Leucine zipper EF hand-containing transmembrane protein 1 (Letm1) and uncoupling proteins 2 and 3 (UCP2/3) contribute to two distinct mitochondrial Ca2+ uptake pathways

Cytosolic Ca(2+) signals are transferred into mitochondria over a huge concentration range. In our recent work we described uncoupling proteins 2 and 3 (UCP2/3) to be fundamental for mitochondrial uptake of high Ca(2+) domains in mitochondria-ER junctions. On the other hand, the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca(2+)/H(+) antiporter that achieved mitochondrial Ca(2+) sequestration at small Ca(2+) increases. Thus, the contributions of Letm1 and UCP2/3 to mitochondrial Ca(2+) uptake were compared in endothelial cells. Knock-down of Letm1 did not affect the UCP2/3-dependent mitochondrial uptake of intracellularly released Ca(2+) but strongly diminished the transfer of entering Ca(2+) into mitochondria, subsequently, resulting in a reduction of store-operated Ca(2+) entry (SOCE). Knock-down of Letm1 and UCP2/3 did neither impact on cellular ATP levels nor the membrane potential. The enhanced mitochondrial Ca(2+) signals in cells overexpressing UCP2/3 rescued SOCE upon Letm1 knock-down. In digitonin-permeabilized cells, Letm1 exclusively contributed to mitochondrial Ca(2+) uptake at low Ca(2+) conditions. Neither the Letm1- nor the UCP2/3-dependent mitochondrial Ca(2+) uptake was affected by a knock-down of mRNA levels of mitochondrial calcium uptake 1 (MICU1), a protein that triggers mitochondrial Ca(2+) uptake in HeLa cells. Our data indicate that Letm1 and UCP2/3 independently contribute to two distinct, mitochondrial Ca(2+) uptake pathways in intact endothelial cells.     

β₃-Adrenergic activation of sequential Ca(2+) release from mitochondria and the endoplasmic reticulum and the subsequent Ca(2+) entry in rodent brown adipocytes

We studied how mitochondrial uncoupling by β(3)-adrenergic stimulation elicits Ca(2+) signals in rodent brown adipocytes by fluorometry of Ca(2+) concentrations ([Ca(2+)](i), [Ca(2+)](m) and [Ca(2+)](ER)) in the cytoplasm, mitochondria and the endoplasmic reticulum (ER), respectively, and mitochondrial membrane potential, using fura-2, rhod-5N, cameleon and rhodamine 123. Immunoblotting demonstrated α(1A)- and β(3)-adrenergic receptor and UCP1 in adipocytes, while RT-PCR revealed the mRNA of type 3, 7 and 9 adenylate cyclase, UCP1, UCP2, UCP3 and type 1 and 2 inositoltrisphosphate receptors. Isoproterenol and BRL37344, β-agonist, caused triphasic rises in [Ca(2+)](i) (β-responses) with mitochondrial depolarization in adipocytes. BRL37344 transiently decreased [Ca(2+)](m). β-Responses were blocked by propranolol, β-antagonist, H-89, protein kinase A blocker, and knockout of UCP1 gene. The late phase of β-responses was depressed by a Ca(2+) free, EGTA solution, U73122, a phospholipase C blocker, and thapsigargin, ER-Ca(2+) pump blocker, and by transfecting siRNA for type 2 IP(3)R. Intracellular loading of BAPTA/AM depressed the late phase more strongly than the initial phase. β-Agonists, phenylephrine, α-agonist, and cyclopiazonic acid, ER-Ca(2+) pump blocker, decreased [Ca(2+)](ER). Thus, the mitochondrial uncoupling by β(3)-adrenergic activation causes Ca(2+) release from mitochondria and subsequently from the ER and further evokes plasmalemmal Ca(2+) entries, including the store-operated Ca(2+) entry.     

