Saccharomyces cerevisiae). In this report, we found that a yeast mutant, pmc1, lacking the vacuolar calcium ion (Ca2+) pump Ca2+-ATPase (Pmc1p), was more sensitive to Al treatment than the wild-type strain. Overexpression of either PMC1 or an anti-apoptotic factor, such as Bcl-2, Ced-9 or PpBI-1, decreased cytoplasmic Ca2+ levels and rescued yeast from Al sensitivity in both the wild-type and pmc1 mutant. Moreover, pretreatment with the Ca2+ chelator BAPTA-AM sustained cytoplasmic Ca2+ at low levels in the presence of Al, effectively making the cells more tolerant to Al exposure. Quantitative RT-PCR revealed that the expression of calmodulin (CaM) and phospholipase C (PLC), which are in the Ca2+ signaling pathway, was down-regulated under Al stress. This effect was largely counteracted when cells overexpressed anti-apoptotic Ced-9 or were pretreated with BAPTA-AM. Taken together, our results suggest that the negative regulation of Al-induced cytoplasmic Ca signaling is a novel mechanism underlying internal resistance to Al toxicity.Our previous study suggested that increased cytoplasmic calcium (Ca) signals may mediate aluminum (Al) toxicity in yeast ( Aluminum (Al) toxicity has been implicated as a major cause of severe loss of crops grown in acidic soils. In recent years, significant progress has been made in understanding the molecular mechanisms of Al toxicity and tolerance in the area of stress phytophysiology. Al targets multiple cellular sites, resulting in disruption of the structure and function of the cell wall, plasma membrane, cytoskeleton, signal transduction pathways, and calcium (Ca) uptake capability ALMT1, HvAACT1 and SbMATEArabidopsis genes ALS1 and ALS3 or the rice genes STAR1, STAR2 and ART1 can confer Al tolerance Al exclusion and intracellular resistance are two important mechanisms that promote Al tolerance in plants. External mechanisms defined to date include the release of organic acid anions and phenolic compounds, increased pH in the rhizosphere, modifications in cell wall components, and redistribution and internalization of Al. Organic acid anions, such as citrate, oxalate, and malate, are secreted by roots in response to Al 2+) levels in yeast, which can be decreased by overexpressing anti-apoptotic members Apoptosis, a typical form of PCD, facilitates the rapid removal of potentially threatening or undesired cells and plays a central role in normal development and homeostasis of metazoan organisms 2+, as a vital intracellular second messenger, governs countless cellular functions. To maintain basal levels of cytoplasmic Ca2+ under various and ever-changing conditions, cells have evolved mechanisms to carefully regulate Ca2+ entry and removal 2+ into their internal stores 2+ is stored in the cell wall and in intracellular organelles, including the endoplasmic reticulum (ER), mitochondria, and vacuoles. In plant and yeast cells, the vacuole serves as the principal Ca2+ sequestration site and contains >95% of total cellular Ca2+ stores 2+ store is maintained through the action of two specialized transporters: the high-affinity Ca2+-ATPase Pmc1p and the low-affinity Ca2+/H+ exchanger Vcx1p PMC1 gene effectively decreases the ability of yeast cells to grow in high Ca2+ environments, whereas the deletion of VCX1 decreases tolerance to Ca2+ only slightly; these findings suggest that Pmc1p plays a more significant role in vacuolar Ca2+ sequestration.Abiotic and biotic stresses induce cell death via the disruption of Ca homeostasis in yeast, plant, and animal cells 2+-ATPases (PMCAs) and functions as a P-type ion pump. Calcineurin activation by Calmodulin (CaM) and elevated cytosolic Ca2+ leads to increased expression of PMC1 2+ sensors 2+ signaling pathways are controlled by the Ca2+-mediated activation of CaM. CaM is essential for all eukaryotic life and participates in Ca2+-dependent stress response pathways through activation of CaM kinases Cmk1 and Cmk2 and the phosphatase calcineurin 3), which in turn releases Ca from internal stores Pmc1p has approximately 40% identity to mammalian plasma membrane Ca2+ regulates Al stress will provide insights into the process of reducing Al toxicity in plants. To this end, we investigated the functional roles of Pmc1p, the Ca2+ chelator BAPTA-AM and anti-apoptotic members in modulating cytosolic Ca2+ and facilitating Al tolerance.Al can induce both PCD and Ca burst in yeast, and anti-apoptotic members can enhance Al tolerance coincident with decreased Ca signals Mata ade2-1 can1-100 his3-11,15 leu2-3112 trp1-1 ura3-1) and its isogenic derivative mutants K605 (pmc1::TRP1), K661 (vcx1△) and K609 (pmr1::HIS3) pmr1::HIS3 pmc1::TRP1 cnb1::LEU2) 3 and CaCl2 stock solutions at concentrations of 1 M and 2.5 M, respectively, were filter sterilized and added to liquid medium at room temperature or to plates at <50°C. For the BAPTA-AM test, the cells were incubated with BAPTA-AM  in liquid medium for 30 min, and then other stresses were added.The budding yeast wild-type strain K601/W303-1A ; aliquots (5 µL) of each dilution were spotted onto a 1/2 SD/Gal-Raf/-Ura or 1/2 SD/Gal-Raf/-His plate with or without treatment. Plates were incubated at 30°C for 3 days. Cell survival was evaluated by counting colony-forming units (cfu). The cultured cells were harvested at defined intervals, diluted to cell density of 0.0005 at OD600, and a 30 µL aliquot of each was plated onto YPD plates Cells were preincubated in the appropriate liquid medium and allowed to grow twice to the exponential phase. For growth assays, the concentration at ODPMC1, we designed primers to PMC1-P1 (5′-CCC AAG CTT ATG TCT AGA CAA GAC GAA AAT TC-3′) with a HindIII site and PMC1-P2 (5′-CGC GGA TCC TTA ATA AAA GGC GGT GGA-3′) with a BamHI site. The resulting fragment was inserted into the pYES2 vector, in order to express PMC1 under the control of the GAL1 promoter and generated the plasmid pYES2-PMC1. After confirmation of the fidelity of the constructs by sequencing, the wild-type and pmc1 mutant yeast strains were, respectively, transformed with plasmids pYES2-PMC1 and the pYES2 vector by the lithium acetate (LiAc)/polyethylene glycol method Bcl-2, Ced-9 and PpBI-1 were inserted into the yeast-inducible expression vector pGilda, which expresses LexA fusion proteins Bcl-2, pGilda-Ced-9, and pGilda-PpBI-1) or the empty vector (pGilda) were transformed into the wild-type yeast and the pmc1 mutant. Transformants were selected for