Uncoupling protein 3 adjusts mitochondrial Ca(2+) uptake to high and low Ca(2+) signals.

Uncoupling proteins 2 and 3 (UCP2/3) are essential for mitochondrial Ca(2+) uptake but both proteins exhibit distinct activities in regard to the source and mode of Ca(2+) mobilization. In the present work, structural determinants of their contribution to mitochondrial Ca(2+) 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 Ca(2+), while they mimicked the wild-type proteins by potentiating mitochondrial sequestration of entering Ca(2+). 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 Ca(2+) sensitivities in regard to mitochondrial Ca(2+) sequestration. In intact cells, these mutants established different activities in mitochondrial uptake of either intracellularly released (UCP3(R171,E172)) or entering (UCP3(R167)) Ca(2+). Our data demonstrate that distinct sites in the IML2 of UCP3 effect mitochondrial uptake of high and low Ca(2+) signals.