treatment. Differentiation along the monocyte/macrophage lineage by phorbol ester resulted in an increased expression of both SERCA isoforms. In addition, when cells were treated by phorbol ester in the presence of the glucocorticoid dexamethasone, a known inhibitor of monocyte differentiation, a selective blockage of the induction of SERCAPLIM was observed. Altered SERCA expression modified the functional characteristics of calcium transport into the endoplasmic reticulum. These observations show for the first time that the modulation of calcium pump expression is an integral component of the differentiation program of myeloid precursors and indicate that a lineage-specific remodelling of the endoplasmic reticulum occurs during cell maturation. In addition, these data show that SERCA isoforms may serve as useful markers for the study of myeloid differentiation.
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Aulestia, Francisco J., Pedro C. Redondo, Arancha Rodríguez-García, Juan A. Rosado, Ginés M. Salido, Maria Teresa Alonso y Javier García-Sancho. "Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells". Biochemical Journal 435, n.º 1 (15 de marzo de 2011): 227–35. http://dx.doi.org/10.1042/bj20101427.
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Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms.
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Papp, Bela, Sophie Launay, Pascal Gélébart, Atousa Arbabian, Agnes Enyedi, Jean-Philippe Brouland, Edgardo D. Carosella y Homa Adle-Biassette. "Endoplasmic Reticulum Calcium Pumps and Tumor Cell Differentiation". International Journal of Molecular Sciences 21, n.º 9 (9 de mayo de 2020): 3351. http://dx.doi.org/10.3390/ijms21093351.
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Endoplasmic reticulum (ER) calcium homeostasis plays an essential role in cellular calcium signaling, intra-ER protein chaperoning and maturation, as well as in the interaction of the ER with other organelles. Calcium is accumulated in the ER by sarco/endoplasmic reticulum calcium ATPases (SERCA enzymes) that generate by active, ATP-dependent transport, a several thousand-fold calcium ion concentration gradient between the cytosol (low nanomolar) and the ER lumen (high micromolar). SERCA enzymes are coded by three genes that by alternative splicing give rise to several isoforms, which can display isoform-specific calcium transport characteristics. SERCA expression levels and isoenzyme composition vary according to cell type, and this constitutes a mechanism whereby ER calcium homeostasis is adapted to the signaling and metabolic needs of the cell, depending on its phenotype, its state of activation and differentiation. As reviewed here, in several normal epithelial cell types including bronchial, mammary, gastric, colonic and choroid plexus epithelium, as well as in mature cells of hematopoietic origin such as pumps are simultaneously expressed, whereas in corresponding tumors and leukemias SERCA3 expression is selectively down-regulated. SERCA3 expression is restored during the pharmacologically induced differentiation of various cancer and leukemia cell types. SERCA3 is a useful marker for the study of cell differentiation, and the loss of SERCA3 expression constitutes a previously unrecognized example of the remodeling of calcium homeostasis in tumors.
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Gilchrist, James S. C., Chris Palahniuk, Bernard Abrenica, Penelope Rampersad, Mark Mutawe y Tom Cook. "RyR1/SERCA1 cross-talk regulation of calcium transport in heavy sarcoplasmic reticulum vesicles". Canadian Journal of Physiology and Pharmacology 81, n.º 3 (1 de marzo de 2003): 220–33. http://dx.doi.org/10.1139/y03-035.
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We investigated the functional interdependence of sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1 in heavy sarcoplasmic reticulum membranes by synchronous fluorescence determination of extravesicular Ca2+ transients and catalytic activity. Under conditions of dynamic Ca2+ exchange ATPase catalytic activity was well coordinated to ryanodine receptor activation/inactivation states. Ryanodine-induced activation of Ca2+ release channel leaks also produced marked ATPase activation in the absence of measurable increases in bulk free extra vesicular Ca2+. This suggested that Ca2+ pumps are highly sensitive to Ca2+ release channel leak status and potently buffer Ca2+ ions exiting cytoplasmic openings of ryanodine receptors. Conversely, ryanodine receptor activation was dependent on Ca2+-ATPase pump activity. Ryanodine receptor activation by cytosolic Ca2+ was (i) inversely proportional to luminal Ca2+ load and (ii) dependent upon the rate of presentation of cytosolic Ca2+. Progressive Ca2+ filling coincided with progressive loss of Ca2+ sequestration rates and at a threshold loading, ryanodine-induced Ca2+ release produced small transient reversals of catalytic activity. These data indicate that attainment of threshold luminal Ca2+ loads coordinates sensitization of Ca2+ release channels with autogenic inhibition of Ca2+ pumping. This suggests that Ca2+-dependent control of Ca2+ release in intact heavy sarcoplasmic reticulum membranes involves a Ca2+- mediated "cross-talk" between sarco-endoplasmic reticulum Ca2+ ATPase isoform1 and ryanodine receptor isoform 1.Key words: Ca2+, sarcoplasmic reticulum, RyR, SERCA, calsequestrin, ryanodine.
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Hoon Cho, Jeong, Jaya Bandyopadhyay, Jiyeon Lee, Chul-Seung Park y Joohong Ahnn. "Two isoforms of sarco/endoplasmic reticulum calcium ATPase (SERCA) are essential in Caenorhabditis elegans". Gene 261, n.º 2 (diciembre de 2000): 211–19. http://dx.doi.org/10.1016/s0378-1119(00)00536-9.
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Chandrasekera, P. Charukeshi, Margaret E. Kargacin, Julie P. Deans y Jonathan Lytton. "Determination of apparent calcium affinity for endogenously expressed human sarco(endo)plasmic reticulum calcium-ATPase isoform SERCA3". American Journal of Physiology-Cell Physiology 296, n.º 5 (mayo de 2009): C1105—C1114. http://dx.doi.org/10.1152/ajpcell.00650.2008.
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The sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) play a crucial role in regulating free cytosolic Ca2+ concentration in diverse cell types. It has been shown that recombinant SERCA3, when measured in heterologous systems, exhibits low apparent affinity for Ca2+; however, Ca2+ affinity of native SERCA3 in an endogenous setting has not been examined. Such a measurement is complicated, because SERCA3 is always coexpressed with the housekeeping isoform SERCA2b. We used a fluorescence-based assay for monitoring continuous Ca2+ uptake into microsomes to examine the properties of endogenous human SERCA3 and SERCA2b. The kinetic parameters were derived using a cooperative two-component uptake model for Ca2+ activation, and the values assigned to SERCA3 were confirmed using the highly specific human SERCA3 inhibitory antibody PL/IM430. First, using recombinant human SERCA3 and SERCA2b proteins transiently expressed in HEK-293 cells, we confirmed the previously observed low apparent Ca2+ affinity for SERCA3 compared with SERCA2b (1.10 ± 0.04 vs. 0.26 ± 0.01 μM), and using mixtures of recombinant protein isoforms, we validated the two-component uptake model. Then we determined apparent Ca2+ affinity for SERCA proteins present endogenously in cultured Jurkat T lymphocytes and freshly isolated human tonsil lymphocytes. The apparent Ca2+ affinity in these two preparations was 1.04 ± 0.07 and 1.1 ± 0.2 μM for SERCA3 and 0.27 ± 0.02 and 0.26 ± 0.01 μM for SERCA2b, respectively. Our data demonstrate, for the first time, that affinity for Ca2+ is inherently lower for SERCA3 expressed in situ than for other SERCA isoforms.
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Green, Kim N., Angelo Demuro, Yama Akbari, Brian D. Hitt, Ian F. Smith, Ian Parker y Frank M. LaFerla. "SERCA pump activity is physiologically regulated by presenilin and regulates amyloid β production". Journal of Cell Biology 181, n.º 7 (30 de junio de 2008): 1107–16. http://dx.doi.org/10.1083/jcb.200706171.
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In addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca2+ is maintained at low levels by pumping Ca2+ into stores in the endoplasmic reticulum (ER) via the sarco ER Ca2+-ATPase (SERCA) pumps. We show that SERCA activity is diminished in fibroblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca2+, whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca2+ clearance and exaggerating inositol 1,4,5-trisphosphate–mediated Ca2+ liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer's disease is underscored by our findings that modulating SERCA activity alters amyloid β production. Our results point to a physiological role for the presenilins in Ca2+ signaling via regulation of the SERCA pump.
