Single-channel biophysical properties of the left ventricular mitochondrial calcium uniporter complex

Abstract

Mitochondrial calcium uniporter (MCU) forms a macromolecular complex composed by MCU, MICU1, MICU2, EMRE and MCUb, which confers its tissue specificity. However, the native single-channel properties and its regulation by kaempferol, a polyphenol known to enhance mitochondrial Ca 2+ uptake has never been investigated in left ventricles. Here, we aimed to characterize the single-channel properties of mouse left ventricular MCU complex incorporated in planar lipid bilayer (PLB) and its regulation by kaempferol. MCU was purified from mouse left ventricle isolated mitochondria, treated with proteinase K and digitonin to disrupt the outer mitochondrial membrane. Submitochondrial particles were subjected to BN-Page to verify the macromolecular complex integrity. MCU was then incorporated in PLB for electrophysiological recording. Voltage steps were applied from −60 mV to +60 mV and Ca 2+ concentration was increased from 1 μM to 1 mM at −40 mV. Currents were recorded in presence of kaempferol (10 μM) and ruthenium red was used to inhibit the single-channel activity. At 1 μM Ca 2+ , MCU displayed 4 opening states with a single-channel open probability (Po) of 0.10 ± 0.05 and a single-channel conductance of 0.07 ± 0.01 nS. In presence of kaempferol, MCU Po and conductance were slightly increased (0.18 ± 0.08 and 0.10 ± 0.02 nS, respectively) and the number of opening states remained unchanged. At 100 μM Ca 2+ , the Po (0.06 ± 0.004) and conductance (0.05 ± 0.01) were not significantly different from those with low Ca 2+ condition whereas the single-channel current displayed only the first two opening states. Kaempferol increased both the conductance (0.11 ± 0.02 nS) and Po (0.27 ± 0.10) and maintained the MCU in 4 opening states. To conclude, we recorded for the first time MCU single-channel isolated from mouse ventricle and demonstrated that Ca 2+ impacts the channel opening states while kaempferol increases Po and conductance in presence of high Ca 2+ .