Uncoupling proteins 2 and 3 are fundamental for mitochondrial Ca2+ uniport

Mitochondrial Ca(2+) uptake is crucial for the regulation of the rate of oxidative phosphorylation, the modulation of spatio-temporal cytosolic Ca(2+) signals and apoptosis. Although the phenomenon of mitochondrial Ca(2+) sequestration, its characteristics and physiological consequences have been convincingly reported, the actual protein(s) involved in this process are unknown. Here, we show that the uncoupling proteins 2 and 3 (UCP2 and UCP3) are essential for mitochondrial Ca(2+) uptake. Using overexpression, knockdown (small interfering RNA) and mutagenesis experiments, we demonstrate that UCP2 and UCP3 are elementary for mitochondrial Ca(2+) sequestration in response to cell stimulation under physiological conditions - observations supported by isolated liver mitochondria of Ucp2(-/-) mice lacking ruthenium red-sensitive Ca(2+) uptake. Our results reveal a novel molecular function for UCP2 and UCP3, and may provide the molecular mechanism for their reported effects. Moreover, the identification of proteins fundemental for mitochondrial Ca(2+) uptake expands our knowledge of the physiological role for mitochondrial Ca(2+) sequestration.     

Sorting of calcium signals at the junctions of endoplasmic reticulum and mitochondria

Calcium signal transmission between endoplasmic reticulum (ER) and mitochondria is supported by a local [Ca(2+)] control that operates between IP(3)receptor Ca(2+)release channels (IP(3)R) and mitochondrial Ca(2+)uptake sites, and displays functional similarities to synaptic transmission. Activation of IP(3)R by IP(3)is known to evoke quantal Ca(2+)mobilization that is associated with incremental elevations of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)). Here we report that activation of IP(3)R by adenophostin-A (AP) yields non-quantal Ca(2+)mobilization in mast cells. We also show that the AP-induced continuous Ca(2+)release causes relatively small [Ca(2+)](m)responses, in particular, the sustained phase of Ca(2+)release is not sensed by the mitochondria. Inhibition of ER Ca(2+)pumps by thapsigargin slightly increases IP(3)-induced [Ca(2+)](m)responses, but augments AP-induced [Ca(2+)](m)responses in a large extent. In adherent permeabilized cells exposed to elevated [Ca(2+)], ER Ca(2+)uptake fails to affect global cytosolic [Ca(2+)], but attenuates [Ca(2+)](m)responses. Moreover, almost every mitochondrion exhibits a region very close to ER Ca(2+)pumps visualized by BODIPY-FL-thapsigargin or SERCA antibody. Thus, at the ER-mitochondrial junctions, localized ER Ca(2+)uptake provides a mechanism to attenuate the mitochondrial response during continuous Ca(2+)release through the IP(3)R or during gradual Ca(2+)influx to the junction between ER and mitochondria.     

Mitochondrial uncoupling protein-4 regulates calcium homeostasis and sensitivity to store depletion-induced apoptosis in neural cells

An increase in the cytoplasmic-free Ca(2+) concentration mediates cellular responses to environmental signals that influence a range of processes, including gene expression, motility, secretion of hormones and neurotransmitters, changes in energy metabolism, and apoptosis. Mitochondria play important roles in cellular Ca(2+) homeostasis and signaling, but the roles of specific mitochondrial proteins in these processes are unknown. Uncoupling proteins (UCPs) are a family of proteins located in the inner mitochondrial membrane that can dissociate oxidative phosphorylation from respiration, thereby promoting heat production and decreasing oxyradical production. Here we show that UCP4, a neuronal UCP, influences store-operated Ca(2+) entry, a process in which depletion of endoplasmic reticulum Ca(2+) stores triggers Ca(2+) influx through plasma membrane "store-operated" channels. PC12 neural cells expressing human UCP4 exhibit reduced Ca(2+) entry in response to thapsigargin-induced endoplasmic reticulum Ca(2+) store depletion. The elevations of cytoplasmic and intramitochondrial Ca(2+) concentrations and mitochondrial oxidative stress induced by thapsigargin were attenuated in cells expressing UCP4. The stabilization of Ca(2+) homeostasis and preservation of mitochondrial function by UCP4 was correlated with reduced mitochondrial reactive oxygen species generation, oxidative stress, and Gadd153 up-regulation and increased resistance of the cells to death. Reduced Ca(2+)-dependent cytosolic phospholipase A2 activation and oxidative metabolism of arachidonic acid also contributed to the stabilization of mitochondrial function in cells expressing human UCP4. These findings demonstrate that UCP4 can regulate cellular Ca(2+) homeostasis, suggesting that UCPs may play roles in modulating Ca(2+) signaling in physiological and pathological conditions.     

