Abstract:Mitochondrial Ca2+ uptake is crucial for coupling receptor stimulation to cellular bioenergetics. Further, Ca2+ uptake by respiring mitochondria prevents Ca2+-dependent inactivation (CDI) of store-operated Ca2+ release-activated Ca2+ (CRAC) channels and inhibits Ca2+ extrusion to sustain cytosolic Ca2+ signaling. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we generated several cell lines with MCU-knockout (MCU-KO) as… Show more
“…Growth was diminished in MCU KO MEFs ( 47 ) but unaltered in siRNA-treated MD-MB-231 breast carcinoma cells ( 42 ). In agreement with our findings, knockdown of MCU in primary B lymphocytes enhanced proliferation in response to anti-immunoglobulin M ( 69 ). Figure 9 Schematic summarizing some of the pathways altered by the chronic loss of IP 3 Rs and MCU.…”
Section: Discussionsupporting
confidence: 93%
“…Growth was diminished in MCU KO MEFs (47) but unaltered in siRNA-treated MD-MB-231 breast carcinoma cells (42). In agreement with our findings, knockdown of MCU in primary B lymphocytes enhanced proliferation in response to anti-IgM (70).…”
Section: J O U R N a L P R E -P R O O Fsupporting
confidence: 90%
“…Thus, mechanisms acting upstream of IP 3 synthesis/degradation, or affecting Ca 2+ extrusion from the cytosol via plasma membrane or ER Ca 2+ pumps, are more likely to be involved. Increased rates of both ER and plasma membrane Ca 2+ pumping have been reported in several MCU KO cell types ( 69 ). Further studies are required to establish a definitive mechanism for the decreased cytosolic Ca 2+ signaling observed in our MCU KO cells.…”
Section: Discussionmentioning
confidence: 99%
“…However, an increased agonist-mediated Ca 2+ transient has been reported in other MCU KO models including MEFs ( 47 ) and vascular smooth muscle cells ( 46 ), where the result has been attributed to a lack of mitochondrial Ca 2+ buffering. The intracellular store Ca 2+ content or the dynamics of IP 3 -mediated Ca 2+ puffs were not altered in MCU KO HEK293 cells ( 69 ). Thus, mechanisms acting upstream of IP 3 synthesis/degradation, or affecting Ca 2+ extrusion from the cytosol via plasma membrane or ER Ca 2+ pumps, are more likely to be involved.…”
Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP
3
Rs) and the mitochondrial Ca
2+
uniporter (MCU) are key proteins that regulate intracellular Ca
2+
concentration. Mitochondrial Ca
2+
accumulation activates Ca
2+
-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP
3
R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca
2+
signaling. Our results show that TKO cells (exhibiting total loss of Ca
2+
signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca
2+
signaling is dispensable for the bioenergetic needs of both IP
3
R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.
“…Growth was diminished in MCU KO MEFs ( 47 ) but unaltered in siRNA-treated MD-MB-231 breast carcinoma cells ( 42 ). In agreement with our findings, knockdown of MCU in primary B lymphocytes enhanced proliferation in response to anti-immunoglobulin M ( 69 ). Figure 9 Schematic summarizing some of the pathways altered by the chronic loss of IP 3 Rs and MCU.…”
Section: Discussionsupporting
confidence: 93%
“…Growth was diminished in MCU KO MEFs (47) but unaltered in siRNA-treated MD-MB-231 breast carcinoma cells (42). In agreement with our findings, knockdown of MCU in primary B lymphocytes enhanced proliferation in response to anti-IgM (70).…”
Section: J O U R N a L P R E -P R O O Fsupporting
confidence: 90%
“…Thus, mechanisms acting upstream of IP 3 synthesis/degradation, or affecting Ca 2+ extrusion from the cytosol via plasma membrane or ER Ca 2+ pumps, are more likely to be involved. Increased rates of both ER and plasma membrane Ca 2+ pumping have been reported in several MCU KO cell types ( 69 ). Further studies are required to establish a definitive mechanism for the decreased cytosolic Ca 2+ signaling observed in our MCU KO cells.…”
Section: Discussionmentioning
confidence: 99%
“…However, an increased agonist-mediated Ca 2+ transient has been reported in other MCU KO models including MEFs ( 47 ) and vascular smooth muscle cells ( 46 ), where the result has been attributed to a lack of mitochondrial Ca 2+ buffering. The intracellular store Ca 2+ content or the dynamics of IP 3 -mediated Ca 2+ puffs were not altered in MCU KO HEK293 cells ( 69 ). Thus, mechanisms acting upstream of IP 3 synthesis/degradation, or affecting Ca 2+ extrusion from the cytosol via plasma membrane or ER Ca 2+ pumps, are more likely to be involved.…”
Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP
3
Rs) and the mitochondrial Ca
2+
uniporter (MCU) are key proteins that regulate intracellular Ca
2+
concentration. Mitochondrial Ca
2+
accumulation activates Ca
2+
-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP
3
R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca
2+
signaling. Our results show that TKO cells (exhibiting total loss of Ca
2+
signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca
2+
signaling is dispensable for the bioenergetic needs of both IP
3
R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.
