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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]

A response by Michael Trenker and colleagues to a letter to the editor about their article discussing the role of uncoupling proteins (UCP2/3) in mitochondrial Ca2+ uniport in the November 2008 issue is presented.

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]

Mitochondrial Ca2+ uptake is crucial for the regulation of the rate of oxidative phosphorylation, the modulation of spatio-temporal cytosolic Ca2+ signals and apoptosis. Although the phenomenon of mitochondrial Ca2+ 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 Ca2+ uptake. Using overexpression, knockdown (small interfering RNA) and mutagenesis experiments, we demonstrate that UCP2 and UCP3 are elementary for mitochondrial Ca2+ sequestration in response to cell stimulation under physiological conditions — observations supported by isolated liver mitochondria of Ucp2−/− mice lacking ruthenium red-sensitive Ca2+ 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 Ca2+ uptake expands our knowledge of the physiological role for mitochondrial Ca2+ sequestration. [ABSTRACT FROM AUTHOR]