G37R SOD1 mutant alters mitochondrial complex I activity, Ca(2+) uptake and ATP production

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective death of motor neurons. Mutations in Cu/Zn superoxide dismutase-1 (SOD1) cause familial ALS but the molecular mechanisms whereby these mutations induce motor neuron death remain controversial. Here, we show that stable overexpression of mutant human SOD1 (G37R) - but not wild-type SOD1 (wt-SOD1) - in mouse neuroblastoma cells (N2a) results in morphological abnormalities of mitochondria accompanied by several dysfunctions. Activity of the oxidative phosphorylation complex I was significantly reduced in G37R cells and correlated with lower mitochondrial membrane potential and reduced levels of cytosolic ATP. Using targeted chimeric aequorin we further analyzed the consequences of mitochondrial dysfunction on cellular Ca(2+) handling. Mitochondrial Ca(2+) uptake, elicited by IP(3)-induced Ca(2+) release from endoplasmic reticulum (ER) was significantly reduced in G37R cells, while uptake induced by a brief Ca(2+) pulse was not affected in permeabilized cells. The decreased mitochondrial Ca(2+) uptake resulted in increased cytosolic Ca(2+) transients, whereas ER Ca(2+) load and resting cytosolic Ca(2+) levels were not affected. Together, these findings suggest that the mechanism linking mutant G37R SOD1 and ALS involves mitochondrial respiratory chain deficiency resulting in ATP loss and impairment of mitochondrial and cytosolic Ca(2+) homeostasis.     

Taurine increases glucose sensitivity of UCP2-overexpressing beta-cells by ameliorating mitochondrial metabolism

A low-taurine diet during fetal or early postnatal life causes abnormal pancreatic beta-cell development. Tissue and plasma taurine concentrations can also be low in diabetic patients. We examined the effect of taurine on impaired glucose responses in diabetic rat beta-cells adenovirally overexpressing uncoupling protein (UCP)2, which is upregulated in obesity-related type 2 diabetes. We found that taurine pretreatment restored the ATP-to-ADP (ATP/ADP) ratio and glucose-stimulated insulin secretion in UCP2-infected islets. ATP-sensitive K(+) channel sensitivity to dihydroxyacetone, another insulin secretagogue, was similar in both UCP2-infected and control beta-cells. In freshly isolated mitochondria from UCP2-overexpressing insulin-secreting (INS)-1 beta-cells, methyl pyruvate-mediated mitochondrial Ca(2+) increase was significantly ameliorated by taurine. A mitochondrial Ca(2+) uniporter blocker, ruthenium red, inhibited the action of taurine. This study suggests that taurine enhances the glucose sensitivity of UCP2-overexpressing beta-cells, probably by increasing mitochondrial Ca(2+) influx through the Ca(2+) uniporter, thereby enhancing mitochondrial metabolic function and increasing the ATP/ADP ratio.     

