UCP2 modulates single-channel properties of a MCU-dependent Ca2+ inward current in mitochondria

The mitochondrial Ca2+ uniporter is a highly Ca2+-selective protein complex that consists of the pore-forming mitochondrial Ca2+ uniporter protein (MCU), the scaffolding essential MCU regulator (EMRE), and mitochondrial calcium uptake 1 and 2 (MICU1/2), which negatively regulate mitochondrial Ca2+ uptake. We have previously reported that uncoupling proteins 2 and 3 (UCP2/3) are also engaged in the activity of mitochondrial Ca2+ uptake under certain conditions, while the mechanism by which UCP2/3 facilitates mitochondrial Ca2+ uniport remains elusive. This work was designed to investigate the impact of UCP2 on the three distinct mitochondrial Ca2+ currents found in mitoplasts isolated from HeLa cells, the intermediate- (i-), burst- (b-) and extra-large (xl-) mitochondrial/mitoplast Ca2+ currents (MCC). Using the patch clamp technique on mitoplasts from cells with reduced MCU and EMRE unveiled a very high affinity of MCU for xl-MCC that succeeds that for i-MCC, indicating the coexistence of at least two MCU/EMRE-dependent Ca2+ currents. The manipulation of the expression level of UCP2 by either siRNA-mediated knockdown or overexpression changed exclusively the open probability (NPo) of xl-MCC by approx. 38 % decrease or nearly a 3-fold increase, respectively. These findings confirm a regulatory role of UCP2 in mitochondrial Ca2+ uptake and identify UCP2 as a selective modulator of just one distinct MCU/EMRE-dependent mitochondrial Ca2+ inward current.