Voltage-Dependent Membrane Insertion and Translocation of α-Synuclein: Liposome Studies

Alpha-synuclein (αSyn), a 140 amino acid protein enriched in neuronal tissue, is implicated in the pathogenesis of Parkinson's Disease (PD), one of the most common neurodegenerative disorders. The membrane appears to be a potential target of αSyn. Studies with oligomeric or aggregated “protofibrillar” or amyloid forms of αSyn, in which β-structure is dominant, have been shown to permeabilize membranes in cell-free systems.1,2 A monomeric αSyn preparation, which could allow determination of a defined structure in membrane provided an alternative perspective.3 αSyn, disordered in solution, assumes a predominately α-helical conformation when bound to liposomes with an anionic surface and high degree of curvature.3,4 Using cysteine scanning mutagenesis to probe the membrane interaction of different regions of αSyn, the interaction of monomeric αSyn with membranes has been studied, thus far with mutants that span the polypeptide, from Val3 to Ala124. Two membrane systems, planar bilayer membranes and liposomes, have utilized an array of tagged cysteines to determine voltage-dependent topology of αSyn in membranes. Voltage-dependent membrane insertion into, and translocation across, liposome anionic phospholipid membranes, was studied through the response of the fluorescence probe 7-nitro-2,1-benzoxadiazole (NBD), covalently attached to individual Cys residues, to a potassium diffusion potential generated by addition of valinomycin to K+-loaded liposomes. Translocation was assessed through quenching of NBD fluorescence by entrapped bromide. A map of the voltage-dependent membrane topology of αSyn was obtained, in response to a potential of −118 mV, in which a segment of αSyn containing Val48Cys is translocated. In liposomes and planar bilayer membranes, translocation with the PD familial mutant E46K was greater than that of wild type. (Support: Michael J. Fox Foundation for Parkinson's Research). References: (1) Volles et al. 2001; (2) Quist et al. 2005; (3) Zakharov et al. 2007, (4) Davidson et al. 1998.