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Papp, Béla y Jean-Philippe Brouland. "Altered Endoplasmic Reticulum Calcium Pump Expression during Breast Tumorigenesis". Breast Cancer: Basic and Clinical Research 5 (enero de 2011): BCBCR.S7481. http://dx.doi.org/10.4137/bcbcr.s7481.
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Endoplasmic reticulum calcium homeostasis is involved in several essential cell functions including cell proliferation, protein synthesis, stress responses or secretion. Calcium uptake into the endoplasmic reticulum is performed by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). In order to study endoplasmic reticulum calcium homeostasis in situ in mammary tissue, in this work SERCA3 expression was investigated in normal breast and in its benign and malignant lesions in function of the cell type, degree of malignancy, and histological and molecular parameters of the tumors. Our data indicate, that although normal breast acinar epithelial cells express SERCA3 abundantly, its expression is strongly decreased already in very early non-malignant epithelial lesions such as adenosis, and remains low in lobular carcinomas. Whereas normal duct epithelium expresses significant amounts of SERCA3, its expression is decreased in several benign ductal lesions, as well as in ductal adenocarcinoma. The loss of SERCA3 expression is correlated with Elston-Ellis grade, negative hormone receptor expression or triple negative status in ductal carcinomas. The concordance between decreased SERCA3 expression and several histological, as well as molecular markers of ductal carcinogenesis indicates that endoplasmic reticulum calcium homeostasis is remodeled during tumorigenesis in the breast epithelium.
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Ushioda, Ryo, Akitoshi Miyamoto, Michio Inoue, Satoshi Watanabe, Masaki Okumura, Ken-ichi Maegawa, Kaiku Uegaki et al. "Redox-assisted regulation of Ca2+ homeostasis in the endoplasmic reticulum by disulfide reductase ERdj5". Proceedings of the National Academy of Sciences 113, n.º 41 (30 de septiembre de 2016): E6055—E6063. http://dx.doi.org/10.1073/pnas.1605818113.
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Calcium ion (Ca2+) is an important second messenger that regulates numerous cellular functions. Intracellular Ca2+ concentration ([Ca2+]i) is strictly controlled by Ca2+ channels and pumps on the endoplasmic reticulum (ER) and plasma membranes. The ER calcium pump, sarco/endoplasmic reticulum calcium ATPase (SERCA), imports Ca2+ from the cytosol into the ER in an ATPase activity-dependent manner. The activity of SERCA2b, the ubiquitous isoform of SERCA, is negatively regulated by disulfide bond formation between two luminal cysteines. Here, we show that ERdj5, a mammalian ER disulfide reductase, which we reported to be involved in the ER-associated degradation of misfolded proteins, activates the pump function of SERCA2b by reducing its luminal disulfide bond. Notably, ERdj5 activated SERCA2b at a lower ER luminal [Ca2+] ([Ca2+]ER), whereas a higher [Ca2+]ER induced ERdj5 to form oligomers that were no longer able to interact with the pump, suggesting [Ca2+]ER-dependent regulation. Binding Ig protein, an ER-resident molecular chaperone, exerted a regulatory role in the oligomerization by binding to the J domain of ERdj5. These results identify ERdj5 as one of the master regulators of ER calcium homeostasis and thus shed light on the importance of cross talk among redox, Ca2+, and protein homeostasis in the ER.
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Poulsen, J. C. J., C. Caspersen, D. Mathiasen, J. M. East, R. E. A. Tunwell, F. A. Lai, N. Maeda, K. Mikoshiba y M. Treiman. "Thapsigargin-sensitive Ca2+-ATPases account for Ca2+ uptake to inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive Ca2+ stores in adrenal chromaffin cells". Biochemical Journal 307, n.º 3 (1 de mayo de 1995): 749–58. http://dx.doi.org/10.1042/bj3070749.
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In chromaffin cells of adrenal medulla, heterogeneity of Ca2+ stores has been suggested with respect to the mechanisms of Ca2+ uptake and release. We have examined Ca(2+)-ATPases responsible for loading of Ca2+ stores in these cells for their sensitivity to thapsigargin, a highly selective inhibitor of the SERCA [sarco(endo)plasmic reticulum calcium ATPase] family of intracellular Ca2+ pumps. Using immunostaining, we studied the distribution of Ca(2+)-ATPases, and of receptors for inositol 1,4,5-trisphosphate (InsP3) and ryanodine, in the density-gradient fractions of microsomes from bovine adrenal medulla. In parallel, we examined distribution profiles of ATP-dependent Ca2+ uptake in the same fractions, along with subcellular markers for plasma membranes and endoplasmic reticulum (ER). Two Ca(2+)-ATPase-like proteins (116 and 100 kDa) were detected, consistent with the presence of SERCA 2b and SERCA 3 isoenzymes of Ca2+ pumps. The distribution of these putative Ca(2+)-ATPase iso-enzymes paralleled that of InsP3 and ryanodine receptors. This distribution of ER Ca(2+)-ATPases, as determined immunologically, was consistent with that of thapsigargin-sensitive, but not of thapsigargin-insensitive, ATP-dependent Ca2+ uptake. In contrast, the distribution profile of the thapsigargin-insensitive Ca2+ uptake was strongly correlated to that of plasma membranes, and co-distributed with plasma membrane Ca(2+)-ATPase detected immunologically. In isolated, permeabilized chromaffin cells, InsP3 and caffeine induced Ca2+ release following an ATP-dependent Ca2+ accumulation to the stores. This accumulation was abolished by thapsigargin. Together, these data strongly indicate that the thapsigargin-sensitive, presumably SERCA-type Ca(2+)-ATPases account for Ca2+ uptake to InsP3-sensitive, as well as to caffeine-sensitive, Ca2+ stores in bovine adrenal chromaffin cells.
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Ravishankar, Harsha, Martin Nors Pedersen, Mattias Eklund, Aljona Sitsel, Chenge Li, Annette Duelli, Matteo Levantino et al. "Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering". Science Advances 6, n.º 12 (marzo de 2020): eaaz0981. http://dx.doi.org/10.1126/sciadv.aaz0981.
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Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca2]E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.
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Gruber, Simon J., Razvan L. Cornea, Ji Li, Kurt C. Peterson, Tory M. Schaaf, Gregory D. Gillispie, Russell Dahl, Krisztina M. Zsebo, Seth L. Robia y David D. Thomas. "Discovery of Enzyme Modulators via High-Throughput Time-Resolved FRET in Living Cells". Journal of Biomolecular Screening 19, n.º 2 (16 de enero de 2014): 215–22. http://dx.doi.org/10.1177/1087057113510740.
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We have used a “two-color” SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.
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Manjarrés, Isabel M., Arancha Rodríguez-García, María Teresa Alonso y Javier García-Sancho. "The sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) is the third element in capacitative calcium entry". Cell Calcium 47, n.º 5 (mayo de 2010): 412–18. http://dx.doi.org/10.1016/j.ceca.2010.03.001.
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Launay, Sophie, Maurizio Giannı̀, Luisa Diomede, Laura M. Machesky, Jocelyne Enouf y Béla Papp. "Enhancement of ATRA-induced cell differentiation by inhibition of calcium accumulation into the endoplasmic reticulum: cross-talk between RARα and calcium-dependent signaling". Blood 101, n.º 8 (15 de abril de 2003): 3220–28. http://dx.doi.org/10.1182/blood-2002-09-2730.