STIM1 knockdown reveals that store-operated Ca2+ channels located close to sarco/endoplasmic Ca2+ ATPases (SERCA) pumps silently refill the endoplasmic reticulum

Stromal interaction molecule (STIM) proteins are putative ER Ca2+ sensors that recruit and activate store-operated Ca2+ (SOC) channels at the plasma membrane, a process triggered by the Ca2+ depletion of the endoplasmic reticulum (ER). To test whether STIM1 is required for ER refilling, we used RNA interference and measured Ca2+ signals in the cytosol, the ER, and the mitochondria of HeLa cells. Knockdown of STIM1 (mRNA levels, 73%) reduced SOC entry by 73% when sarco/endoplasmic Ca2+ ATPases (SERCA) were inhibited by thapsigargin but did not prevent Ca2+ stores refilling when cells were stimulated by physiological agonists. Stores could be fully refilled by increasing the external Ca2+ concentration above physiological values, but no cytosolic Ca2+ signals were detected during store refilling even at very high Ca2+ concentrations. [Ca2+](ER) measurements revealed that the basal activity of SERCA was not affected in STIM1 knockdown cells and that [Ca2+](ER) levels were restored within 2 min in physiological saline following store depletion. Mitochondrial inhibitors reduced ER refilling in wild-type but not in STIM1 knockdown cells, indicating that ER refilling does not require functional mitochondria at low STIM1 levels. Our data show that ER refilling is largely preserved at reduced STIM1 levels, despite a drastic reduction of store-operated Ca2+ entry, because Ca2+ ions are directly transferred from SOC channels to SERCA. These findings are consistent with the formation of microdomains containing not only SOC channels on the plasma membrane and STIM proteins on the ER but also SERCA pumps and mitochondria to refill the ER without perturbing the cytosol.     

Ca(2+) sources for the exocytotic release of glutamate from astrocytes

Astrocytes can exocytotically release the gliotransmitter glutamate from vesicular compartments. Increased cytosolic Ca(2+) concentration is necessary and sufficient for this process. The predominant source of Ca(2+) for exocytosis in astrocytes resides within the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate and ryanodine receptors of the ER provide a conduit for the release of Ca(2+) to the cytosol. The ER store is (re)filled by the store-specific Ca(2+)-ATPase. Ultimately, the depleted ER is replenished by Ca(2+) which enters from the extracellular space to the cytosol via store-operated Ca(2+) entry; the TRPC1 protein has been implicated in this part of the astrocytic exocytotic process. Voltage-gated Ca(2+) channels and plasma membrane Na(+)/Ca(2+) exchangers are additional means for cytosolic Ca(2+) entry. Cytosolic Ca(2+) levels can be modulated by mitochondria, which can take up cytosolic Ca(2+) via the Ca(2+) uniporter and release Ca(2+) into cytosol via the mitochondrial Na(+)/Ca(2+) exchanger, as well as by the formation of the mitochondrial permeability transition pore. The interplay between various Ca(2+) sources generates cytosolic Ca(2+) dynamics that can drive Ca(2+)-dependent exocytotic release of glutamate from astrocytes. An understanding of this process in vivo will reveal some of the astrocytic functions in health and disease of the brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Effects of PMCA and SERCA pump overexpression on the kinetics of cell Ca(2+) signalling

The dynamic interactions of the main pathways for active Ca(2+) transport have been analysed in living cells by altering the expression of their components. The plasma membrane (PMCA) and the endoplasmic reticulum (ER) (SERCA) Ca(2+) pumps were transiently overexpressed in CHO cells, and the Ca(2+) homeostasis in the subcellular compartments was investigated using specifically targeted chimaeras of the Ca(2+)- sensitive photoprotein aequorin. In resting cells, overexpression of the PMCA and SERCA pumps caused a reduction and an increase in ER [Ca(2+)] levels, respectively, while no significant differences were detected in cytosolic and mitochondrial [Ca(2+)]. Upon stimulation with an inositol 1,4, 5-trisphosphate (IP(3))-generating agonist, the amplitude of the mitochondrial and cytosolic Ca(2+) rises correlated with the ER [Ca(2+)] only up to a threshold value, above which the feedback inhibition of the IP(3) channel by Ca(2+) appeared to be limiting.     

