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Tightly regulated Ca(2+) homeostasis is a prerequisite for proper cardiac function. To dissect the regulatory network of cardiac Ca(2+) handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca(2+) extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca(2+) uptake and accelerates the transfer of Ca(2+) from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca(2+) sparks and thereby inhibits Ca(2+) overload-induced erratic Ca(2+) waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca(2+) uptake in the regulation of cardiac rhythmicity.

The article examines the regulatory network of cardiac Ca2+ handling through the creation of a chemical suppressor screen on zebrafish tremblor embryos. Topics discussed include the identification of efsevin based on its ability to restore coordinated contractions in tremblor and its capacity to bind to VDAC2 which in turn allows mitochondrial Ca2+ uptake and transfer acceleration from intracellular stores into mitochondria.

Abstract: Uncoupling proteins 2 and 3 (UCP2/3) are essential for mitochondrial Ca2+ uptake but both proteins exhibit distinct activities in regard to the source and mode of Ca2+ mobilization. In the present work, structural determinants of their contribution to mitochondrial Ca2+ uptake were explored. Previous findings indicate the importance of the intermembrane loop 2 (IML2) for the contribution of UCP2/3. Thus, the IML2 of UCP2/3 was substituted by that of UCP1. These chimeras had no activity in mitochondrial uptake of intracellularly released Ca2+, while they mimicked the wild-type proteins by potentiating mitochondrial sequestration of entering Ca2+. Alignment of the IML2 sequences revealed that UCP1, UCP2 and UCP3 share a basic amino acid in positions 163, 164 and 167, while only UCP2 and UCP3 contain a second basic residue in positions 168 and 171, respectively. Accordingly, mutants of UCP3 in positions 167 and 171/172 were made. In permeabilized cells, these mutants exhibited distinct Ca2+ sensitivities in regard to mitochondrial Ca2+ sequestration. In intact cells, these mutants established different activities in mitochondrial uptake of either intracellularly released (UCP3R171,E172) or entering (UCP3R167) Ca2+. Our data demonstrate that distinct sites in the IML2 of UCP3 effect mitochondrial uptake of high and low Ca2+ signals. [ABSTRACT FROM AUTHOR]

Abstract: The transmission of Ca2+ signals to mitochondria is an important phenomenon in cell signaling. We have recently reported that the novel uncoupling proteins UCP2 and UCP3 (UCP2/3) are fundamental for mitochondrial Ca2+ uniport (MCU). In the present study we investigate the contribution of UCP2/3 to mitochondrial accumulation of Ca2+ either exclusively released from the ER or entering the cell via the store-operated Ca2+ entry (SOCE) pathway. Using siRNA we demonstrate that constitutively expressed UCP2/3 are essentially involved in mitochondrial sequestration of intracellularly released Ca2+ but not of that entering the cells via SOCE. However, overexpression of UCP2/3 yielded elevated mitochondrial Ca2+ uptake from both sources, though it was more pronounced in case of entering Ca2+, indicating that the expression levels of UCP2/3 are crucial for the capacity of mitochondria to sequester entering Ca2+. Our data point to distinct UCP2/3-dependent and UCP2/3-independent modes of mitochondrial Ca2+ sequestration, which may meet the various demands necessary for an adequate organelle Ca2+ loading from different Ca2+ sources in intact cells. [Copyright &y& Elsevier]

The stromal interacting molecule (STIM1) is pivotal for store-operated Ca2+ entry (SOC). STIM1 proteins sense the Ca2+ concentration within the lumen of the endoplasmic reticulum (ER) via an EF-hand domain. Dissociation of Ca2+ from this domain allows fast oligomerization of STIM1 and the formation of spatially discrete clusters close to the plasma membrane. By lifetime-imaging of STIM1 interaction, the rearrangement of STIM1, ER Ca2+ concentration ([Ca2+]ER) and cytosolic Ca2+ signals ([Ca2+]cyto) we show that [Ca2+]cyto affects the subcellular distribution of STIM1 oligomers and prevents subplasmalemmal STIM clustering, even if the ER is depleted. These data indicate that [Ca2+]cyto, independently of the ER Ca2+ filling state, crucially tunes the formation and disassembly of subplasmalemmal STIM1 clusters, and, thus, protects cells against Ca2+ overload resulting from excessive SOC activity. [ABSTRACT FROM AUTHOR]

Background: Analysis of many proteins produced during the transition into the stationary phase and under stress conditions (including starvation stress) demonstrated that a number of novel proteins were induced in common to each stress and could be the reason for cross-protection in bacterial cells. It is necessary to investigate the synthesis of these proteins during different stress conditions., Methods: The changes in protein profile of Flexibacter chinensis at various stages of the starvation process and the other stresses were investigated using two-dimensional gel electrophoresis which has proven to be a powerful tool for investigation of the changes in protein profiles under such conditions., Results: Most starvation proteins were synthesized during the early stationary phase and many of these proteins remained during long-term starvation. Some of these proteins were transiently synthesized. The sequencing result of one of the proteins showed that there was a 62.5% identity in 8 amino acids overlapped with the 5' residue of a 10 kDa chaperon protein which is known to be involved in the starvation stress response in other organisms., Conclusion: There are many proteins synthesized in common with many stresses in Flexibacter chinensis. Some of these proteins must play a major role in the stability of the cell under different stresses.