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Abstract Sarco–endoplasmic reticulum calcium ATPase (SERCA) enzymes control calcium-induced cellular activation by accumulating calcium from the cytosol into the endoplasmic reticulum (ER). To better understand the role of SERCA proteins and cellular calcium homeostasis in all-trans retinoic acid (ATRA)–induced differentiation, we investigated the effect of pharmacologic inhibition of SERCA-dependent calcium uptake into the ER on ATRA-induced differentiation of the HL-60 myelogenous and the NB4 promyelocytic cell lines. SERCA inhibitors di-tert-butyl-benzohydroquinone (tBHQ), thapsigargin, and cyclopiazonic acid significantly enhanced the induction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and CD11b marker expression induced by suboptimal concentrations of ATRA (50 nM) in both cell lines. Analysis of cellular calcium homeostasis revealed that a 60% mobilization of the total SERCA-dependent intracellular calcium pool was necessary to obtain enhancement of ATRA-dependent differentiation by tBHQ. Moreover, after 3 days of ATRA treatment in combination with tBHQ, NB4 cells showed a significantly decreased calcium mobilization compared with treatments with tBHQ or ATRA alone, suggesting that enhanced differentiation and calcium mobilization are causally related. Interestingly, several ATRA-resistant NB4-derived cell lines were partially responsive to the differentiation-inducing effect of the combination of the 2 drugs. In addition, we found that retinoic acid receptor α (RARα) and PML-RARα proteins are protected from ATRA-induced proteolytic degradation by SERCA inhibition, indicating that cellular calcium homeostasis may interact with signaling systems involved in the control of ATRA-dependent transcriptional activity. By linking calcium to ATRA-dependent signaling, our data open new avenues in the understanding of the mechanisms of differentiation-induction therapy of leukemia.
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Stammers, Andrew N., Shanel E. Susser, Naomi C. Hamm, Michael W. Hlynsky, Dustin E. Kimber, D. Scott Kehler y Todd A. Duhamel. "The regulation of sarco(endo)plasmic reticulum calcium-ATPases (SERCA)". Canadian Journal of Physiology and Pharmacology 93, n.º 10 (octubre de 2015): 843–54. http://dx.doi.org/10.1139/cjpp-2014-0463.
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The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is responsible for transporting calcium (Ca2+) from the cytosol into the lumen of the sarcoplasmic reticulum (SR) following muscular contraction. The Ca2+ sequestering activity of SERCA facilitates muscular relaxation in both cardiac and skeletal muscle. There are more than 10 distinct isoforms of SERCA expressed in different tissues. SERCA2a is the primary isoform expressed in cardiac tissue, whereas SERCA1a is the predominant isoform expressed in fast-twitch skeletal muscle. The Ca2+ sequestering activity of SERCA is regulated at the level of protein content and is further modified by the endogenous proteins phospholamban (PLN) and sarcolipin (SLN). Additionally, several novel mechanisms, including post-translational modifications and microRNAs (miRNAs) are emerging as integral regulators of Ca2+ transport activity. These regulatory mechanisms are clinically relevant, as dysregulated SERCA function has been implicated in the pathology of several disease states, including heart failure. Currently, several clinical trials are underway that utilize novel therapeutic approaches to restore SERCA2a activity in humans. The purpose of this review is to examine the regulatory mechanisms of the SERCA pump, with a particular emphasis on the influence of exercise in preventing the pathological conditions associated with impaired SERCA function.
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Lee, Andy K. y Amy Tse. "Dominant Role of Mitochondria in Calcium Homeostasis of Single Rat Pituitary Corticotropes". Endocrinology 146, n.º 11 (1 de noviembre de 2005): 4985–93. http://dx.doi.org/10.1210/en.2005-0358.
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The rise in cytosolic free Ca2+ concentration ([Ca2+]i) is the major trigger for secretion of ACTH from pituitary corticotropes. To better understand the shaping of the Ca2+ signal in corticotropes, we investigated the mechanisms regulating the depolarization-triggered Ca2+ signal using patch-clamp techniques and indo-1 fluorometry. The rate of cytosolic Ca2+ clearance was unaffected by inhibitors of Na+/Ca2+ exchanger or plasma membrane Ca2+-ATPase (PMCA), slightly slowed by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, but dramatically slowed by mitochondrial uncouplers or inhibitor of mitochondrial uniporter. Measurements with rhod-2 revealed that depolarization-triggered increase in mitochondrial Ca2+ concentration. Thus, mitochondria have a dominant role in cytosolic Ca2+ clearance. Using the Mn2+ quench technique, we found the presence of a continuous basal Ca2+ influx in corticotropes. This basal Ca2+ influx was balanced by the combined actions of mitochondrial uniporter and PMCA and SERCA pumps. Inhibition of the mitochondrial uniporter or PMCA or SERCA pumps elevated basal [Ca2+]i. Using membrane capacitance measurement, we found that the change in the shape of the depolarization-triggered Ca2+ signal after mitochondrial inhibition was associated with enhancement of the exocytotic response. Thus, mitochondria have a dominant role in the regulation of Ca2+ signal and exocytosis in corticotropes.
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Martins, Samantha M., Christiane R. Torres y Sérgio T. Ferreira. "Inhibition of the Ecto-ATPdiphosphohydrolase of Schistosoma mansoni by Thapsigargin". Bioscience Reports 20, n.º 5 (1 de octubre de 2000): 369–81. http://dx.doi.org/10.1023/a:1010330017583.
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ATPdiphosphohydrolases (ATPDases) are ubiquitous enzymes capable ofhydrolyzing nucleoside di- and triphosphates. Although a number ofpossible physiological roles have been proposed for ATPDases, detailedstudies on structure-function relationships have generally been hamperedby the lack of specific inhibitors of these enzymes. We have previouslycharacterized a Ca2+-activated ATPDase on the external surface ofthe tegument of Schistosoma mansoni, the etiologic agent of humanschistosomiasis. In the present work, we have examined the effectsof thapsigargin, a sesquiterpene lactone known as a high affinityinhibitor of sarco-endoplasmic reticulum calcium transport (SERCA)ATPase, on ATPDase activity. Whereas other lactones tested had littleor no inhibitory action, thapsigargin inhibited ATP hydrolysis by the ATPDase (Ki∼20 μM). Interestingly, hydrolysis of ADP was notinhibited by thapsigargin. The lack of inhibition of ATPase activityby orthovanadate, a specific inhibitor of P-type ATPases, and theinhibition of the Mg2+-stimulated ATP hydrolysis by thapsigarginruled out the possibility that the observed inhibition of the ATPDaseby thapsigargin could be due to the presence of contaminating SERCAATPases in our preparation. Kinetic analysis indicated that a singleactive site in the ATPDase is responsible for hydrolysis of both ATPand ADP. Thapsigargin caused changes in both Vmax and Km for ATP, indicating a mixed type of inhibition. Inhibition by thapsigarginwas little or not affected by changes in free Ca2+ or Mg2+concentrations. These results suggest that interaction of thapsigarginwith the S. mansoni ATPDase prevents binding of ATP or its hydrolysisat the active site, while ADP can still undergo catalysis.
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Li, Yun y Patricia Camacho. "Ca2+-dependent redox modulation of SERCA 2b by ERp57". Journal of Cell Biology 164, n.º 1 (29 de diciembre de 2003): 35–46. http://dx.doi.org/10.1083/jcb.200307010.
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We demonstrated previously that calreticulin (CRT) interacts with the lumenal COOH-terminal sequence of sarco endoplasmic reticulum (ER) calcium ATPase (SERCA) 2b to inhibit Ca2+ oscillations. Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins. In this paper, we demonstrate that ERp57 overexpression reduces the frequency of Ca2+ oscillations enhanced by SERCA 2b. In contrast, overexpression of SERCA 2b mutants defective in cysteines located in intralumenal loop 4 (L4) increase Ca2+ oscillation frequency. In vitro, we demonstrate a Ca2+-dependent and -specific interaction between ERp57 and L4. Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site. Overexpression of CRT domains that disrupt the interaction of CRT with ERp57 behave as dominant negatives in the Ca2+ oscillation assay. Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.
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Kirsh, Aleah J., Emma S. Juracic, Heather L. Petrick, Cynthia M. F. Monaco, Pierre-Andre Barbeau, A. Russell Tupling y Graham P. Holloway. "Dietary nitrate does not alter cardiac function, calcium handling proteins, or SERCA activity in the left ventricle of healthy rats". Applied Physiology, Nutrition, and Metabolism 45, n.º 9 (septiembre de 2020): 1049–53. http://dx.doi.org/10.1139/apnm-2020-0078.
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Dietary nitrate has been shown to increase cytosolic calcium concentrations within the heart, which would necessitate greater calcium sequestration for relaxation. In the present study we demonstrate that while nitrate supplementation reduced blood pressure, calcium-handling protein content, sarco(endo)plasmic reticulum Ca-ATPase 2a (SERCA) enzymatic properties, and left ventricular function were not altered. In addition, nitrite did not alter in vitro SERCA activity. Combined, these data suggest that in healthy rats, dietary nitrate does not increase left ventricle SERCA-related calcium-handling properties. Novelty Dietary nitrate decreases blood pressure but does not alter left ventricular calcium-handling protein content or SERCA activity in healthy rats.