Biphasic regulation of mitochondrial Ca2+ uptake by cytosolic Ca2+ concentration

A rise in cytosolic Ca(2+) concentration is used as a key activation signal in virtually all animal cells, where it triggers a range of responses including neurotransmitter release, muscle contraction, and cell growth and proliferation [1]. During intracellular Ca(2+) signaling, mitochondria rapidly take up significant amounts of Ca(2+) from the cytosol, and this stimulates energy production, alters the spatial and temporal profile of the intracellular Ca(2+) signal, and triggers cell death [2-10]. Mitochondrial Ca(2+) uptake occurs via a ruthenium-red-sensitive uniporter channel found in the inner membrane [11]. In spite of its critical importance, little is known about how the uniporter is regulated. Here, we report that the mitochondrial Ca(2+) uniporter is gated by cytosolic Ca(2+). Ca(2+) uptake into mitochondria is a Ca(2+)-activated process with a requirement for functional calmodulin. However, cytosolic Ca(2+) subsequently inactivates the uniporter, preventing further Ca(2+) uptake. The uptake pathway and the inactivation process have relatively low Ca(2+) affinities of approximately 10-20 microM. However, numerous mitochondria are within 20-100 nm of the endoplasmic reticulum, thereby enabling rapid and efficient transmission of Ca(2+) release into adjacent mitochondria by InsP(3) receptors on the endoplasmic reticulum. Hence, biphasic control of mitochondrial Ca(2+) uptake by Ca(2+) provides a novel basis for complex physiological patterns of intracellular Ca(2+) signaling.     

Ruthenium red-mediated suppression of Bcl-2 loss and Ca(2+) release initiated by photodamage to the endoplasmic reticulum: scavenging of reactive oxygen species

The photosensitizer 9-capronyloxytetrakis (methoxyethyl) porphycene localizes predominantly in the endoplasmic reticulum (ER) and, to a lesser extent, in mitochondria of murine leukemia L1210 cells. Subsequent irradiation results in the loss of ER > mitochondrial Bcl-2 and an apoptotic response. Although an increase in cytosolic Ca(2+) was observed after irradiation, apoptosis was not inhibited by either the presence of the calcium chelator BAPTA or by the mitochondrial uniporter inhibitor ruthenium amino binuclear complex (Ru360). Moreover, neither reagent prevented the loss of Bcl-2. Ruthenium red (RR) devoid of Ru360 prevented Bcl-2 loss, release of Ca(2+) from the ER and the initiation of apoptosis. Since RR was significantly more sensitive than Ru360 to oxidation by singlet oxygen, we attribute the protective effect of RR to the quenching of reactive oxygen species. Although cytosolic and (to a lesser extent) mitochondrial Ca(2+) levels were elevated after photodynamic therapy, these changes were apparently insufficient to contribute to the development of apoptosis.     

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

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

The interplay of mitochondria with calcium: an historical appraisal

Indirect findings in the 1950s had indicated that mitochondria could accumulate Ca(2+), but only in 1961 isolated mitochondria were directly shown to take it up in a process driven by the activity of the respiratory chain or by the hydrolysis of added ATP. The uptake of Ca(2+) could be accompanied by the simultaneous uptake of inorganic phosphate, leading to the precipitation of hydroxyapatite in the matrix and to the effective buffering of the free Ca(2+) concentration in it. The uptake of Ca(2+) occurred via an electrophoretic uniporter that has been molecularly identified only recently. Ca(2+) was then released through a Na(+)/Ca(2+) exchanger that has also been identified very recently (a H(+)/Ca(2+) antiporter has also been described in some mitochondrial types). In the matrix two TCA cycle dehydrogenases and pyruvate dehydrogenase phosphate phosphatase were found to be regulated by Ca(2+), providing a rationale for the Ca(2+) cycling process. The affinity of the uptake uniporter was found to be too low to efficiently regulate Ca(2+) in the low to mid nM concentration in the cytosol. However, a number of findings showed that energy linked transport of Ca(2+) did nevertheless occur in mitochondria in situ. The enigma was solved in the 1990s, when it was found that perimitochondrial Ca(2+) pools are created by the discharge of Ca(2+) from vicinal endoplasmic reticulum stores in which the concentration of Ca(2+) is high enough to satisfy the poor affinity of the uniporter. Thus, mitochondria have now regained a key role in the regulation of cytosolic Ca(2+) (not only of their own internal Ca(2+)).     