histidine prototrophy by plating on 1/2 SD/Glu/-His (Clontech) medium.The yeast-inducible expression vector pYES2 To examine the expression pattern of transformants, the overnight cultures were harvested. The frozen cells were mechanically disrupted using a ball mill ACT1 was used as an internal control (27 cycles). PCR was performed with an initial incubation at 94°C for 5 min, followed by 27 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The reaction was terminated by a final incubation at 72°C for 10 min. Primers for LexA are shown in 0.5 µL of cDNA was used as a template for the PCR amplification of target genes, and Ex Taq (Takara). The cycling program was as follows: an initial cycle of 5 min at 95°C, followed by 45 cycles of 15 s at 95°C, 10 s at 60°C and 15 s at 72°C. Data were collected and analyzed by the real-time PCR system . ACT1 was used as an internal control. Primers for PMC1, CMD1, CNB1 and PLC1 are shown in For quantitative RT-PCR, 0.4 µL of a 5-fold dilution of cDNA from each sample was used to analyze gene expression by the SYBR Premix in situ cell death detection kit; Roche, Mannheim, Germany) To visualize DNA strand breaks, cells were fixed in 4% formaldehyde in PBS (pH 7.4) then treated with lyticase  and stained with fluorescein isothiocyanate-labeled TUNEL reagent  at 37°C for 30 min with 10 µM Fluo-3-acetoxymethyl ester  prepared with a 1 mM stock solution in dimethyl sulfoxide. A non-cytotoxic detergent, pluronic F-127 (0.1%), was added to increase solubility of the Fluo-3/AM. Fluo-3 fluorescence was measured by FACSCalibur with 488-nm (blue) argon  in the FL1 channel. Data acquisition was performed using CellQuest (3.1f) software and data analysis by ModFit LT (3.0) software (Variety Software House). 2,000 to 10,000 cells were measured for each analysis Yeast cells were grown in 1/2 SD/Gal-Raf/-Ura or 1/2 SD/Gal-Raf/-His medium with or without treatment for 6 h. After harvesting, cells were resuspended in PBS (pH 7.4) and vortexed briefly. For determination of intracellular CaData were calculated as the mean of results from at least three independent experiments or one representative result of parallel experiments. The Origin 8 program was used for calculation. Error bars represent standard deviation (SD).PMC1 in yeast cells leads to a failure in pumping Ca2+ into the vacuole; thus, the cells failed to survive high Ca2+ stress 2+ transporters in Al toxicity, we tested the sensitivity of pmc1 and vcx1 mutants under a series of exogenous Ca2+ and aluminum ion (Al3+) treatments. As shown in 2+ and Al3+, the pmc1 mutant was more sensitive than wild-type yeast and the vcx1 mutant. Because of the variant status of other mutants tested under normal conditions as well as their reduced sensitivity to Al treatment  but grew at a much slower pace under conditions of high concentrations of Ca2+ (200–800 mM). Furthermore, compared to the wild-type control, the pmc1 mutant was more sensitive to moderate levels (0.5–1 mM) of Al3+ in liquid medium. We then performed a time-course assay to monitor yeast growth with 1 mM Al treatment. As shown in pmc1 mutant displayed more sensitivity to Al treatment. The difference in growth (OD600) appeared early, between the 10th–15th h, and became increasingly significant thereafter when mutant cells completely ceased to grow. In addition, both strains exhibited positive effects of Al toxicity within the first 10 h. This result indicated that the loss of vacuolar PMC1 in the mutant cells led to a failure in the regulative response to Al-elicited Ca2+ afflux.As shown in pmc1 mutant was not an artifact, we tested the vulnerable specificity of the pmc1 mutant. As shown in 2+) or cadmium ion (Cd2+). The pmc1 mutant displayed enhanced sensitivity to only Ca2+ and Al3+ but not to any of the other substances.To ensure that the Al susceptibility of the 2+ pool and, consequently, by activating Ca signaling.Taking into account all of these results along with those from our previous studies 2+ level in yeast pmc1 mutant under Al stress. As shown in pmc1 mutant cells was much higher than that of the wild-type cells, indicating Al-elicited Ca signaling is an early mechanism of Al-induced PCD.Al can induce both apoptotic-like cell death and an increase of CaPMC1 assumes an Al tolerance function in complementation tests, the exogenous PMC1 gene was transformed into wild-type yeast or back into the pmc1 mutant. Quantitative RT-PCR analysis showed that the levels of PMC1 mRNA in wild-type overexpressing exogenous PMC1 was 12 times higher than wild-type, and pmc1 mutant overexpressing PMC1 exhibited a level of PMC1 mRNA that was about 8 times higher than wild-type yeast . The chelator had little effect on unstressed cells. Furthermore, all the concentrations of BAPTA-AM used in this study effectively enhanced the viability of Al-exposed cells  but almost no PCR signal in uninduced medium (Glu)  . As show2+ levels in the wild-type, pmc1 mutant and transgenic cells expressing anti-apoptotic members under Al stress. Al3+ treatment (1 mM) resulted in increased Ca2+ levels in both wild-type and pmc1 mutant cells compared to the unstressed cells. Al-induced cytoplasmic Ca2+ levels in cells expressing anti-apoptotic members was distinctly decreased compared to the cells transformed with vector only  and CNB1  decreased upon exposure to Al  concentration may be too high and may affect both the H+ gradient and the pH of the vacuole or other organelles. The growth properties of wild-type yeast and the pmc1 mutant were similar when cells were exposed to different pH values  expression pmc1 mutant growth under Al and Ca stresses  and Al (B) stresses.(TIF)Click here for additional data file.Figure S2Al and Ca stress-increased cytoplasmic Ca signals can be alleviated by BAPTA-AM. Flow cytometry analysis of 0.5 mM Al3+- and 0.2 M Ca2+-challenged cytosolic Ca2+ levels in wt yeast.(TIF)Click here for additional data file.Figure S3Ca signals and growth properties of wt and pmc1 mutant strains in response to pH variations. A. Ca2+ changed little in response to pH variations. Flow cytometry analysis of pH-challenged cytosolic Ca2+ levels in wt and pmc1 mutant strains. B. Growth properties of yeast cells under different pH values.(TIF)Click here for additional data file.Figure S42O2 sensitivity in the Hpmc1 mutant. Growth properties of the wt strain and the pmc1 mutant under H2O2 stress.(TIF)Click here for additional data file.


