Mitochondrial VDAC as an Electromechanical Probe of Lipid-Dependent Membrane Binding of Alpha-Synuclein

Abstract

Alpha-synuclein (α-syn) is a small, intrinsically disordered cytoplasmic protein highly expressed in the central nervous system and implicated in Parkinson disease. α-Syn was found to specifically bind to native mitochondrial membranes in live cells and is known to be involved in neurodegenerative mitochondrial dysfunction and in neuroapoptosis. Thus, understanding the molecular mechanisms of α-syn interaction with model membranes mimicking the mitochondrial outer membrane (MOM) is of particular importance. Here we study the α-syn-membrane interaction on a single-molecule level using the voltage dependent anion channel (VDAC) to probe the kinetic and structural rearrangements of α-syn at the lipid membrane. We find that both the frequency and duration of the VDAC blockage by α-syn depend strongly on lipid composition. The highest interaction frequency of α-syn with VDAC is observed in anionic lipids; it drops by 100-fold in cationic lipids. The interaction frequency also increases with nonlamellar phosphatidylethanolamine content in neutral membranes. The duration of blockage is also affected by lipid composition, particularly the onset of α-syn translocation through the VDAC pore. These results support a model in which α-syn's N-terminus binding to the membrane is followed by C-terminal blockage of the VDAC pore. Comparison of electrophysiological data with macroscopic measurements of α-syn-lipid binding using Fluorescence Correlation Spectroscopy and Bilayer Overtone Analysis methods suggests that three domains of α-syn play different roles in membrane interactions and, consequently, with VDAC pore. Our results establish VDAC as a suitable electromechanical probe to report on monomeric α-syn membrane binding kinetics and dynamic conformational adaptations to the lipid charges and lipid packing stress. This system will facilitate further exploration of the well-recognized, but not well understood, regulatory roles of mitochondrial lipids in bioenergetics and cell metabolism.