Mitochondrial calcium transients in adult rabbit cardiac myocytes: inhibition by ruthenium red and artifacts caused by lysosomal loading of Ca(2+)-indicating fluorophores

A cold/warm loading protocol was used to ester-load Rhod 2 into mitochondria and other organelles and Fluo 3 into the cytosol of adult rabbit cardiac myocytes for confocal fluorescence imaging. Transient increases in both cytosolic Fluo 3 and mitochondrial Rhod 2 fluorescence occurred after electrical stimulation. Ruthenium red, a blocker of the mitochondrial Ca(2+) uniporter, inhibited mitochondrial Rhod 2 fluorescence transients but not cytosolic Fluo 3 transients. Thus the ruthenium red-sensitive mitochondrial Ca(2+) uniporter catalyzes Ca(2+) uptake during beat-to-beat transients of mitochondrial free Ca(2+), which in turn may help match mitochondrial ATP production to myocardial ATP demand. After ester loading, substantial amounts of Ca(2+)-indicating fluorophores localized into an acidic lysosomal/endosomal compartment. This lysosomal fluorescence did not respond to electrical stimulation. Because fluorescence arose predominantly from lysosomes after the cold loading/warm incubation procedure, total cellular fluorescence failed to track beat-to-beat changes of mitochondrial fluorescence. Only three-dimensionally resolved confocal imaging distinguished the relatively weak mitochondrial signal from the bright lysosomal fluorescence.     

Intracellular alkalinization induces cytosolic Ca2+ increases by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)

Intracellular pH (pHi) and Ca(2+) regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca(2+). The sources of the Ca(2+) increase are from the endoplasmic reticulum (ER) Ca(2+) pools as well as from Ca(2+) influx. The store-mobilization component of the Ca(2+) increase induced by the pHi rise was not sensitive to antagonists for either IP(3)-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), leading to depletion of the ER Ca(2+) store. We further showed that the physiological consequence of depletion of the ER Ca(2+) store by pHi rise is the activation of store-operated channels (SOCs) of Orai1 and Stim1, leading to increased Ca(2+) influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca(2+) leak from ER pools followed by Ca(2+) influx via SOCs.     

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

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

Functional coupling between ryanodine receptors, mitochondria and Ca(2+) ATPases in rat submandibular acinar cells

Agonist stimulation of exocrine cells leads to the generation of intracellular Ca(2+) signals driven by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) that rapidly become global due to propagation throughout the cell. In many types of excitable cells the intracellular Ca(2+) signal is propagated by a mechanism of Ca(2+)-induced Ca(2+) release (CICR), mediated by ryanodine receptors (RyRs). Expression of RyRs in salivary gland cells has been demonstrated immunocytochemically although their functional role is not clear. We used microfluorimetry to measure Ca(2+) signals in the cytoplasm, in the endoplasmic reticulum (ER) and in mitochondria. In permeabilized acinar cells caffeine induced a dose-dependent, transient decrease of Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)). This decrease was inhibited by ryanodine but was insensitive to heparin. Application of caffeine, however, did not elevate cytosolic Ca(2+) concentration ([Ca(2+)](i)) suggesting fast local buffering of Ca(2+) released through RyRs. Indeed, activation of RyRs produced a robust mitochondrial Ca(2+) transient that was prevented by addition of Ca(2+) chelator BAPTA but not EGTA. When mitochondrial Ca(2+) uptake was blocked, activation of RyRs evoked only a non-transient increase in [Ca(2+)](i) and substantially smaller Ca(2+) release from the ER. Upon simultaneous inhibition of mitochondrial Ca(2+) uptake and either plasmalemmal or ER Ca(2+) ATPase, activation of RyRs caused a transient rise in [Ca(2+)](i). Collectively, our data suggest that Ca(2+) released through RyRs is mostly "tunnelled" to mitochondria, while Ca(2+) ATPases are responsible for the fast initial sequestration of Ca(2+). Ca(2+) uptake by mitochondria is critical for maintaining continuous CICR. A complex interplay between RyRs, mitochondria and Ca(2+) ATPases is accomplished through strategic positioning of mitochondria close to both Ca(2+) release sites in the ER and Ca(2+) pumping sites of the plasmalemma and the ER.     