Background: T-type Ca2+ (Cav3.2) channel inhibitors prevent neuronal death during ischemia, but the cytotoxic signals have not been resolved.Results: Cav3.2 channels generate cytotoxic Ca2+ elevations in the cytosol and mitochondria of PC12 cells deprived of oxygen and glucose.Conclusion: Ca2+ transfer from Cav3.2 channels to mitochondria contributes to ischemic toxicity.Significance: Cav3.2 channels and the mitochondrial Ca2+ uniporter (MCU) are potential targets for the treatment of stroke. 2+ channel inhibitors protect hippocampal CA1 neurons from delayed death after global ischemia in rats, suggesting that Cav3.1, Cav3.2, or Cav3.3 channels generate cytotoxic Ca2+ elevations during anoxia. To test this hypothesis, we measured the Ca2+ concentration changes evoked by oxygen and glucose deprivation (OGD) in the cytosol and in the mitochondria of PC12 cells. OGD evoked long-lasting cytosolic Ca2+ elevations that were reduced by Cav3.2 inhibition (50 μm Ni2+) and Cav3.1/Cav3.2 silencing and potentiated by Cav3.2 overexpression. The kinetics of the sustained cytosolic Ca2+ elevations occurring during OGD directly correlated to the extent of cell death measured 20 h after reoxygenation, which was decreased by Ni2+ and Cav3.1/Cav3.2 silencing and increased by Cav3.2 overexpression. Ni2+ and Cav3.1/Cav3.2 silencing delayed the decline of cellular ATP during OGD, consistent with a reduction in the Ca2+ load actively extruded by plasma membrane Ca2+ pumps. The cytosolic Ca2+ elevations were paralleled by mitochondrial Ca2+ elevations that were also increased by Cav3.2 overexpression and decreased by Ni2+ but not by Cav3.1/Cav3.2 silencing. Overexpression and silencing of the mitochondrial Ca2+ uniporter, the major mitochondrial Ca2+ uptake protein, revealed that the cytotoxicity was correlated to the amplitude of the mitochondrial, rather than the cytosolic, Ca2+ elevations. Selective activation of T-type Ca2+ channels evoked both cytosolic and mitochondrial Ca2+ elevations, but only the mitochondrial responses were reduced by Cav3.1/Cav3.2 silencing. We conclude that the opening of Cav3.2 channels during ischemia contribute to the entry of Ca2+ ions that are transmitted to mitochondria, resulting in a deleterious mitochondrial Ca2+ overload.T-type Ca Increased Ca2+ release from the endoplasmic reticulum (ER), increased Ca2+ influx across plasma membrane (PM) channels, and decreased Ca2+ extrusion have all been implicated in the pathogenesis of the [Ca2+]cyt overload during ischemia  were long considered to be the main non-excitotoxic Ca2+ entry pathway, as Cav1.2 opening upon neuronal depolarization can cause massive Ca2+ entry during ischemia. However, clinical studies showed little benefit of dihydropyridine analogues in acute ischemic stroke patients (2+ channels (Cav3.1 and Cav3.2), on the other hand, was shown to protect rat organotypic hippocampal slice cultures from ischemia-induced delayed cell death  that was identified recently  activate enzymes of the tricarboxylic acid cycle to boost ATP production  and Roger Tsien . Cytosolic FRET-based ATP indicators  were provided by Drs. Hiromi Imamura  and Hiroyuki Noji (Osaka University).l-glutamine medium containing 5% fetal calf serum, 10% horse serum, and 100 units/ml of penicillin and streptomycin. Cells were cultured in 95% air and 5% CO2 at 37 °C and subcultured every 7 days at a 1:10 dilution. Cells were plated on poly-L-lysine-coated coverslips or plates and grown for 24 h before transfection with siRNA and/or fluorescent proteins using Lipofectamine 2000. Experiments were performed 48 h after transfection. Cav3.1 and Cav3.2 silencing was achieved with a pool of three target-specific siRNAs , and knockdown efficiency was verified by quantitative PCR.PC12 cells were grown in RPMI 1640 m sucrose, 10 mm HEPES, 0.5 mm EGTA (pH 7.6)) and lysed for 20 min on ice in lysis buffer (25 mm Tris-HCl (pH 7.6), 150 mm NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (Roche) as described in Ref. g for 20 min, and the protein content of the supernatant was determined using the Bradford protein assay (Bio-Rad). 30 μg of total protein/lane was loaded on an SDS-PAGE gel. For immunoblotting, proteins were transferred onto a nitrocellulose membrane and probed with 1/500 anti-CCDC109A/MCU and anti-Tom20 . Horseradish peroxidase-conjugated secondary antibodies 1/20,000 (Amersham Biosciences) were used, followed by detection by chemiluminescence.Mitochondria were isolated by differential centrifugation in isolation buffer  with NaOH at 37 °C, placed into an anoxic chamber , and flushed continuously with a mixture of 95% N2/5% CO2 at 37 °C . A similar solution containing 2 mm of sodium dithionite and equilibrated with the N2/CO2 mixture for 5 min was injected into the chamber to initiate the OGD. Oxygen levels were monitored with a titanium-coated probe and a Neo Fox 1 channel oxy monitor system . After the OGD period, cells were washed twice with RPMI 1640 medium and cultured for 24 h in a normoxic environment. Control (non-ischemic) cells were exposed to a HEPES-buffered solution containing 10 mm glucose in an incubator with 95% air and 5% CO2 at 37 °C.Cells were washed twice in a HEPES-buffered solution containing 140 m2+]cyt and [Ca2+]mit were measured with D3cpv and 4mtD3cpv, respectively, and cytosolic [ATP] was measured with ATeamcyto, all FRET-based indicators. Cells were excited at 430 nm through a 455DRLP dichroic filter and alternately imaged with 480AF30 and 535DF25 emission filters . Image pairs were acquired every 10 s. Fluorescence ratios were calculated in MetaFluor 6.3  and analyzed in Excel (Microsoft) and GraphPad Prism 4 (GraphPad). Ca2+ slopes were determined by a linear fit to the steepest part of the Ca2+ increase, recorded between 20 and 60 min after OGD initiation.Cells were imaged on an Axiovert s100 TV microscope using a ×40, 1.3 numerical aperture oil immersion objective  and a cooled, 16-bit charge-coupled device back-illuminated frame transfer MicroMax camera . cyt was measured by fluorescence microscopy during a 3-h long OGD generated by replacing the saline solution with a glucose-free medium bubbled with 95% nitrogen and 5% CO2. OGD triggered an immediate [Ca2+]cyt increase followed within 20 min by a monotonic elevation that reached a steady-state plateau after 2 h (A). Subsequent oxygen/glucose readmission evoked a further, but transient, [Ca2+]cyt increase. This procedure was associated with ∼80% cytotoxicity, as determined by measuring LDH release 20 h after the insult (B). A 3-h OGD was as efficient as thapsigargin in evoking LDH release and slightly less efficient than staurosporine, two commonly used cytotoxic agents (B). Depriving cells of oxygen and glucose for 90 min caused ∼25% cytotoxicity, whereas 30 min of OGD did not promote LDH release (not shown). Consistent with a causal role for Ca2+ entry in this cell death, cytotoxicity decreased by 30% when OGD was performed in the absence of extracellular Ca2+ (A). Strikingly, a similar protection was observed when OGD was performed in the presence of 50 μm Ni2+ (A), a concentration that selectively inhibits Cav3.2 channels . Knockdown of T-type channels reduced cytotoxicity by 40% to the level observed in the absence of external Ca2+ or in the presence of Ni2+ (A). Concordantly, overexpression of the major endogenous T-type channel isoform of PC12 cells Cav3.2 (A). Transfection of cells with scrambled siRNA or with an empty vector did not alter OGD-evoked cytotoxicity (not shown). Functional overexpression was verified by immunofluorescence (C) and by recordings of transient inward currents activated by depolarizing voltage steps above −50 mV that were of large amplitude and inhibited by Ni2+ (D). These data indicate that, in PC12 cells, T-type calcium channels contribute to the cytotoxicity resulting from OGD.To study the impact of oxygen and glucose deprivation (OGD) on cytosolic Cafter 2 h A. Subseqe insult B. A 3-h c agents B. Deprivlar Ca2+ A. Striki μm Ni2+ A, a concectively ). To cond Cav3.2 , using ad Cav3.2 B. Knockd of Ni2+ A. Concors Cav3.2  increases Cav3.2 