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Altshuler, Ianina, James J. Vaillant, Sen Xu y Melania E. Cristescu. "The Evolutionary History of Sarco(endo)plasmic Calcium ATPase (SERCA)". PLoS ONE 7, n.º 12 (20 de diciembre de 2012): e52617. http://dx.doi.org/10.1371/journal.pone.0052617.
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Rodríguez, Yoel y Magdaléna Májeková. "Structural Changes of Sarco/Endoplasmic Reticulum Ca2+-ATPase Induced by Rutin Arachidonate: A Molecular Dynamics Study". Biomolecules 10, n.º 2 (1 de febrero de 2020): 214. http://dx.doi.org/10.3390/biom10020214.
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Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) maintains the level of calcium concentration in cells by pumping calcium ions from the cytoplasm to the lumen while undergoing substantial conformational changes, which can be stabilized or prevented by various compounds. Here we attempted to clarify the molecular mechanism of action of new inhibitor rutin arachidonate, one of the series of the acylated rutin derivatives. We performed molecular dynamics simulations of SERCA1a protein bound to rutin arachidonate positioned in a pure dipalmitoylphosphatidylcholine bilayer membrane. Our study predicted the molecular basis for the binding of rutin arachidonate towards SERCA1a in the vicinity of the binding site of calcium ions and near the location of the well-known inhibitor thapsigargin. The stable hydrogen bond between Glu771 and rutin arachidonate plays a key role in the binding. SERCA1a is kept in the E2 conformation preventing the formation of important salt bridges between the side chains of several residues, primarily Glu90 and Lys297. All in all, the structural changes induced by the binding of rutin arachidonate to SERCA1a may shift proton balance near the titrable residues Glu771 and Glu309 into neutral species, hence preventing the binding of calcium ions to the transmembrane binding sites and thus affecting calcium homeostasis. Our results could lead towards the design of new types of inhibitors, potential drug candidates for cancer treatment, which could be anchored to the transmembrane region of SERCA1a by a lipophilic fatty acid group.
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Satoh, Kanayo, Toru Matsu-ura, Masahiro Enomoto, Hideki Nakamura, Takayuki Michikawa y Katsuhiko Mikoshiba. "Highly Cooperative Dependence of Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) 2a Pump Activity on Cytosolic Calcium in Living Cells". Journal of Biological Chemistry 286, n.º 23 (22 de abril de 2011): 20591–99. http://dx.doi.org/10.1074/jbc.m110.204685.
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Watson, William D., Stephen L. Facchina, Maurizio Grimaldi y Ajay Verma. "Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors identify a novel calcium pool in the central nervous system". Journal of Neurochemistry 87, n.º 1 (12 de septiembre de 2003): 30–43. http://dx.doi.org/10.1046/j.1471-4159.2003.01962.x.
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Nagamune, Kisaburo, Wandy L. Beatty y L. David Sibley. "Artemisinin Induces Calcium-Dependent Protein Secretion in the Protozoan Parasite Toxoplasma gondii". Eukaryotic Cell 6, n.º 11 (31 de agosto de 2007): 2147–56. http://dx.doi.org/10.1128/ec.00262-07.
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ABSTRACT Intracellular calcium controls several crucial cellular events in apicomplexan parasites, including protein secretion, motility, and invasion into and egress from host cells. The plant compound thapsigargin inhibits the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA), resulting in elevated calcium and induction of protein secretion in Toxoplasma gondii. Artemisinins are natural products that show potent and selective activity against parasites, making them useful for the treatment of malaria. While the mechanism of action is uncertain, previous studies have suggested that artemisinin may inhibit SERCA, thus disrupting calcium homeostasis. We cloned the single-copy gene encoding SERCA in T. gondii (TgSERCA) and demonstrate that the protein localizes to the endoplasmic reticulum in the parasite. In extracellular parasites, TgSERCA partially relocalized to the apical pole, a highly active site for regulated secretion of micronemes. TgSERCA complemented a calcium ATPase-defective yeast mutant, and this activity was inhibited by either thapsigargin or artemisinin. Treatment of T. gondii with artemisinin triggered calcium-dependent secretion of microneme proteins, similar to the SERCA inhibitor thapsigargin. Artemisinin treatment also altered intracellular calcium in parasites by increasing the periodicity of calcium oscillations and inducing recurrent, strong calcium spikes, as imaged using Fluo-4 labeling. Collectively, these results demonstrate that artemisinin perturbs calcium homeostasis in T. gondii, supporting the idea that Ca2+-ATPases are potential drug targets in parasites.
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FRANKLIN, Isobel K., Robert A. WINZ y Michael J. HUBBARD. "Endoplasmic reticulum Ca2+-ATPase pump is up-regulated in calcium-transporting dental enamel cells: a non-housekeeping role for SERCA2b". Biochemical Journal 358, n.º 1 (8 de agosto de 2001): 217–24. http://dx.doi.org/10.1042/bj3580217.
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Dental enamel-forming cells face a major challenge to avoid the cytotoxic effects of excess calcium. We have characterized sarcoplasmic/endoplasmic reticulum calcium-ATPase pumps (SERCA) in rat enamel cells to address the proposal that non-mitochondrial calcium stores play a dominant role in transcellular calcium transport. A single major isoform, SERCA2b, was detected during the protein-secretory and calcium-transport stages of enamel formation using reverse-transcriptase PCR, cDNA cloning, Northern analysis and immunoblotting. Most importantly, SERCA2b exhibited a specific 3-fold up-regulation to high expression levels during calcium transport, as determined by quantitative immunoblotting and ATPase assays. Sensitivity of the calcium-dependent ATPase to thapsigargin and three other SERCA inhibitors was characterized. These findings indicate that enamel cells are well-equipped to sequester calcium in endoplasmic reticulum stores and so protect against calcium toxicity, associate SERCA with transcellular calcium transport for the first time, and establish SERCA2b as a molecular and pharmacological target for future investigations of calcium transcytosis. The observed physiological regulation in enamel cells contradicts the widespread perception that SERCA2b is restricted to general housekeeping duties.
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Duman, Joseph G., Liangyi Chen y Bertil Hille. "Calcium Transport Mechanisms of PC12 Cells". Journal of General Physiology 131, n.º 4 (17 de marzo de 2008): 307–23. http://dx.doi.org/10.1085/jgp.200709915.
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Many studies of Ca2+ signaling use PC12 cells, yet the balance of Ca2+ clearance mechanisms in these cells is unknown. We used pharmacological inhibition of Ca2+ transporters to characterize Ca2+ clearance after depolarizations in both undifferentiated and nerve growth factor-differentiated PC12 cells. Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), plasma membrane Ca2+ ATPase (PMCA), and Na+/Ca2+ exchanger (NCX) account for almost all Ca2+ clearance in both cell states, with NCX and PMCA making the greatest contributions. Any contribution of mitochondrial uniporters is small. The ATP pool in differentiated cells was much more labile than that of undifferentiated cells in the presence of agents that dissipated mitochondrial proton gradients. Differentiated PC12 cells have a small component of Ca2+ clearance possessing pharmacological characteristics consistent with secretory pathway Ca2+ ATPase (SPCA), potentially residing on Golgi and/or secretory granules. Undifferentiated and differentiated cells are similar in overall Ca2+ transport and in the small transport due to SERCA, but they differ in the fraction of transport by PMCA and NCX. Transport in neurites of differentiated PC12 cells was qualitatively similar to that in the somata, except that the ER stores in neurites sometimes released Ca2+ instead of clearing it after depolarization. We formulated a mathematical model to simulate the observed Ca2+ clearance and to describe the differences between these undifferentiated and NGF-differentiated states quantitatively. The model required a value for the endogenous Ca2+ binding ratio of PC12 cell cytoplasm, which we measured to be 268 ± 85. Our results indicate that Ca2+ transport in undifferentiated PC12 cells is quite unlike transport in adrenal chromaffin cells, for which they often are considered models. Transport in both cell states more closely resembles that of sympathetic neurons, for which differentiated PC12 cells often are considered models. Comparison with other cell types shows that different cells emphasize different Ca2+ transport mechanisms.