Mitochondrial Ca(2+) uptake depends on the spatial and temporal profile of cytosolic Ca(2+) signals

Using confocal imaging of Rhod-2-loaded HeLa cells, we examined the ability of mitochondria to sequester Ca(2+) signals arising from different sources. Mitochondrial Ca(2+) (Ca(2+)mit) uptake was stimulated by inositol 1,4,5-trisphosphate (InsP(3))-evoked Ca(2+) release, capacitative Ca(2+) entry, and Ca(2+) leaking from the endoplasmic reticulum. For each Ca(2+) source, the relationship between cytosolic Ca(2+) (Ca(2+)cyt) concentration and Ca(2+)mit was complex. With Ca(2+)cyt < 300 nm, a slow and persistent Ca(2+)mit uptake was observed. If Ca(2+)cyt increased above approximately 400 nm, Ca(2+)mit uptake accelerated sharply. For equivalent Ca(2+)cyt increases, the rate of Ca(2+)mit rise was greater with InsP(3)-evoked Ca(2+) signals than any other source. Spatial variation of the Ca(2+)mit response was observed within individual cells. Both the fraction of responsive mitochondria and the amplitude of the Ca(2+)mit response were graded in direct proportion to stimulus concentration. Trains of repetitive Ca(2+) oscillations did not maintain elevated Ca(2+)mit levels. Only low frequency Ca(2+) transients (<1/15 min) evoked repetitive Ca(2+)mit signals. Our data indicate that there is a lag between Ca(2+)cyt and Ca(2+)mit increases but that mitochondria will accumulate calcium when it is elevated over basal levels regardless of its source. Furthermore, in addition to the characteristics of Ca(2+) signals, Ca(2+) uniporter desensitization and proximity of mitochondria to InsP(3) receptors modulate mitochondrial Ca(2+) responses.     

Ca2+ homeostasis and exocytosis in carotid glomus cells: role of mitochondria

In oxygen sensing carotid glomus (type 1) cells, the hypoxia-triggered depolarization can be mimicked by mitochondrial inhibitors. We examined the possibility that, other than causing glomus cell depolarization, mitochondrial inhibition can regulate transmitter release via changes in Ca(2+) dynamics. Under whole-cell voltage clamp conditions, application of the mitochondrial inhibitors, carbonyl cyanide m-chlorophenylhydrazone (CCCP) or cyanide caused a dramatic slowing in the decay of the depolarization-triggered Ca(2+) signal in glomus cells. In contrast, inhibition of the Na(+)/Ca(2+) exchanger (NCX), plasma membrane Ca(2+)-ATPase (PMCA) pump or sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump had much smaller effects. Consistent with the notion that mitochondrial Ca(2+) uptake is the dominant mechanism in cytosolic Ca(2+) removal, inhibition of the mitochondrial uniporter with ruthenium red slowed the decay of the depolarization-triggered Ca(2+) signal. Hypoxia also slowed cytosolic Ca(2+) removal, suggesting a partial impairment of mitochondrial Ca(2+) uptake. Using membrane capacitance measurement, we found that the increase in the duration of the depolarization-triggered Ca(2+) signal after mitochondrial inhibition was associated with an enhancement of the exocytotic response. The role of mitochondria in the regulation of Ca(2+) signal and transmitter release from glomus cells highlights the importance of mitochondria in hypoxic chemotransduction in the carotid bodies.