A. Transfrescence C and by 2+ channels on the cytosolic [Ca2+]cyt elevations evoked by OGD. Shorter (30- and 90-min) OGD was applied to streamline the acquisition process because the prototypical biphasic [Ca2+]cyt elevations could be well resolved during these time frames (A and B). Notably, following 90 min of OGD, [Ca2+]cyt did not increase further upon oxygen/glucose readmission but rapidly returned to basal levels (A). Predictably, [Ca2+]cyt elevations were nearly abolished during OGD performed in the absence of external Ca2+, a procedure that also decreased the resting [Ca2+]cyt concentration (B–D). Ni2+, on the other hand, appeared to selectively impact the delayed phase of the [Ca2+]cyt elevation, as it decreased the rates of the secondary [Ca2+]cyt increase by 70% (A and E) without altering resting [Ca2+]cyt levels or the amplitude of the initial [Ca2+]cyt peak (C and D). T-type Ca2+ channel knockdown also decreased the slope by 40% without altering resting [Ca2+]cyt levels and, in addition, slightly increased the initial peak amplitude (C–E). Conversely, Cav3.2 overexpression increased [Ca2+]cyt levels at all times during OGD, increasing the rates of the secondary [Ca2+]cyt elevation by 70%. These data indicate that T-type Ca2+ channels contribute to Ca2+ entry during the sustained phase of the [Ca2+]cyt elevations evoked by OGD. The kinetics of the delayed [Ca2+]cyt elevations measured during 30 min and 90 min of OGD correlated with the expression levels of T type Ca2+ channel isoforms and with cytotoxicity (F), a correlation that was particularly apparent for Ca2+ slopes measured during 90 min of OGD (R2 = 0.97). This tight relationship suggests that the Ca2+ ions entering across T-type channels during the delayed phase of the [Ca2+]cyt elevations evoked by OGD are particularly toxic for cells.To confirm this link, we studied the impact of pharmacological and genetic manipulations of T-type Cae frames , A and Bl levels A. Predicntration , B–D. Nie by 70% , A and Emplitude , C–E. Cotoxicity F, a corr2+]cyt. Reducing the [Ca2+]cyt elevation associated with OGD should, therefore, decrease ATP consumption by plasma membrane Ca2+ pumps and delay ATP depletion during ODG. To test this hypothesis we measured cytosolic ATP levels with the genetically encoded indicators ATeam (A). The values recorded after 20 min of OGD approached the levels measured in cells exposed to 10 μg/ml oligomycin and 10 mm 2-deoxyglucose , indicating that cytosolic ATP contents were fully depleted. Interestingly, pharmacological and genetic invalidation of T-type channel activity markedly delayed the kinetics of ATP decrease, increasing the time required to reach the minimal ATP levels during OGD by ∼50% . Ca2+ removal had an even more pronounced delaying effect (not shown) whereas overexpression of Cav3.2 channels had no significant effect . None of these treatments had any effect on basal ATP levels (not shown), indicating that T-type Ca2+ channel inhibition specifically decreases ATP consumption during OGD. This ATP sparing effect might contribute to the reduced cytotoxicity observed in these conditions.During ischemia, mitochondrial and glycolytic ATP production is prevented, and the residual ATP is rapidly consumed by the activity of plasma membrane pumps that attempt to maintain a normal membrane potential and [Cars ATeam . As expers ATeam A. The va2+ channels also contribute to mitochondrial matrix Ca2+ ([Ca2+]mit) elevations during OGD, using the mitochondria-targeted Cameleon probe 4mtD3cpv. The [Ca2+]mit responses evoked by OGD mimicked the responses observed in the cytosol, with a rapid increase followed by a sustained plateau and, after short OGD, a rapid return to baseline levels (C). As observed in the cytosol, Cav3.2 overexpression increased the amplitude of [Ca2+]mit elevations by accelerating the second phase of the response, whereas Ni2+ had the opposite effect (D). In contrast with the cytosolic data however, Cav3.2 overexpression increased, whereasNi2+ decreased, resting [Ca2+]mit levels, whereas Cav3.1 and Cav3.2 silencing had no measurable impact on basal [Ca2+]mit levels and on the [Ca2+]mit elevations evoked by OGD (D).We next investigated whether T-type Cae levels C. As obse effect D. In cond by OGD D.2+]mit elevations contributed to OGD toxicity, we altered the expression levels of the major Ca2+ uptake protein of mitochondria, the mitochondrial Ca2+ uniporter (MCU) (E), increased the delayed phase of the [Ca2+]mit elevation evoked by OGD as well as resting [Ca2+]mit levels (A and B) without altering the [Ca2+]cyt elevations evoked by OGD (E) Conversely, MCU silencing, validated by Western blot (E), decreased the [Ca2+]mit elevation evoked by OGD but did not alter basal [Ca2+]mit levels (A and B). The increased [Ca2+]mit elevation of MCU overexpressers was prevented by Ni2+, suggesting that a fraction of the Ca2+ taken up by mitochondria originated from T-type Ca2+ channels. Consistent with a role for mitochondrial Ca2+ uptake in cytotoxicity, MCU overexpression increased whereas MCU silencing decreased the cytotoxicity evoked by OGD (C). The deleterious effect of MCU expression was also prevented by Ni2+ (C). The opposite effects of Ni2+ and of MCU overexpression on [Ca2+]mit and cytotoxicity suggests that mitochondria might be located close to T-type Ca2+ channels. Accordingly, expression of a matrix-targeted RFP revealed a high density of mitochondria in PC12 cells, with numerous mitochondria located beneath the plasma membrane that co-localized extensively with Cav3.2 immunoreactivity . This indicates that the majority of Cav3.2 channels were located in close proximity to subplasmalemmal mitochondria. To test whether Ca2+ ions entering across T-type Ca2+ channels reach mitochondria, we used a low potassium concentration (20 mm) to depolarize the plasma membrane to ∼-50 mV and selectively activate T-type Ca2+ channels. This protocol evoked rapid increases in [Ca2+]mit that were reduced by Cav3.1/Cav 3.2 silencing (A). The mitochondrial responses evoked by ER Ca2+ release or by SOCE channel activation were not affected by Cav3.1/Cav 3.2 silencing (C and 6D), validating the specificity of the siRNA. These data indicate that Ca2+ ions entering across T-type Ca2+ channels are taken up by mitochondria. These mitochondria must be located close to the influx channels as mitochondrial Ca2+ uptake only occurs at microdomains of high Ca2+ concentration (2+]cyt elevations evoked by 20 mm K+ were not affected by Cav3.1/Cav 3.2 silencing (B), indicating that incoming Ca2+ ions are preferentially transmitted from T-type Ca2+ channels to mitochondria. These data indicate that Cav3.2 channels are functionally coupled to subplasmalemmal mitochondria and contribute to pathological [Ca2+]mit elevations during OGD.To test whether the cyt and [Ca2+]mit elevations during this recovery phase. Importantly, the [Ca2+]mit elevations evoked by OGD were increased by Cav3.2 overexpression and decreased by their invalidation or pharmacological inhibition, indicating that mitochondria take up Ca2+ ions flowing across T-type channels.Our results indicate that Call death , 36. We poptotic , but whe minutes A. Furthe2+ ions entering across voltage-gated Ca2+ channels in chromaffin cells, an uptake facilitated by the generation of high Ca2+ microdomains around the large capacity L-type channels  that selectively activate T-type Ca2+ channels. Efficient Ca2+ transfer from PM to mitochondria is also suggested by our observation that Ni2+ specifically reduced resting [Ca2+]mit and not [Ca2+]cyt levels. Ni2+ also inhibits Ca2+ uptake by isolated mitochondria (2+ transfer from T-type Ca2+ channels to subplasmalemmal mitochondria contributes significantly to the toxicity during ischemia.Subplasmalemmal mitochondria were shown previously to take up Cachannels . In contactivate . Here, wchondria , but succhondria , 39. Our