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Hiranandani, Nitisha, Sripriya Raman, Anuradha Kalyanasundaram, Muthu Periasamy y Paul M. L. Janssen. "Frequency-dependent contractile strength in mice over- and underexpressing the sarco(endo)plasmic reticulum calcium-ATPase". American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 293, n.º 1 (julio de 2007): R30—R36. http://dx.doi.org/10.1152/ajpregu.00508.2006.
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One of the prominent markers of end-stage heart failure at the molecular level is a decrease in function and/or expression of the sarcoplasmic reticulum ATPase protein [sarco(endo)plasmic reticulum calcium-ATPase, SERCA]. It has been often postulated that a decrease in SERCA pump activity can contribute in a major way to decreased cardiac function. To establish a functional relationship, we assessed how alterations in SERCA activity level affect basic contractile function in healthy myocardium devoid of other significant molecular changes. We investigated baseline contractile function, frequency-dependent activation, and β-adrenergic response in ultrathin trabeculae isolated from hearts of mice overexpressing SERCA (transgenic, TG), underexpressing SERCA2a (heterozygous knockout, Het), and their respective wild-type (WT) littermates. At physiological temperature and frequency, compared with their respective WT littermates, SERCA1a mice displayed increased developed force at frequencies of 4–8 Hz (∼90% increase at 4 Hz) and force equal to WT mice at 10–14 Hz. Force development at 4 Hz in presence of 1 μM isoproterenol was similar in TG and WT mice. In Het mice, developed force was nearly identical at the lower end of the frequency range (4–8 Hz) but slightly depressed at higher frequency ( P < 0.05 at 14 Hz). In presence of 1 μM isoproterenol, developed force at 4 Hz was equal to that in WT mice. Compared with normal levels, increased SERCA activity enhanced force development only at subphysiological frequencies. A reduction in SERCA activity only showed a depression of force at the higher frequency range. Thus generalizations regarding the correlation between SERCA activity and contractility can be highly ambiguous, because this relationship is critically dependent on other factors including stimulation frequency.
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Chami, Mounia y Frédéric Checler. "Alterations of the Endoplasmic Reticulum (ER) Calcium Signaling Molecular Components in Alzheimer’s Disease". Cells 9, n.º 12 (1 de diciembre de 2020): 2577. http://dx.doi.org/10.3390/cells9122577.
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Sustained imbalance in intracellular calcium (Ca2+) entry and clearance alters cellular integrity, ultimately leading to cellular homeostasis disequilibrium and cell death. Alzheimer’s disease (AD) is the most common cause of dementia. Beside the major pathological features associated with AD-linked toxic amyloid beta (Aβ) and hyperphosphorylated tau (p-tau), several studies suggested the contribution of altered Ca2+ handling in AD development. These studies documented physical or functional interactions of Aβ with several Ca2+ handling proteins located either at the plasma membrane or in intracellular organelles including the endoplasmic reticulum (ER), considered the major intracellular Ca2+ pool. In this review, we describe the cellular components of ER Ca2+ dysregulations likely responsible for AD. These include alterations of the inositol 1,4,5-trisphosphate receptors’ (IP3Rs) and ryanodine receptors’ (RyRs) expression and function, dysfunction of the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity and upregulation of its truncated isoform (S1T), as well as presenilin (PS1, PS2)-mediated ER Ca2+ leak/ER Ca2+ release potentiation. Finally, we highlight the functional consequences of alterations of these ER Ca2+ components in AD pathology and unravel the potential benefit of targeting ER Ca2+ homeostasis as a tool to alleviate AD pathogenesis.
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Schaaf, Tory M., Kurt C. Peterson, Benjamin D. Grant, Prachi Bawaskar, Samantha Yuen, Ji Li, Joseph M. Muretta, Gregory D. Gillispie y David D. Thomas. "High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA". SLAS DISCOVERY: Advancing the Science of Drug Discovery 22, n.º 3 (13 de diciembre de 2016): 262–73. http://dx.doi.org/10.1177/1087057116680151.
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A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green fluorescent protein (GFP, donor) and red fluorescent protein (RFP, acceptor) fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS.
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Ma, Hailun, Carlota M. Sumbilla, Iain K. G. Farrance, Michael G. Klein y Giuseppe Inesi. "Cell-specific expression of SERCA, the exogenous Ca2+transport ATPase, in cardiac myocytes". American Journal of Physiology-Cell Physiology 286, n.º 3 (marzo de 2004): C556—C564. http://dx.doi.org/10.1152/ajpcell.00328.2003.
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We evaluated various constructs to obtain cell-specific expression of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) gene in cardiac myocytes after cDNA transfer by means of transfections or infections with adenovirus vectors. Expression of exogenous enhanced green fluorescent protein (EGFP) and SERCA genes was studied in cultured chicken embryo and neonatal rat cardiac myocytes, skeletal and smooth muscle cells, fibroblasts, and hepatocytes. Whereas the cytomegalovirus (CMV) promoter yielded high levels of protein expression in all cells studied, cardiac troponin T (cTnT) promoter segments demonstrated high specificity for cardiac myocytes. Their efficiency for protein expression was lower than that of the CMV promoter, but higher than that of cardiac myosin light chain or β-myosin heavy chain promoter segments. A double virus system for Cre-dependent expression under control of the CMV promoter and Cre expression under control of a cardiac-specific promoter yielded high protein levels in cardiac myocytes, but only partial cell specificity due to significant Cre expression in hepatocytes. Specific intracellular targeting of gene products was demonstrated in situ by specific immunostaining of exogenous SERCA1 and endogenous SERCA2 and comparative fluorescence microscopy. The -374 cTnT promoter segment was the most advantageous of the promoters studied, producing cell-specific SERCA expression and a definite increase over endogenous Ca2+-ATPase activity as well as faster removal of cytosolic calcium after membrane excitation. We conclude that analysis of promoter efficiency and cell specificity is of definite advantage when cell-specific expression of exogenous SERCA is wanted in cardiac myocytes after cDNA delivery to mixed cell populations.
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Boczek, Tomasz, Tomasz Radzik, Bozena Ferenc y Ludmila Zylinska. "The Puzzling Role of Neuron-Specific PMCA Isoforms in the Aging Process". International Journal of Molecular Sciences 20, n.º 24 (16 de diciembre de 2019): 6338. http://dx.doi.org/10.3390/ijms20246338.
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The aging process is a physiological phenomenon associated with progressive changes in metabolism, genes expression, and cellular resistance to stress. In neurons, one of the hallmarks of senescence is a disturbance of calcium homeostasis that may have far-reaching detrimental consequences on neuronal physiology and function. Among several proteins involved in calcium handling, plasma membrane Ca2+-ATPase (PMCA) is the most sensitive calcium detector controlling calcium homeostasis. PMCA exists in four main isoforms and PMCA2 and PMCA3 are highly expressed in the brain. The overall effects of impaired calcium extrusion due to age-dependent decline of PMCA function seem to accumulate with age, increasing the susceptibility to neurotoxic insults. To analyze the PMCA role in neuronal cells, we have developed stable transfected differentiated PC12 lines with down-regulated PMCA2 or PMCA3 isoforms to mimic age-related changes. The resting Ca2+ increased in both PMCA-deficient lines affecting the expression of several Ca2+-associated proteins, i.e., sarco/endoplasmic Ca2+-ATPase (SERCA), calmodulin, calcineurin, GAP43, CCR5, IP3Rs, and certain types of voltage-gated Ca2+ channels (VGCCs). Functional studies also demonstrated profound changes in intracellular pH regulation and mitochondrial metabolism. Moreover, modification of PMCAs membrane composition triggered some adaptive processes to counterbalance calcium overload, but the reduction of PMCA2 appeared to be more detrimental to the cells than PMCA3.
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Wheatly, M., Z. Zhang, J. Weil, J. Rogers y L. Stiner. "Novel subcellular and molecular tools to study Ca(2+) transport mechanisms during the elusive moulting stages of crustaceans: flow cytometry and polyclonal antibodies". Journal of Experimental Biology 204, n.º 5 (1 de marzo de 2001): 959–66. http://dx.doi.org/10.1242/jeb.204.5.959.
