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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]

Summary: The underlying molecular action of the novel uncoupling proteins 2 and 3 (UCP2 and UCP3) is still under debate. The proteins have been implicated in many cell functions, including the regulation of insulin secretion and regulation of reactive oxygen species (ROS) generation. These effects have mainly been explained by suggesting that the proteins establish a proton leak through the inner mitochondrial membrane (IMM). However, accumulating data question this mechanism and suggest that UCP2 and UCP3 may play other roles, including carrying free fatty acids from the matrix towards the intermembrane space, or contributing to the mitochondrial Ca2+ uniport. Accordingly, in this review we reflect on these actions of UCP2/UCP3 and discuss alternative explanations for the molecular mechanisms by which UCP2/UCP3 might contribute to aspects of cell function. Based on the potential role of UCP2/UCP3 in regulating mitochondrial Ca2+ uptake, we propose a scheme whereby these proteins integrate Ca2+-dependent signal transduction and energy metabolism in order to meet the energy demand of the cell for its continuous response, adaptation, and stimulation to environmental input. [Copyright &y& Elsevier]

Summary: Ca2+ entry in endothelial cells is a key signaling event as it prolongs the Ca2+ signal activated by a receptor agonist, and thus allows an adequate production of a variety of compounds. The possible routes that lead to Ca2+ entry in non-excitable cells include the receptor-activated Ca2+ entry (RACE), which requires the presence of an agonist to be activated, and the store-operated Ca2+ entry (SOCE) pathway, whose activation requires the depletion of the ER Ca2+ store. However, the relative importance of these two influx pathways during physiological stimulation is not known. In the present study we experimentally differentiated these two types of influxes and determined under which circumstances they are activated. We show that La3+ (at 10μM) is a discriminating compound that efficiently blocks SOCE but is almost without effect on histamine-induced Ca2+ entry (RACE). In line with this, histamine does not induce massive store depletion when performed in the presence of extracellular Ca2+. In addition, inhibition of mitochondrial respiration significantly reduces SOCE but modestly affects RACE. Thus, agonist-induced Ca2+ entry is insensitive to La3+, and only modestly affected by mitochondrial depolarization. These data shows that agonist relies almost exclusively on RACE for sustained Ca2+ signaling in endothelial cells. [Copyright &y& Elsevier]

Abstract: In this study the relationship between the efficiency of endoplasmic reticulum (ER) Ca2+ refilling and the extent of Ca2+ entry was investigated in endothelial cells. ER and mitochondrial Ca2+ concentration were measured using genetically encoded Ca2+ sensors, while the amount of entering Ca2+ was controlled by varying either the extracellular Ca2+ or the electrical driving force for Ca2+ by changing the plasma membrane potential. In the absence of an agonist, ER Ca2+ replenishment was fully accomplished even if the Ca2+ concentration applied was reduced from 2 to 0.5mM. A similar strong efficiency of ER Ca2+ refilling was obtained under condition of plasma membrane depolarization. However, in the presence of histamine, ER Ca2+ refilling depended on mitochondrial Ca2+ transport and was more susceptible to membrane depolarization. Store-operated Ca2+ entry (SOCE), was strongly reduced under low Ca2+ and depolarizing conditions but increased if ER Ca2+ uptake was blocked or if ER Ca2+ was released continuously by IP3. A correlation of the kinetics of ER Ca2+ refilling with cytosolic Ca2+ signals revealed that termination of SOCE is a rapid event that is not delayed compared to ER refilling. Our data indicate that ER refilling occurs in priority to, and independently from the cytosolic Ca2+ elevation upon Ca2+ entry and that this important process is widely achieved even under conditions of diminished Ca2+ entry. [Copyright &y& Elsevier]