Pre-treatment with NLF or Ruthenium red (RuR) significantly improved viability of murine hippocampal HT-22 cells, reduced Ca2+ content and preserved membrane potential (Ψm) in mitochondria following oxygen-glucose deprivation (OGD). Following histamine-stimulated Ca2+ release from endoplasmic reticulum, in contrast to the vehicle-treated cells, the cells treated with NLF or RuR also demonstrated reduced Ca2+ content in their mitochondria, the event associated with preserved Ψm. Because RuR inhibits mitochondrial Ca2+ uniporter, we tested whether the NLF acts via the mechanism similar to the RuR. However, in contrast to the RuR, in the experiment with direct interaction of these agents with mitochondria isolated from naïve mice, the NLF did not alter mitochondrial Ca2+ influx, and did not prevent Ca2+ induced collapse of the Ψm. These data strongly argues against interaction of NLF and mitochondrial Ca2+ uniporter. Although the exact mechanism remains unclear, our study is the first to show that NLF inhibits intramitochondrial Ca2+ flux and protects developing brain against HI-reperfusion injury. This novel action of NLF has important clinical implication, because it targets a fundamental mechanism of post-ischemic cell death: intramitochondrial Ca2+ overload → mitochondrial membrane permeabilization → secondary energy failure.Nelfinavir (NLF), an antiretroviral agent, preserves mitochondrial membranes integrity and protects mature brain against ischemic injury in rodents. Our study demonstrates that in neonatal mice NLF significantly limits mitochondrial calcium influx, the event associated with protection of the brain against hypoxic-ischemic insult (HI). Compared to the vehicle-treated mice, cerebral mitochondria from NLF-treated mice exhibited a significantly greater tolerance to the Ca An opening of mPTP results in a loss of the proton-motive force for ATP synthesis, and eventuates in a release of pro-apoptotic proteins 2+ channel antagonist, nimodipine Neonatal hypoxic-ischemic brain injury (HI) is a leading cause of permanent neurological deficit in children. The mechanisms of cerebral damage following severe HI-insult are not fully understood. Therefore, there are no mechanism-targeted strategies in the management of infants with HI encephalopathy. Mitochondria have been recognized as organelles mediating cellular injury in the HI-reperfusion brain injury 2+ triggered opening of mPTP during post-HI reperfusion.Recently, Weaver at al. have shown a robust neuroprotective effect of antiretroviral agent, Nelfinavir (NLF) in the model of a focal ischemic injury in mature rodents In accordance with the National Institute of Health Guidelines for animal research, all animal procedures for these experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the Columbia University (Protocol # AC-AAAA6294). Surgical procedures were performed under isoflurane anesthesia. Injections, functional testing and live handling of mice were done with all efforts to minimize distress.In all experiments we used neonatal, 9–10 days old (P9– P10) C57Bl/6J mice purchased at P5 with their dams . The following study groups were formed: 1) Control group (naïve), mice with no interventions performed; 2) NLF-treated group; mice pre-treated with NLF and exposed to HI and 3) Vehicle-treated group; mice pre-treated with vehicle and exposed to HI.2+92% N2) for 15 min in the hypoxic chamber placed in the temperature-controlled (37°C) neonatal isolette (Airshield Inc. NC). After hypoxic exposure pups were returned to their dams and were kept overnight in the isolette at 32°C to minimize a temperature-related variability in the extent of brain injury HI-insult was produced according to the Rice-Vannucci model of HI-brain injury adapted to P9–10 neonatal mice NLF was administered by intraperitoneal injection of 50 µl of solution  at 20 hours prior to HI-insult. This dose was adapted from the study that demonstrated neuroprotective effect of the agent in adult mice with focal stroke Quantification of brain infarct volume was performed in 24 hours after HI as described previously Mitochondria isolation was done as described before Mitochondrial respiration was examined using the Clark electrode . In brief, 0.05 mg of mitochondrial protein were added to 0.5 ml of the respiration buffer composed of 200 mM sucrose, 25 mM KCl, 2 mM K2HPO4, 5 mM HEPES-KOH (pH 7.2), 5 mM MgCl2, 0.2 mg/ml of BSA, 30 µM Ap5A -pentaphosphate - an inhibitor of adenylate kinase). The buffer was supplemented with substrates, 10 mM sodium glutamate, and 5 mM sodium malate. ADP (100 nmoles) was added to initiate a phosphorylating respiration (State 3). Rates of O2 consumption were expressed in nmol O2/mg mitochondrial protein/min. 70 µM 2′-4′dinitro-phenol (DNP) was used to estimate a maximal respiration rate.Mitochondrial calcium buffering capacity was measured with the fluorescent, membrane impermeable dye 5N-Calcium Green as described 2 pulses repeatedly added every 50 seconds until mitochondrial membranes permeabilization was detected as spontaneous release of calcium. The amount of calcium required for mPTP opening was measured in nmoles/mg of mitochondrial protein.2+ uptake ability. Then, the same mitochondria were incubated with 4.4 µM of NLF for 5 min in the cuvette, followed by Ca2+ pulses (10 nmoles). Ruthenium Red , an inhibitor of mitochondrial calcium uniporter, was used in the same experimental paradigm.For assessment of direct influence of the NLF on mitochondrial calcium uptake, mitochondria were isolated from naïve mice. Initially, mitochondria (0.05 mg/ml) were tested for CaMitochondrial membrane potential (Ψm) was measured using fluorescent dye, Safranin (1 µM) which quenches fluorescence interacting with intact Ψm. Briefly, once the baseline of the Safranin fluorescence in the buffer (10 mM Tris-MOPS buffer (pH 7.4), 120 mM KCl, 1 mM K2HPO4, 5 mM sodium succinate, 2.5 mM sodium glutamate, 0.2 mg/ml BSA, 1 mM ATP) was reached, mitochondria (0.05 mg/ml) was added and an immediate quenching in the Safranin fluorescence was recorded. Once the minimal Safranin fluorescence was reached, at 100 seconds following supplementation with mitochondria, 50 nmoles of Ca pulses were given every 50 seconds until the Safranin fluorescence reached the baseline again. At this point, the absence of any changes in Safranin fluorescence following supplementation with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone  confirmed that the Ψm is fully collapsed. Mitochondrial samples were pre-incubated for five minutes either with NLF (4.4 µM) or RuR (1 µM) or vehicle (0.001% DMSO in the respiratory buffer).Ex-vivo quantification of mitochondrial calcium load was determined with fluorescent dye, 5N-Calcium Green. Briefly, mitochondria were isolated without EGTA. Mitochondria (0.1 mg/ml) were incubated in 10 mM Tris-MOPS buffer (pH 7.4), containing 120 mM KCl, 1 mM K2HPO4, 10 µM EGTA, 5 mM sodium succinate, 2.5 mM sodium glutamate, 1 µM 5N-Calcium Green. Following 200 sec of the stabilization of fluorescence, 0.1% digitonin was added to disrupt mitochondrial membranes and to release Ca2+. An increase in calcium fluorescence was recorded until it reached a plateau. The amount of the released mitochondrial calcium was quantified as an increase in the Ca2+ fluorescence normalized to the digitonin-induced non-specific fluorescence and expressed in nmoles per mg of mitochondrial protein.2 atmosphere.Mouse hippocampal neural/glial immortalized HT-22 cell line  was grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 25 mM glucose, 2 mM L-glutamine, 2 mM pyruvate, penicillin (10 units/ml), streptomycin (10 mg/ml), and 10% (vol/vol) heat inactivated fetal bovine serum (FBS) in a humidified cell incubator  at 37°C under a 5% CO2<0.1%) level was maintained for 12 hours by continuous flow of N2 95% and 5% CO2 under automatic O2 and CO2 control. Normoxic incubation was done in the DMEM (pyruvate-free) containing 25 mM glucose at 37°C under a 5% CO2/21% O2.Oxygen-Glucose Deprivation (OGD) was performed in DMEM (glucose and pyruvate-free) with 1% (vol/vol) FBS. Hypoxic . Calcium was detected with fluorescent dye, Rhod-2 (0.5 µM) for mitochondrial Ca2+ content or Fluo 4 (1 µM) for total cellular Ca2+ content. Mitochondrial membrane potential (Ψm) was assessed with fluorescent probes, Rhodamine 123  