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Our understanding of calcium homeostasis during the crustacean moulting cycle derives from research on intermoult animals that has been extrapolated to other stages. In terms of transepithelial Ca(2+) flux, the more interesting stages are those surrounding ecdysis since crustaceans experience a sizeable negative calcium balance in immediate premoult and a significant positive calcium balance in immediate postmoult. These stages are elusive in the sense that larger species such as lobsters are rarely captured at this time, and smaller species such as blue crabs and crayfish are seldom synchronized in their moulting cycle. The reductionist approaches employed in cellular physiology, such as vesicle techniques, employ pooling of fresh tissues from many organisms. Examination of the elusive moulting stages requires more sensitive approaches that can utilize tissue from an individual crustacean to characterize Ca(2+) pumps (Sarco/Endoplasmic Reticulum Ca(2+)-ATPase, SERCA; Plasma Membrane Ca(2+)-ATPase, PMCA) and the Na(+)/Ca(2+) eXchanger (NCX). An emerging subcellular approach described in this paper is to use flow cytometry as a technique to monitor Ca(2+) uptake into Fluo-3-loaded membrane vesicles. This paper illustrates the utility of this technique for measuring ATP-dependent Ca(2+) uptake into hepatopancreatic basolateral membrane vesicles. Obstacles to progress in molecular studies have not been limited by synchronization of moulting since tissue can be snap-frozen and collected from many animals over time. Here, the problem has been the lack of specific antibodies that hybridize with the Ca(2+) transporters of interest so that they can be localized within epithelia. In this paper, we introduce polyclonal antibodies raised in rabbits against crayfish SERCA, PMCA and NCX. Immunocytochemistry of SERCA in muscle, PMCA in antennal gland and NCX in heart confirms the specificity of the antibodies.
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Talmadge, R. J., M. J. Castro, D. F. Apple y G. A. Dudley. "Phenotypic adaptations in human muscle fibers 6 and 24 wk after spinal cord injury". Journal of Applied Physiology 92, n.º 1 (1 de enero de 2002): 147–54. http://dx.doi.org/10.1152/japplphysiol.000247.2001.
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10.1152/japplphysiol.000247.2001.—The effects of spinal cord injury (SCI) on the profile of sarco(endo) plasmic reticulum calcium-ATPase (SERCA) and myosin heavy chain (MHC) isoforms in individual vastus lateralis (VL) muscle fibers were determined. Biopsies from the VL were obtained from SCI subjects 6 and 24 wk postinjury ( n = 6). Biopsies from nondisabled (ND) subjects were obtained at two time points 18 wk apart ( n = 4). In ND subjects, the proportions of VL fibers containing MHC I, MHC IIa, and MHC IIx were 46 ± 3, 53 ± 3, and 1 ± 1%, respectively. Most MHC I fibers contained SERCA2. Most MHC IIa fibers contained SERCA1. All MHC IIx fibers contained SERCA1 exclusively. SCI resulted in significant increases in fibers with MHC IIx (14 ± 4% at 6 wk and 16 ± 2% at 24 wk). In addition, SCI resulted in high proportions of MHC I and MHC IIa fibers with both SERCA isoforms (29% at 6 wk and 54% at 24 wk for MHC I fibers and 16% at 6 wk and 38% at 24 wk for MHC IIa fibers). Thus high proportions of VL fibers were mismatched for SERCA and MHC isoforms after SCI (19 ± 3% at 6 wk and 36 ± 9% at 24 wk) compared with only ∼5% in ND subjects. These data suggest that, in the early time period following SCI, fast fiber isoforms of both SERCA and MHC are elevated disproportionately, resulting in fibers that are mismatched for SERCA and MHC isoforms. Thus the adaptations in SERCA and MHC isoforms appear to occur independently.
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Forcaia, Greta, Beatrice Formicola, Giulia Terribile, Sharon Negri, Dmitry Lim, Gerardo Biella, Francesca Re, Francesco Moccia y Giulio Sancini. "Multifunctional Liposomes Modulate Purinergic Receptor-Induced Calcium Wave in Cerebral Microvascular Endothelial Cells and Astrocytes: New Insights for Alzheimer’s disease". Molecular Neurobiology 58, n.º 6 (29 de enero de 2021): 2824–35. http://dx.doi.org/10.1007/s12035-021-02299-9.
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AbstractIn light of previous results, we assessed whether liposomes functionalized with ApoE-derived peptide (mApoE) and phosphatidic acid (PA) (mApoE-PA-LIP) impacted on intracellular calcium (Ca2+) dynamics in cultured human cerebral microvascular endothelial cells (hCMEC/D3), as an in vitro human blood-brain barrier (BBB) model, and in cultured astrocytes. mApoE-PA-LIP pre-treatment actively increased both the duration and the area under the curve (A.U.C) of the ATP-evoked Ca2+ waves in cultured hCMEC/D3 cells as well as in cultured astrocytes. mApoE-PA-LIP increased the ATP-evoked intracellular Ca2+ waves even under 0 [Ca2+]e conditions, thus indicating that the increased intracellular Ca2+ response to ATP is mainly due to endogenous Ca2+ release. Indeed, when Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) activity was blocked by cyclopiazonic acid (CPA), the extracellular application of ATP failed to trigger any intracellular Ca2+ waves, indicating that metabotropic purinergic receptors (P2Y) are mainly involved in the mApoE-PA-LIP-induced increase of the Ca2+ wave triggered by ATP. In conclusion, mApoE-PA-LIP modulate intracellular Ca2+ dynamics evoked by ATP when SERCA is active through inositol-1,4,5-trisphosphate-dependent (InsP3) endoplasmic reticulum Ca2+ release. Considering that P2Y receptors represent important pharmacological targets to treat cognitive dysfunctions, and that P2Y receptors have neuroprotective effects in neuroinflammatory processes, the enhancement of purinergic signaling provided by mApoE-PA-LIP could counteract Aβ-induced vasoconstriction and reduction in cerebral blood flow (CBF). Our obtained results could give an additional support to promote mApoE-PA-LIP as effective therapeutic tool for Alzheimer’s disease (AD).
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Fischer, L., A. S. Gukovskaya, S. H. Young, I. Gukovsky, A. Lugea, P. Buechler, J. M. Penninger, H. Friess y S. J. Pandol. "Phosphatidylinositol 3-kinase regulates Ca2+ signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase". American Journal of Physiology-Gastrointestinal and Liver Physiology 287, n.º 6 (diciembre de 2004): G1200—G1212. http://dx.doi.org/10.1152/ajpgi.00212.2004.
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Calcium is a key mediator of hormone-induced enzyme secretion in pancreatic acinar cells. At the same time, abnormal Ca2+ responses are associated with pancreatitis. We have recently shown that inhibition of phosphatidylinositol 3-kinase (PI3-kinase) by LY-294002 and wortmannin, as well as genetic deletion of PI3-kinase-γ, regulates Ca2+ responses and the Ca2+-sensitive trypsinogen activation in pancreatic acinar cells. The present study sought to determine the mechanisms of PI3-kinase involvement in Ca2+ responses induced in these cells by CCK and carbachol. The PI3-kinase inhibitors inhibited both Ca2+ influx and mobilization from intracellular stores induced by stimulation of acini with physiological and pathological concentrations of CCK, as well as with carbachol. PI3-kinase inhibition facilitated the decay of cytosolic free Ca2+ concentration ([Ca2+]i) oscillations observed in individual acinar cells. The PI3-kinase inhibitors decreased neither CCK-induced inositol 1,4,5-trisphosphate [Ins( 1 , 4 , 5 )P3] production nor Ins( 1 , 4 , 5 )P3-induced Ca2+ mobilization, suggesting that the effect of PI3-kinase inhibition is not through Ins( 1 , 4 , 5 )P3 or Ins( 1 , 4 , 5 )P3 receptors. PI3-kinase inhibition did not affect Ca2+ mobilization induced by thapsigargin, a specific inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). Moreover, SERCA blockade with thapsigargin abolished the effects of pharmacological and genetic PI3-kinase inhibition on [Ca2+]i signals, suggesting SERCA as a downstream target of PI3-kinase. Both pharmacological PI3-kinase inhibition and genetic deletion of PI3-kinase-γ increased the amount of Ca2+ in intracellular stores during CCK stimulation. Finally, addition of the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate to permeabilized acini significantly attenuated Ca2+ reloading into the endoplasmic reticulum. The results indicate that PI3-kinase regulates Ca2+ signaling in pancreatic acinar cells through its inhibitory effect on SERCA.
