Translocon closure to Ca<sup>2+</sup> leak in proliferating vascular smooth muscle cells

Amer, Mona G.; Li, Jing; O’Regan, David; Steele, Derek S.; Porter, Karen E.; Sivaprasadarao, Asipu; Beech, David J.

doi:10.1152/ajpheart.00984.2008

Cited by 24 publications

(27 citation statements)

References 35 publications

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“…Using established procedures, calcium leakage was studied directly after thapsigargin addition as well as indirectly as a reduction in thapsigargin-induced leakage following puromycin pre-treatment. [2][3][4][5][6][7] Both analyses were carried out using untreated cells, cells treated with either one or both of the two SEC61A1-specific siRNAs, and with cells treated with the negative control siRNA. Compared to untreated cells and to cells treated with the negative control subsequent store operated calcium entry (SOCE) in the presence of extracellular calcium.…”

Section: Discussionmentioning

confidence: 99%

“…The drug thapsigargin inhibits SERCAs and, thereby, reveals the calcium leak pathway. [2][3][4][5][6][7] Leakage of Ca 2+ from the ER can be visualized as a reduction of Ca 2+ concentration in the ER by employing an ER calcium indicator, such as Mag-FURA-2, in digitonin-permeabilized cells. Alternatively, it can be visualized as an increase in the cytosolic Ca 2+ concentration by employing a cytosolic calcium indicator, such as FURA-2, in intact cells in the presence or absence of extracellular Ca 2+ .…”

Section: Introductionmentioning

confidence: 99%

“…[8][9][10][11] Indirect evidence from various studies suggests that the major entry site of newly synthesized polypeptides in the ER membrane, the Sec61 complex (also called translocon or protein translocase) contributes to the Ca 2+ leak. [2][3][4][5][6][7] The indirect evidence is as follows. First, puromycin-induced release of nascent polypeptides from ribosomes leads to a drop in ER lumenal Ca 2+ that cannot necessarily be seen as a concomitant increase in cytosolic Ca 2+ due to PMCA action, 2,3 in turn, the drop in ER lumenal Ca 2+ leads to a reduced thapsigargin-induced increase in cytosolic Ca 2+ in the absence of extracellular Ca 2+ .…”

Section: Introductionmentioning

confidence: 99%

“…First, puromycin-induced release of nascent polypeptides from ribosomes leads to a drop in ER lumenal Ca 2+ that cannot necessarily be seen as a concomitant increase in cytosolic Ca 2+ due to PMCA action, 2,3 in turn, the drop in ER lumenal Ca 2+ leads to a reduced thapsigargin-induced increase in cytosolic Ca 2+ in the absence of extracellular Ca 2+ . 7 Second, pactamycin-induced inhibition of the initiation of protein synthesis results in calcium leakage from the ER (that is not detectable as a concomitant increase in cytosolic Ca 2+ due to PMCA) and in ©2 0 1 1 L a n d e s B i o s c i e n c e .…”

Section: Introductionmentioning

confidence: 99%

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Sec61 complexes form ubiquitous ER Ca²⁺leak channels

Lang

Erdmann²,

Jung³

et al. 2011

Channels

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Sec61 complexes form ubiquitous ER Ca²⁺leak channels

Lang

Erdmann²,

Jung³

et al. 2011

Channels

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“…The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca 2+ leak channels [2][3][4][5][6][7][8] . The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca 2+ from the ER.…”

Section: Introductionmentioning

confidence: 99%

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca<sup>2+</sup> Leak Channels in the ER Membrane and their Regulatory Mechanisms

