Dynamic plasticity of mitochondrial VDAC2 revealed by single-molecule electrophysiology

Abstract

The voltage-dependent anion channel (VDAC), the most ubiquitous mitochondrial outer membrane channel, exists as three mammalian isoforms. VDAC2 is the only isoform whose knockout is essentially lethal in mice embryos, is required for BAX/BAK-induced apoptosis, and thought to regulate mitochondrial calcium flux. Despite different physiological roles, VDAC2 is similar to VDAC1 in structure and forms voltage-gated channels of comparable conductance when reconstituted into planar lipid membranes. Previously, two populations of VDAC2 channels differing by selectivity and conductance were reported. Here, using single-channel electrophysiology of recombinant human VDAC2, we found that, unlike VDAC1 characterized by one unique "open" high-conducting state, VDAC2 fluctuates between at least two distinctive high-conducting states of conductance differing by ∼10% and selectivity differing by ∼15%. Both states manifest themselves as spontaneous transitions in the current through the single reconstituted channel observed in time. These results demonstrate that the two populations of VDAC2 described earlier are, in fact, substates of the same channel. Alpha-synuclein (aSyn), a neuronal protein related to the pathogenesis of Parkinson's disease, is a potent regulator of VDAC1. Using aSyn as a molecular probe of VDAC2 pore, we find that VDAC2 substates differ by their interaction kinetics with aSyn, exhibiting up to 10x different on-rates within the same channel. Furthermore, ∼20% of the channels showed no interaction with aSyn from one side of the channel. The appearance of distinct substates within the same channel reveals the dynamic plasticity of VDAC2. The role of cysteines in the occurrence of VDAC2 substates is explored using cysteine-less VDAC2 mutant. Our results shed light on the distinctive physiological role of VDAC2 and suggest that its dynamic plasticity could be a key feature regulating its interaction with cytosolic and mitochondrial proteins and calcium, thus explaining the versatility of this multifaced channel.