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Goodman, Jena B., Fuzhong Qin, Robert J. Morgan, Jordan M. Chambers, Dominique Croteau, Deborah A. Siwik, Ion Hobai et al. "Redox-Resistant SERCA [Sarco(endo)plasmic Reticulum Calcium ATPase] Attenuates Oxidant-Stimulated Mitochondrial Calcium and Apoptosis in Cardiac Myocytes and Pressure Overload–Induced Myocardial Failure in Mice". Circulation 142, n.º 25 (22 de diciembre de 2020): 2459–69. http://dx.doi.org/10.1161/circulationaha.120.048183.
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Background: SERCA [sarco(endo)plasmic reticulum calcium ATPase] is regulated by oxidative posttranslational modifications at cysteine 674 (C674). Because sarcoplasmic reticulum (SR) calcium has been shown to play a critical role in mediating mitochondrial dysfunction in response to reactive oxygen species, we hypothesized that SERCA oxidation at C674 would modulate the effects of reactive oxygen species on mitochondrial calcium and mitochondria-dependent apoptosis in cardiac myocytes. Methods: Adult rat ventricular myocytes expressing wild-type SERCA2b or a redox-insensitive mutant in which C674 is replaced by serine (C674S) were exposed to H 2 O 2 (100 µmol/Lμ). Free mitochondrial calcium concentration was measured in adult rat ventricular myocytes with a genetically targeted fluorescent probe, and SR calcium content was assessed by measuring caffeine-stimulated release. Mice with heterozygous knock-in of the SERCA C674S mutation were subjected to chronic ascending aortic constriction. Results: In adult rat ventricular myocytes expressing wild-type SERCA, H 2 O 2 caused a 25% increase in mitochondrial calcium concentration that was associated with a 50% decrease in SR calcium content, both of which were prevented by the ryanodine receptor inhibitor tetracaine. In cells expressing the C674S mutant, basal SR calcium content was decreased by 31% and the H 2 O 2 -stimulated rise in mitochondrial calcium concentration was attenuated by 40%. In wild-type cells, H 2 O 2 caused cytochrome c release and apoptosis, both of which were prevented in C674S-expressing cells. In myocytes from SERCA knock-in mice, basal SERCA activity and SR calcium content were decreased. To test the effect of C674 oxidation on apoptosis in vivo, SERCA knock-in mice were subjected to chronic ascending aortic constriction. In wild-type mice, ascending aortic constriction caused myocyte apoptosis, LV dilation, and systolic failure, all of which were inhibited in SERCA knock-in mice. Conclusions: Redox activation of SERCA C674 regulates basal SR calcium content, thereby mediating the pathologic reactive oxygen species–stimulated rise in mitochondrial calcium required for myocyte apoptosis and myocardial failure.
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Henrich, Michael y Keith J. Buckler. "Effects of Anoxia and Aglycemia on Cytosolic Calcium Regulation in Rat Sensory Neurons". Journal of Neurophysiology 100, n.º 1 (julio de 2008): 456–73. http://dx.doi.org/10.1152/jn.01380.2007.
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Nociceptive neurons play an important role in ischemia by sensing and transmitting information to the CNS and by secreting peptides and nitric oxide, which can have local effects. While these responses are probably primarily mediated by acid sensing channels, other events occurring in ischemia may also influence neuron function. In this study, we have investigated the effects of anoxia and anoxic aglycemia on Ca2+ regulation in sensory neurons from rat dorsal root ganglia. Anoxia increased [Ca2+]i by evoking Ca2+ release from two distinct internal stores one sensitive to carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and one sensitive to caffeine, cyclopiazonic acid (CPA), and ryanodine [assumed to be the endoplasmic reticulum (ER)]. Anoxia also promoted progressive decline in ER Ca2+ content. Despite partially depolarizing mitochondria, anoxia had relatively little effect on mitochondrial Ca2+ uptake when neurons were depolarized but substantially delayed mitochondrial Ca2+ release and subsequent Ca2+ clearance from the cytosol on repolarization. Anoxia also reduced both sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity and Ca2+ extrusion [probably via plasma membrane Ca2+-ATPase (PMCA)]. Thus anoxia has multiple effects on [Ca2+]i homeostasis in sensory neurons involving internal stores, mitochondrial buffering, and Ca2+ pumps. Under conditions of anoxic aglycemia, there was a biphasic and more profound elevation of [Ca2+]i, which was associated with complete ER Ca2+ store emptying and progressive, and eventually complete, inhibition of Ca2+ clearance by PMCA and SERCA. These data clearly show that loss of oxygen, and exhaustion of glycolytic substrates, can profoundly affect many aspects of cell Ca2+ regulation, and this may play an important role in modulating neuronal responses to ischemia.
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Zhang, Yuxia, Michio Inoue, Akihisa Tsutsumi, Satoshi Watanabe, Tomohiro Nishizawa, Kazuhiro Nagata, Masahide Kikkawa y Kenji Inaba. "Cryo-EM structures of SERCA2b reveal the mechanism of regulation by the luminal extension tail". Science Advances 6, n.º 33 (agosto de 2020): eabb0147. http://dx.doi.org/10.1126/sciadv.abb0147.
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Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps Ca2+ from the cytosol into the ER and maintains the cellular calcium homeostasis. Herein, we present cryo–electron microscopy (cryo-EM) structures of human SERCA2b in E1∙2Ca2+–adenylyl methylenediphosphonate (AMPPCP) and E2-BeF3− states at 2.9- and 2.8-Å resolutions, respectively. The structures revealed that the luminal extension tail (LE) characteristic of SERCA2b runs parallel to the lipid-water boundary near the luminal ends of transmembrane (TM) helices TM10 and TM7 and approaches the luminal loop flanked by TM7 and TM8. While the LE served to stabilize the cytosolic and TM domain arrangement of SERCA2b, deletion of the LE rendered the overall conformation resemble that of SERCA1a and SERCA2a and allowed multiple conformations. Thus, the LE appears to play a critical role in conformational regulation in SERCA2b, which likely explains the different kinetic properties of SERCA2b from those of other isoforms lacking the LE.
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Belke, Darrell D., Eric Swanson, Jorge Suarez, Brian T. Scott, Antine E. Stenbit y Wolfgang H. Dillmann. "Increased expression of SERCA in the hearts of transgenic mice results in increased oxidation of glucose". American Journal of Physiology-Heart and Circulatory Physiology 292, n.º 4 (abril de 2007): H1755—H1763. http://dx.doi.org/10.1152/ajpheart.00884.2006.
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While several transgenic mouse models exhibit improved contractile characteristics in the heart, less is known about how these changes influence energy metabolism, specifically the balance between carbohydrate and fatty acid oxidation. In the present study we examine glucose and fatty acid oxidation in transgenic mice, generated to overexpress sarco(endo)plasmic reticulum calcium-ATPase (SERCA), which have an enhanced contractile phenotype. Energy substrate metabolism was measured in isolated working hearts using radiolabeled glucose and palmitate. We also examined oxygen consumption to see whether SERCA overexpression is associated with increased oxygen utilization. Since SERCA is important in calcium handling within the cardiac myocyte, we examined cytosolic calcium transients in isolated myocytes using indo-1, and mitochondrial calcium levels using pericam, an adenovirally expressed, mitochondrially targeted ratiometric calcium indicator. Oxygen consumption did not differ between wild-type and SERCA groups; however, we were able to show an increased utilization of glucose for oxidative metabolism and a corresponding decreased utilization of fatty acids in the SERCA group. Cytosolic calcium transients were increased in myocytes isolated from SERCA mice, and they show a faster rate of decay of the calcium transient. With these observations we noted increased levels of mitochondrial calcium in the SERCA group, which was associated with an increase in the active form of the pyruvate dehydrogenase complex. Since an increase in mitochondrial calcium levels leads to activation of the pyruvate dehydrogenase complex (the rate-limiting step for carbohydrate oxidation), the increased glucose utilization observed in isolated perfused hearts in the SERCA group may reflect a higher level of mitochondrial calcium.