or tetra-methylrhodamine, ethyl ester perchlorate, . Cells were examined under NIKON A1R MP confocal microscope equipped with temperature, gas and humidity control station for live-cell studies. Semi-quantitative analysis of the total cellular or mitochondrial Ca2+ content and Ψm was performed based on optical density (OD) of the merged images  with Image-J software (public domain). All data were expressed in arbitrary fluorescent units (FU). Changes in the Ca2+ and Ψm specific fluorescence in response to histamine or OGD challenge were expressed in % in the relation to controls (mean = 100%). Assessment of cellular viability was performed at six hours following OGD. Cells were stained with propidium iodide  and Hoechst (10 ug/ml). Amount of PI positive cells and a number of nuclei were calculated in the images . The cellular mortality was estimated as a ratio of PI positive cells to a total number of cells detected with Hoechst. Each experiment for live imaging was repeated at list four times and at list of 15 images have been taken from each dish for quantitative analysis.Histamine-induced CaCytosolic fractions were obtained during mitochondrial isolation. The degradation of cytosolic proteins was preserved using 0.5% protease inhibitor cocktail. Samples were run on 12% polyacrilamide NuPAGE Bis-Tris gels and transferred to 0.22 µn nitrocellulose membranes. After blocking in 5% nonfat milk membranes were probed with mouse anti-cytochrome C , mouse anti-β-actin-HRP-conjugated , or mouse anti-COX IV  primary antibodies followed by incubation with secondary peroxidase-conjugated donkey anti-mouse antibodies . The visualization of bands was performed using ECL-plus western blotting detection system . Images were quantified and analyzed using Image-J software. Bands intensity was normalized to β-actin in the cytosolic fractions and expressed in Optical Density (OD) Arbitrary Units.Chemicals and reagents were obtained from Sigma, St. Louis, CA, USA, except of D-sucrose; 0.9% sodium chloride; MOPS; K2HPO4 ; 5N-calcium green; Rhod-2; Fluo-4; TMRE; Rhodamine 123; Mitotracker green; Hoechst; Dimethyl sulfoxide (DMSO); 12% polyacrylamide NuPAGE Bis-Tris gels; DMEM; Fetal bovine serum (FBS) heat inactivated; penicillin; streptomycin; . Nelfinavir was a generous gift from Dr. Dennis PA, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.Student’s T-test, one-or two ways ANOVA or ANOVA for repeated measures with Fisher’s post-hoc analysis were used when appropriate. All data were present as mean ± SE. A difference was considered significant with p ≤ 0.05.2+ content compared to the vehicle-treated littermates  improved mitochondrial tolerance against Ca2+ induced permeabilization of mitochondrial membranes in reperfusion, associated with (c) partially preserved ADP-phosphorylating activity, and (d) a better-preserved mitochondrial membrane integrity, evidenced by the decreased release of the cytochrome c from mitochondria. All these effects of NLF pre-treatment were coupled with a significant reduction of the infarct volumes in mice pre-treated with NLF compared to the vehicle-treated littermates  pre-treatment with NLF significantly reduced the HI-brain injury in neonatal mice and (2) this neuroprotection was associated with attenuation of Ca2+ overload results in a loss of the mitochondrial membrane integrity secondary to an opening of mitochondrial permeability transition pore. An opening of mPTP dissipates the H+ gradient across the inner membrane, causing a loss of the proton motive force for ATP synthesis. This mechanism has been suggested to explain secondary energy failure in reperfusion 2+ influx into mitochondria is the main triggering factor in the opening of mPTP in vivo contained significantly less Ca2+ at the end of the HI-insult is very important, because the organelles preloaded with Ca2+ during HI-insult quickly exhaust their Ca2+ buffering capacity during reperfusion when the major Ca2+ influx into mitochondria takes place 2+ buffering capacity, the parameter which defines a threshold of mitochondrial resistance against permeabilization of their membranes. A better-preserved mitochondrial membrane integrity in mice treated with NLF could be supported with data showing a markedly reduced cytochrome C release into cytosol in the mice treated with NLF, compared to that in the vehicle-controls. This better-preserved cytochrome C content in the mitochondria may account for a significantly better-preserved ADP-phosphorylating activity compared to the vehicle-treated counterparts. It has been shown that an opening of mPTP resulted in substantial loss of the cytochrome C, associated with significant inhibition of ADP-phosphorylating activity which was prevented by the cyclosporine A 2+ overload, the event upstream of the key cell-death mechanism – an opening of mPTP.Mitochondrial calcium overload during and, especially, following cerebral or cardiac ischemia has been considered as one of the key pathogenic events in the post-ischemic cellular death pathway 2+ influx into mitochondria is Ca2+ uniporter 2+ uniporter. We have employed an “analogy approach” to obtain evidence to support this hypothesis. Our experiments with the use of HT-22 cells demonstrated a stunning similarity in mitochondrial response to the cytosolic Ca2+ overload in the presence of NLF or RuR. Of note, cellular Ca2+ overload was induced by two different paradigms; the OGD or histamine challenge. In both experimental settings the NLF or RuR significantly limited Ca2+ load to mitochondria and prevented a collapse of Ψm secondary to the cyclosporine A-sensitive mPTP opening. However, in the experiments with isolated mitochondria, NLF exerted absolutely no effect on mitochondrial Ca2+ upload and, most importantly, did not prevent a loss of the Ψm in response to Ca2+. In contrast, as expected, RuR fully preserved Ψm during Ca2+ challenge and completely blocked mitochondrial Ca2+ up-take. Thus, our data demonstrate that in vivo and in vitro the pre-treatment with NLF significantly limits intramitochondrial Ca2+ flux, but the mechanism of this novel pharmacological action is unrelated to the inhibition of the mitochondrial Ca2+ uniporter. It has been reported that NLF limits intracellular Ca2+ flux in beta-cells 2+ content following OGD in the cells pre-treated with NLF compared to that pre-treated with RuR  subunit in the formation of the mitochondrial permeability transition pore complex 2+-induced mPTP, since ANT1 and ANT2 knock-out mouse liver mitochondria were less sensitive to Ca2+ compared to wild-type mitochondria. Regardless whether NLF interacts with ANT or not, the inhibiting effect of NLF on intra-mitochondrial Ca2+ flux cannot be overestimated, because Ca2+ overload is the primary trigger for an opening of mPTP in the ischemia and reperfusion.Although, the exact mechanism for the NLF-driven inhibition of mitochondrial Ca2+ influx into mitochondria during and following ischemia represents a potential neuroprotective strategy, targeting the key event: mitochondrial membrane permeabilization in the cell death pathway.Thus, our study shows that inhibition of Ca