“…CRAC channels subserve slow inactivation processes as well, but SCDI, which is modulated by the uptake of Ca 2+ into mitochondria, seems to be primarily orchestrated in a STIM1-dependent manner and thus is not discussed herein [160][161][162][163].…”
Section: Ca 2+ Dependent Crac Channel Inactivationmentioning
Orai1, the Ca2+-selective pore in the plasma membrane, is one of the key components of the Ca2+release-activated Ca2+ (CRAC) channel complex. Activated by the Ca2+ sensor in the endoplasmic reticulum (ER) membrane, stromal interaction molecule 1 (STIM1), via direct interaction when ER luminal Ca2+ levels recede, Orai1 helps to maintain Ca2+ homeostasis within a cell. It has already been proven that the C-terminus of Orai1 is indispensable for channel activation. However, there is strong evidence that for CRAC channels to function properly and maintain all typical hallmarks, such as selectivity and reversal potential, additional parts of Orai1 are needed. In this review, we focus on these sites apart from the C-terminus; namely, the second loop and N-terminus of Orai1 and on their multifaceted role in the functioning of CRAC channels.
Arginine vasopressin (AVP) induces an increase in intracellular Ca2+ concentration ([Ca2+]i) with an oscillatory pattern in isolated perfused kidney inner medullary collecting duct (IMCD). The AVP-induced Ca2+ mobilization in inner medullary collecting ducts is essential for apical exocytosis and is mediated by the exchange protein directly activated by cyclic adenosine monophosphate (Epac). Murine principal kidney cortical collecting duct cells (mpkCCD) is the cell model used for transcriptomic and phosphoproteomic studies of AVP signaling in kidney collecting duct. The present study examined the characteristics of Ca2+ mobilization in mpkCCD cells, and utilized mpkCCD as a model to investigate the Epac-induced intracellular and intra-organellar Ca2+ mobilization. Ca2+ mobilization in cytosol, endoplasmic reticulum lumen, and mitochondrial matrix were monitored with a Ca2+ sensitive fluorescent probe and site-specific Ca2+ sensitive biosensors. Fluorescence images of mpkCCD cells and isolated perfused inner medullary duct were collected with confocal microscopy. Cell permeant ligands of ryanodine receptors (RyRs) and inositol 1,4,5 trisphosphate receptors (IP3Rs) both triggered increase of [Ca2+]i and Ca2+ oscillations in mpkCCD cells as reported previously in IMCD. The cell permeant Epac-specific cAMP analog Me-cAMP/AM also caused a robust Ca2+ mobilization and oscillations in mpkCCD cells. Using biosensors to monitor endoplasmic reticulum (ER) luminal Ca2+ and mitochondrial matrix Ca2+, Me-cAMP/AM not only triggered Ca2+ release from ER into cytoplasm, but also shuttled Ca2+ from ER into mitochondria. The Epac-agonist induced synchronized Ca2+ spikes in cytosol and mitochondrial matrix, with concomitant declines in ER luminal Ca2+. Me-cAMP/AM also effectively triggered store-operated Ca2+ entry (SOCE), suggesting that Epac-agonist is capable of depleting ER Ca2+ stores. These Epac-induced intracellular and inter-organelle Ca2+ signals were mimicked by the RyR agonist 4-CMC, but they were distinctly different from IP3R activation. The present study hence demonstrated that mpkCCD cells retain all reported features of Ca2+ mobilization observed in isolated perfused IMCD. It further revealed information on the dynamics of Epac-induced RyR-dependent Ca2+ signaling and ER-mitochondrial Ca2+ transfer. ER-mitochondrial Ca2+ coupling may play a key role in the regulation of ATP and reactive oxygen species (ROS) production in the mitochondria along the nephron. Our data suggest that mpkCCD cells can serve as a renal cell model to address novel questions of how mitochondrial Ca2+ regulates cytosolic Ca2+ signals, inter-organellar Ca2+ signaling, and renal tubular functions.
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