Abstract
IntroductionMitochondrial 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 Ca2+ uptake has never been investigated in left ventricles.ObjectiveHere, 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.MethodMCU 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 Ca2+ 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.ResultsAt 1 μM Ca2+, 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 Ca2+, the Po (0.06 ± 0.004) and conductance (0.05 ± 0.01) were not significantly different from those with low Ca2+ 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.ConclusionTo conclude, we recorded for the first time MCU single-channel isolated from mouse ventricle and demonstrated that Ca2+ impacts the channel opening states while kaempferol increases Po and conductance in presence of high Ca2+.