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Simmonds, Alec I. M. y Frank Seebacher. "Histone deacetylase activity modulates exercise-induced skeletal muscle plasticity in zebrafish (Danio rerio)". American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 313, n.º 1 (1 de julio de 2017): R35—R43. http://dx.doi.org/10.1152/ajpregu.00378.2016.
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Aerobic exercise has a positive impact on animals by enhancing skeletal muscle function and locomotor performance. Responses of skeletal muscle to exercise involve changes in energy metabolism, calcium handling, and the composition of contractile protein isoforms, which together influence contractile properties. Histone deacetylases (HDAC) can cause short-term changes in gene expression and may thereby mediate plasticity in contractile properties of skeletal muscle in response to exercise. The aim of this project was to determine (in zebrafish, Danio rerio) the traits that mediate interindividual differences in sustained and sprint performance and to determine whether inhibiting class I and II HDACs mediates exercise-induced changes in these traits. High sustained performers had greater aerobic metabolic capacity [citrate synthase (CS) activity], calcium handling capacity [sarco/endoplasmic reticulum ATPase (SERCA) activity], and slow contractile protein concentration [slow myosin heavy chain (MHC)] compared with low performers. High sprint performers had lower CS activity and slow MHC concentrations compared with low performers, but there were no significant differences in lactate dehydrogenase activity or fast MHC concentrations. Four weeks of aerobic exercise training increased sustained performance, CS activity, SERCA activity, and slow MHC concentration. Inhibiting class I and II HDACs increased slow MHC concentration in untrained fish but not in trained fish. However, inhibiting HDACs reduced SERCA activity, which was paralleled by a reduction in sustained and sprint performance. The regulation of muscle phenotypes by HDACs could be a mechanism underlying the adaptation of sustained locomotor performance to different environmental conditions, and may therefore be of therapeutic and ecological significance.
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Espinoza-Fonseca, L. Michel. "Structural Basis for the Function of the C-Terminal Proton Release Pathway in the Calcium Pump". International Journal of Molecular Sciences 22, n.º 7 (29 de marzo de 2021): 3507. http://dx.doi.org/10.3390/ijms22073507.
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The calcium pump (sarco/endoplasmic reticulum Ca2+-ATPase, SERCA) plays a major role in calcium homeostasis in muscle cells by clearing cytosolic Ca2+ during muscle relaxation. Active Ca2+ transport by SERCA involves the structural transition from a low-Ca2+ affinity E2 state toward a high-Ca2+ affinity E1 state of the pump. This structural transition is accompanied by the countertransport of protons to stabilize the negative charge and maintain the structural integrity of the transport sites and partially compensate for the positive charges of the two Ca2+ ions passing through the membrane. X-ray crystallography studies have suggested that a hydrated pore located at the C-terminal domain of SERCA serves as a conduit for proton countertransport, but the existence and function of this pathway have not yet been fully characterized. We used atomistic simulations to demonstrate that in the protonated E2 state and the absence of initially bound water molecules, the C-terminal pore becomes hydrated in the nanosecond timescale. Hydration of the C-terminal pore is accompanied by the formation of water wires that connect the transport sites with the cytosol. Water wires are known as ubiquitous proton-transport devices in biological systems, thus supporting the notion that the C-terminal domain serves as a conduit for proton release. Additional simulations showed that the release of a single proton from the transport sites induces bending of transmembrane helix M5 and the interaction between residues Arg762 and Ser915. These structural changes create a physical barrier against full hydration of the pore and prevent the formation of hydrogen-bonded water wires once proton transport has occurred through this pore. Together, these findings support the notion that the C-terminal proton release pathway is a functional element of SERCA and also provide a mechanistic model for its operation in the catalytic cycle of the pump.
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Hirakis, Sophia P., Thomas M. Bartol, Terrence J. Sejnowski y Rommie E. Amaro. "Particle-Based Approaches to Clearing Calcium: a Protein Landscape Model of the Sarco/Endoplasmic Reticulum Calcium-ATPase (SERCA) Pump for Sub-Cellular Stochastic Models". Biophysical Journal 112, n.º 3 (febrero de 2017): 484a. http://dx.doi.org/10.1016/j.bpj.2016.11.2618.
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Dixon, Rose Ellen, Fiona C. Britton, Salah A. Baker, Grant W. Hennig, Christina M. Rollings, Kenton M. Sanders y Sean M. Ward. "Electrical slow waves in the mouse oviduct are dependent on extracellular and intracellular calcium sources". American Journal of Physiology-Cell Physiology 301, n.º 6 (diciembre de 2011): C1458—C1469. http://dx.doi.org/10.1152/ajpcell.00293.2011.
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Spontaneous contractions of the myosalpinx are critical for oocyte transport along the oviduct. Slow waves, the electrical events that underlie myosalpinx contractions, are generated by a specialized network of pacemaker cells called oviduct interstitial cells of Cajal (ICC-OVI). The ionic basis of oviduct pacemaker activity is unknown. Intracellular recordings and Ca2+ imaging were performed to examine the role of extracellular and intracellular Ca2+ sources in slow wave generation. RT-PCR was performed to determine the transcriptional expression of Ca2+ channels. Molecular studies revealed most isoforms of L- and T-type calcium channels (Cav1.2,1.3,1.4,3.1,3.2,3.3) were expressed in myosalpinx. Reduction of extracellular Ca2+ concentration ([Ca2+]o) resulted in the abolition of slow waves and myosalpinx contractions without significantly affecting resting membrane potential (RMP). Spontaneous Ca2+ waves spread through ICC-OVI cells at a similar frequency to slow waves and were inhibited by reduced [Ca2+]o. Nifedipine depolarized RMP and inhibited slow waves; however, pacemaker activity returned when the membrane was repolarized with reduced extracellular K+ concentration ([K+]o). Ni2+ also depolarized RMP but failed to block slow waves. The importance of ryanodine and inositol 1,4,5 trisphosphate-sensitive stores were examined using ryanodine, tetracaine, caffeine, and 2-aminoethyl diphenylborinate. Results suggest that although both stores are involved in regulation of slow wave frequency, neither are exclusively essential. The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor cyclopiazonic acid inhibited pacemaker activity and Ca2+ waves suggesting that a functional SERCA pump is necessary for pacemaker activity. In conclusion, results from this study suggest that slow wave generation in the oviduct is voltage dependent, occurs in a membrane potential window, and is dependent on extracellular calcium and functional SERCA pumps.
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Meehan, Sean, Ava J. Wu, Elaine C. Kang, Takayuki Sakai y Indu S. Ambudkar. "Interferon-γ induces a decrease in the intracellular calcium pump in a human salivary gland cell line". American Journal of Physiology-Cell Physiology 273, n.º 6 (1 de diciembre de 1997): C2030—C2036. http://dx.doi.org/10.1152/ajpcell.1997.273.6.c2030.
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Interferon-γ (IFN-γ) ± tumor necrosis factor-α (TNF-α) induces antiproliferation and intracellular Ca2+ store depletion in a human submandibular ductal cell line (HSG), which can be reversed on cytokine removal [A. J. Wu, G. C. Chen, B. J. Baum, and I. S. Ambudkar. Am. J. Physiol. 270 ( Cell Physiol. 39): C514–C521, 1996]. Here we have examined a possible mechanism for the IFN-γ-induced intracellular Ca2+store depletion. There was a time-dependent decrease in thapsigargin-dependent internal Ca2+ release after exposure of the cells to the cytokines. The intracellular Ca2+ pump [sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)] protein in lysates and membranes of cells treated with IFN-γ ± TNF-α, but not with TNF-α alone, showed a similar time-dependent decrease (examined using a SERCA2 antibody). Removal of the cytokines, which resulted in recovery of cell growth and refill of internal Ca2+ stores, also increased the level of SERCA protein. The decrease in SERCA is not a result of decreased cell proliferation, since thapsigargin, 2,5-di-( t-butyl)-1,4-hydroquinone, or serum-free growth conditions induced antiproliferative effects on HSG cells without any corresponding decrease in SERCA. We suggest that the IFN-γ-induced decrease in the level of SERCA accounts for the depleted state of internal Ca2+stores in cytokine-treated HSG cells. These data suggest a novel mechanism for the inhibition of HSG cell growth by IFN-γ.