STIM1  and STIM2 Ca2+ sensors oligomerize upon Ca2+ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K)-rich domains, and activate Ca2+ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca2+ have been studied but responses towards elevated cytosolic Ca2+ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PIP2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Furthermore, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PIP2, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca2+ levels. Concomitant with higher affinity for PM lipids, binding of CaM  inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PIP2 concentration-dependent manner. Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER–PM contacts via CaM binding.Ca This requires low cytosolic [Ca2+] in the range of 10–100 nM and steep Ca2+ gradients across the ER (endoplasmic reticulum) and PM (plasma membrane), which are maintained by Ca2+ pumps [2+ channels in the ER and PM results in Ca2+ influx and elicits a variety of signals.Caunctions . Ca2+ si2+ channels in a process known as store-operated Ca2+ entry [SOCE ] pathway  from residues 233–685 (GFP–STIM1C) and STIM2 (NP_065911.3) from residues 237–746 (GFP–STIM2C) into SalI/BamHI restriction sites downstream of GFP in pET15b vector  and subjected to SDS/PAGE followed by 1 h of staining with 2.5% (w/v) Coomassie Brilliant Blue solution.2] were made as previously described  for 1 h at 4°C, while rotating. GFP fluorescence of 40 μl of each input, unbound and bound fractions was measured using a 384-well polystyrene black microplate (Greiner Bio-One GmbH) in a microplate reader (Thermo Scientific Appliskan). The amount of protein bound was calculated as percentage of input protein bound to CaM beads.The GFP or GFP-fusion proteins  were incubated with 30 μl of CaM beads (Agilent Technologies) after washing thrice with CaM-binding buffer . Therefore we repeated this competition experiment with PM-like liposomes containing 2 mol% PIP2 and tested GFP–STIM2K binding after pre-incubation with 5 μM CaM in presence of 50, 100 or 200 μM CaCl2. Depending on the [Ca2+] CaM will be either semi- or fully Ca2+-loaded, which may influence the degree of competition. At 100 and 200 μM CaCl2 the addition of CaM led to strong reduction of GFP-STIM2K binding P2. Compared with binding at 100 μM CaCl2, pre-incubation with 5 μM CaM in presence of CaCl2 reduced binding by 61±1% , in presence of CaCl2 showed no competition, indicating that Ca2+-loading of CaM is required for competition with lipid binding. Consistently, CaM in presence of EGTA, only had a weak effect on lipid-binding activity of STIM2K dimer and a similar weak effect had the pre-incubation with CaM3–4m , suggesting that Ca2+-loading of the C-terminal lobe is required for competition. Mutation of the N-terminal lobe resulted in a CaM variant that showed an intermediate level of competition, i.e. 31±2% reduction in binding to 2 mol% PIP2-containing PM-like liposomes. Although this effect is weaker compared with fully Ca2+-loaded CaM, it suggests that semi-Ca2+-loaded CaM at an intermediate cytosolic [Ca2+] may have an inhibitory effect on STIM2-mediated ER–PM contact formation.In order to test further whether the competition of CaM depends on Caby 61±1% A. Pre-in2. The absence of cholesterol and sphingolipids should result in an even distribution of PIP2 without formation of local clusters of PIP2. Under these conditions 100 μM CaCl2 reduced the binding of GFP–STIM2K dimer by 25±4%, consistent with weaker binding P2] as well as the oligomerization of STIM2 determine the binding of STIM2-containing ER to the PM lipids.Finally, we repeated these competition experiments with liposomes comprising only PC and 5 mol% PIP binding B. Pre-inated CaM B. Mutati2 than the lipid-binding domain of STIM1. In the case of STIM1, binding to PIP2 requires tetramerization, whereas for STIM2 K-rich domain dimerization is sufficient for PIP2 binding. The increased affinity of the STIM2 K-rich domain for PM lipids is achieved by formation of a short amphipathic α-helix. Consistently, all K-rich domains of mammalian STIM2 proteins lack a C-terminal proline residue, which is conserved in K-rich domains of STIM1. Formation of an α-helix in STIM2 K-rich domain also leads to increased affinity for Ca2+/CaM, which competes with binding to PIP2. Thus, an elevated cytosolic [Ca2+] may dampen STIM2-mediated ER–PM contact formation via binding of Ca2+/CaM to its K-rich domain. This may contribute to a Ca2+-dependent feedback loop, controlling STIM2-mediated Ca2+ influx under conditions with increased cytosolic [Ca2+].STIM2 has evolved with a lipid-binding domain with higher affinity for PIP2 and PIP3 head groups at the PM had been described previously in membrane binding studies with small GTPases . The Kd of the STIM2 EF-hand domain for Ca2+ in the ER lumen is 0.5 mM, which will activate STIM2 at a luminal [Ca2+] about 0.4 mM, which is very similar to the resting [Ca2+] of the ER lumen  and cytosolic [Ca2+], Ca2+/CaM binding in vivo may compete with binding to PM lipids and thereby could help to destabilize and terminate ER–PM contacts, thereby protecting cells against Ca2+ overload. Consistent with a function of Ca2+/CaM in down-regulation of ER–PM contacts, low cytosolic [Ca2+] in neurons after exposure to low Ca2+ media enhanced STIM2–Orai1 association  is sufficient to release the electrostatic interactions between STIM1 K-rich domain and PM lipids is not known but CRACR2A with two EF-hand domains stabilizes STIM1–Orai1 complexes at low cytosolic [Ca2+], suggesting that localized cytosolic Ca2+ elevations may promote the dissociation of STIM1–Orai1 complex  in the cytosol as well as in the ER lumen.In summary, binding of Ca


In the present study we examined Ca2+ homeostasis and excitation–contraction (E-C) coupling in cultured myotubes derived from two patients with Rippling muscle disease with severe reduction in caveolin-3 expression; one patient harboured the heterozygous c.84C>A mutation while the other patient harbored a homozygous splice-site mutation (c.102+ 2T>C) affecting the splice donor site of intron 1 of the CAV3 gene. Our results show that cells from control and rippling muscle disease patients had similar resting [Ca2+]i and 4-chloro-m-cresol-induced Ca2+ release but reduced KCl-induced Ca2+ influx. Detailed analysis of the voltage-dependence of Ca2+ transients revealed a significant shift of Ca2+ release activation to higher depolarization levels in CAV3 mutated cells. High resolution immunofluorescence analysis by Total Internal Fluorescence microscopy supports the hypothesis that loss of caveolin-3 leads to microscopic disarrays in the colocalization of the voltage-sensing dihydropyridine receptor and the ryanodine receptor, thereby reducing the efficiency of excitation–contraction coupling. Hum Mutat 32:309–317, 2011. © 2011 Wiley-Liss, Inc.Rippling muscle disease is caused by mutations in the gene encoding caveolin-3 (CAV3), the muscle-specific isoform of the scaffolding protein caveolin, a protein involved in the formation of caveolae. In healthy muscle, caveolin-3 is responsible for the formation of caveolae, which are highly organized sarcolemmal clusters influencing early muscle differentiation, signalling and Ca CAV3 (MIM♯ 601253) the gene encoding caveolin-3 (CAV3), a caveolin isoform exclusively expressed in skeletal, cardiac, and smooth muscles  and ryanodine receptor-activated Ca2+ release. Interestingly however, cells bearing the mutated CAV3 showed a shift in depolarization-induced Ca2+ release and a decreased depolarization-induced Ca2+ influx, suggesting that lack of caveolin leads to a less efficient excitation–contraction coupling.Although the precise pathomechanism is still elusive, a number of reports have indicated that CAV3 may have a role in CaCAV3 reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence (GenBank NM_033337.1), according to journal guidelines (http://www.hgvs.org/mutnomen). The initiation codon is codon 1.Nucleotide numbering for Primary skeletal muscle cultures were established from fragments of muscle biopsies obtained from patients undergoing diagnostic testing, as previously described i were simultaneously recorded with membrane currents using the fluorescent Ca2+ indicator K5-Fluo-3  and a laser-scanning confocal microscope  with a 60 × water immersion objective lens. Fluo-3 was excited at 488 nm with an argon ion laser, and emitted light was collected above 500 nm. Linescan images were recorded at a rate of 500 lines/sec. Confocal images were analyzed in ImageSXM (free software based on NIH Image) i are expressed as changes in fluorescence (ΔF/F0). All measurements have been performed at room temperature.Human myoblasts were grown on laminin-coated glass coverslips and differentiated into myotubes. Cells were voltage-clamped in the whole-cell patch clamp configuration with low resistance borosilicate glass micropipettes (1–3 MΩ) using an Axopatch 200B amplifier  controlled by a custom-written data-acquisition software developed by LabView . The pipette solution contained (in mM) 100 CsAsp, 20 tetraethylammonium(TEA)-Cl, 10 HEPES, 5 MgClBarrett,  and furt2 were isolated. Proteins were separated by SDS PAGE, blotted onto nitrocellulose, and probed with antibodies against caveolin-3 , RyR1 , α1.1 subunit of the DHPR , SERCA2 , calsequestrin  or depolarization-induced peak Ca2+ release in mouse skeletal muscle cells. A deeper investigation into the electromechanical coupling of caveolin-3-depleted cells, however, revealed a 5-mV shift in the V1/2 activating potential, resulting in reduced Ca2+ release at low depolarizing potentials. Although different from what was reported in mouse skeletal muscle , who showed that RyR1 and caveolin-3 coimmunoprecipitate and that caveolin-3 interacts directly with a transmembrane domain of the RyR1. Thus, the lack of CAV3 seems to derange the microarchitecture of the main protein components of the E-C coupling machinery leading (1) to a less efficient coupling, particularly evident at low depolarizing stimuli and (2) to a decrease in ECCE. We are aware that the TIRF experiments were performed on myotubes and theoretically the colocalization results could reflect a different degree of differentiation of cells from control and RMD patients. To minimalize this possibility, we only analyzed those myotubes containing more than nuclei. In support of our finding, it was demonstrated that skeletal muscles from CAV3 KO mice show abnormalities in the organization of the T-tubules with dilated and longitudinally oriented T-tubules [Galbiati et al., We were interested in investigating whether the reduced E-C coupling efficiency in a et al.  showed t2+ influx may explain, at least in part, the phenotypic characteristics of patients with reduced CAV3 levels. Interestingly, Lamb [Although the results of the present investigation do not explain how the rippling phenomena are induced by passive stretching and percussion, the finding of reduced E-C coupling efficiency and reduced Caly, Lamb  suggesteCAV3 mutations.In conclusion, we show that loss of caveolin-3 leads to a decrease in the E-C coupling efficiency in human myotubes, and this feature may be one of the underlying causes of the rippling phenotype seen in patients harboring


Hlx was first detected via is expression in hematopoietic cells of the myelomonocytic and B lymphocyte lineages and further studies indicated a potential role for Hlx in hematopoietic differentiation [HLX expression correlates with poor overall survival. These findings, combined with the observation that HLX knockdown results in an inhibition of growth and clonogenicity of leukemia cells, suggest that HLX may be a novel promising prognostic and therapeutic target in AML.Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor clinical outcome in most subtypes of the disease. Less than one third of patients achieve durable remission with current treatment regimens, and prognostication and risk stratification are challenging. We recently reported that a new non-clustered homeobox gene, H2.0-like homeobox (HLX), regulates early hematopoiesis and promotes AML in mice and humans . Hlx wasntiation . We now Δ/Δ model of AML [Hlx expression, suggesting that Hlx overexpression is an early event in leukemogenesis and Hlx is involved in malignant transformation [Recent experimental evidence has shown that acute myeloid leukemia (AML) arises from transformed immature hematopoietic cells following the accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells (HSC) and progenitors . In ordel of AML . Using tormation .in vivo we utilized a competitive, congenic transplantation model. Experimental elevation of HLX in HSC resulted in near complete depletion of early HSPC populations  and 16-fold enrichment of myeloid progenitors with a surface phenotype slightly past the GMP stage (CD45+Kit−CD34−CD44highCD49bhighCD11bmid). Overexpression of HLX in HSPC in vitro led to a myeloid differentiation block and to formation of phenotypically similar, aberrant myeloid progenitors with unlimited serial clonogenicity. These results indicate that HLX suppresses the function of normal immature HSC and progenitors but leads to a differentiation block and confers unlimited clonogenic capacity at the level of phenotypically more mature progenitors. Because the loss of HSC did not seem to be mediated by induction of apoptosis or necrosis, we speculate that HLX exerts this dual role by triggering initial differentiation of HSC followed by suppression of terminal differentiation at a more committed progenitor level. Genetic in vivo modeling using an inducible system will be required to definitively clarify the role of HLX elevation at different stages of hematopoietic stem and progenitor cell differentiation in vivo.To assess the effects of HLX overexpression in wild-type HSPC Hlx may function in concert with cofactors that could confer cell type specificity to the effects of HLX overexpression and contribute to leukemic transformation. Combinatorial genetic modeling with other disease alleles in AML will be instrumental to uncover the exact role of Hlx in AML pathogenesis.Of note, although overexpression of HLX leads to the formation of immortalized myeloid progenitors with unlimited serial clonogenicity, we so far have not observed development of overt leukemia upon transplantation, suggesting that HLX elevation alone is not sufficient for full transformation. Like other homeobox genes, HLX is significantly overexpressed (2 to 16 fold) in more than 80% of patients with AML, and this occurs across all major disease subtypes and is independent of blast counts. Higher levels of HLX are associated with poor overall survival in 3 different, large cohorts of AML patients . Importantly, the prognostic value of HLX holds up in a multivariate analysis, indicating HLX overexpression and prognostic value is an independent phenomenon across several molecular subsets of patients. Notably, high HLX expression status seems to overrule several mutations which have been associated with favorable prognosis. Patients with CEBPA or NPM1 mutations for instance only have favorable overall survival if HLX is low, but a poor prognosis if HLX is expressed at high levels. While this needs to be confirmed in independent and, ideally, prospective studies, these observations suggest broad importance of HLX for the disease biology of AML, and a role for determining HLX expression levels in the prognostication of AML patients.Several HOX genes are expressed at high levels in subtypes of AML. Important roles in leukemic transformation have been demonstrated for specific members of the HOX-A and HOX-B cluster, and the nonclustered homeobox gene CDX2 was recently reported to be implicated in leukemogenesis -10. HoweHLX overexpression in both pre-leukemic and bulk AML cells, we studied the effects of HLX reduction in both murine and human AML cells. ShRNA-mediated reduction of HLX significantly inhibits leukemic growth and clonogenic capacity, and overcomes the differentiation block of AML cells. We found that HLX regulates a set of genes which likely mediate its leukemia-promoting functions, including candidates BTG1 (B-cell translocation gene 1) and PAK1 (p21 protein-activated kinase 1). Intersecting the gene expression data from our in vitro overexpression and inhibition studies with data from genome-wide expression analysis of a large cohort of human AML patients, we identified an HLX-dependent molecular signature of genes, which again included PAK1, with prognostic value for AML patients.Based on our findings of the significance of In summary, our studies have identified HLX as a novel key transcription factor involved in the regulation of early hematopoiesis and AML pathogenesis, and suggest HLX as a promising new therapeutic target in AML. Additional mechanistic studies will be required to identify the transcriptional targets of HLX in HSPC and to define its functionally critical downstream pathways, which will improve our understanding of the precise role of HLX in HSPC biology and malignant transformation, and may offer additional opportunities for targeted therapeutic intervention.