Lang

Schäuble

Cavalié

et al. 2011

JoVE

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In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling 1 . The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca 2+ leak channels [2][3][4][5][6][7][8] . The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca 2+ from the ER.Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex 9,10 . We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca 2+ leak channel and to characterize its putative regulatory mechanisms [11][12][13] . This system combines siRNA mediated gene silencing and live cell Ca also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca 2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca 2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca 2+ efflux, and iii) study the effects of disease linked mutations in the relevant components. Video LinkThe video component of this article can be found at https://www.jove.com/video/2730/ Protocol Preparation of stock solutions1. Prepare calcium-free buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 0.5 mM EGTA, 10 mM glucose in 10 mM HEPES-KOH, (pH 7.35)). 2. Solubilize FURA-2 AM in DMSO to obtain an 1 mM stock solution. Mix using a vortex until the solution is homogenously light yellow. Protect cup from light. Dilute the FURA-2 AM stock solution into 1 ml Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin to a final concentration of 4 μM for loading of HeLa cells. 3. Prepare a 12.5 mM puromycin stock solution in distilled water (pH 7.0). Store aliquots at -20°C. Dilute the 12.5 mM puromycin stock solution in the calcium-free buffer to a final concentration of 500 μM puromycin. 4. Dissolve thapsigargin in DMSO to obtain a 1 mM Stock solution. 5. Solubilize 1 mg ophiobolin A in 20 μl chloroform and, subsequently, mix with 25 μl DMSO. The microcentrifuge tube remains open until chloroform is evaporated. Thus the final ophiobolin A concentration is 100 mM. Store aliquots at -20 °C. Prior to use, dilute stock solutions into calcium-free buffer to a final concentration of 100 μM. Thus, final DMSO concentration for live cell imaging does not exceed 0.1%. 6. Dissolve trifluoperazine in DMSO to a concentration of 10 mM and store aliquots at -20 °C. Dilute stock solutions into calcium-free buffer to a final concentration of 10 μM prior to use. Thus final DMSO con...

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Regulators of Ca2+ Signaling in Mast Cells: Potential Targets for Treatment of Mast Cell-Related Diseases?

Beaven

2011

Advances in Experimental Medicine and Biology

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A calcium signal is essential for degranulation, generation of eicosanoids and optimal production of cytokines in mast cells in response to antigen and other stimulants. The signal is initiated by phospholipase C-mediated production of inositol1,4,5-trisphosphate resulting in release of stored Ca(2+) from the endoplasmic reticulum (ER) and Golgi. Depletion of these stores activates influx of extracellular Ca(2+), usually referred to as store-operated calcium entry (SOCE), through the interaction of the Ca(2+)-sensor, stromal interacting molecule-1 (STIM1 ), in ER with Orai1(CRACM1) and transient receptor potential canonical (TRPC) channel proteins in the plasma membrane (PM). This interaction is enabled by microtubular-directed reorganization of ER to form ER/PM contact points or "punctae" in which STIM1 and channel proteins colocalize. The ensuing influx of Ca(2+) replenishes Ca(2+) stores and sustains elevated levels of cytosolic Ca(2+) ions-the obligatory signal for mast-cell activation. In addition, the signal can acquire spatial and dynamic characteristics (e.g., calcium puffs, waves, oscillations) that encode signals for specific functional outputs. This is achieved by coordinated regulation of Ca(2+) fluxes through ATP-dependent Ca(2+)-pumps and ion exchangers in mitochondria, ER and PM. As discussed in this chapter, studies in mast cells revealed much about the mechanisms described above but little about allergic and autoimmune diseases although studies in other types of cells have exposed genetic defects that lead to aberrant calcium signaling in immune diseases. Pharmacologic agents that inhibit or activate the regulatory components of calcium signaling in mast cells are also discussed along with the prospects for development of novel SOCE inhibitors that may prove beneficial in the treatment inflammatory mast-cell related diseases.

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Translocon closure to Ca²⁺ leak in proliferating vascular smooth muscle cells

Abstract: Amer MS, Li J, O'Regan DJ, Steele DS, Porter KE, Sivaprasadarao A, Beech DJ. Translocon closure to Ca 2ϩ leak in proliferating vascular smooth muscle cells.

Cited by 24 publications

References 35 publications

Sec61 complexes form ubiquitous ER Ca²⁺leak channels

Sec61 complexes form ubiquitous ER Ca²⁺leak channels

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca<sup>2+</sup> Leak Channels in the ER Membrane and their Regulatory Mechanisms

Regulators of Ca2+ Signaling in Mast Cells: Potential Targets for Treatment of Mast Cell-Related Diseases?

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Translocon closure to Ca2+ leak in proliferating vascular smooth muscle cells

Abstract: Amer MS, Li J, O'Regan DJ, Steele DS, Porter KE, Sivaprasadarao A, Beech DJ. Translocon closure to Ca 2ϩ leak in proliferating vascular smooth muscle cells.

Cited by 24 publications

References 35 publications

Sec61 complexes form ubiquitous ER Ca2+leak channels

Sec61 complexes form ubiquitous ER Ca2+leak channels

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca<sup>2+</sup> Leak Channels in the ER Membrane and their Regulatory Mechanisms

Regulators of Ca2+ Signaling in Mast Cells: Potential Targets for Treatment of Mast Cell-Related Diseases?

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Product

Resources

About

Translocon closure to Ca²⁺ leak in proliferating vascular smooth muscle cells

Sec61 complexes form ubiquitous ER Ca²⁺leak channels

Sec61 complexes form ubiquitous ER Ca²⁺leak channels