Probing the Motion of the Intrinsically Disordered Neuronal Protein Alpha-Synuclein through the VDAC Pore using a Single-Molecule Approach

Abstract

Recent studies demonstrate the direct interaction of α-synuclein with mitochondrial membranes and point to the role of the voltage-dependent anion channel (VDAC) of the mitochondrial outer membrane (MOM) in facilitating translocation of α-synuclein through the MOM. Single-channel studies of the ionic current through VDAC reconstituted into a planar lipid membrane in the presence of α-synuclein reveal a complex interaction of α-synuclein with the VDAC pore that is characterized by the presence of two blocked states. The duration of both blocked states is biphasic with voltage: at low voltage, the blockage time increases with voltage, while at sufficiently large voltages, it decreases with voltage thus implying translocation. Detailed kinetic analysis of the transitions between the blocked states suggests that the second blocked state arises from simultaneous capture of two α-synuclein molecules. Selectivity studies at asymmetric electrolyte concentrations demonstrate a distinct splitting of the conductance of each blocked state into higher- and lower-selectivity sub-states. Transitions between these sub-states report on the stochastic dynamics of a single α-synuclein molecule within the pore. Finally, the shape of the blockage time distributions over the entire voltage range can be understood using a first-passage approach that reveals the potential experienced by α-synuclein molecules in the VDAC channel. These results highlight the suitability of the α-synuclein/VDAC system as a model for the investigation of the detailed molecular motion of disordered proteins in large β-barrel channels.