Alpha-Synuclein Binding and Translocation by Alpha-Hemolysin Channel

Abstract

Gauging the pathways of a natively unfolded Parkinson disease-related protein, α-synuclein (α-syn), between and within cells, is important for understanding pathogenesis. Here, we use a robust β-barrel ion channel, α-hemolysin (α-HL), as a model system to investigate the possibility of channel-assisted membrane transport of α-syn. The interaction of α-syn with the channel was observed as a transient, ∼95 % block of the channel current when (i) α-syn was applied from the membrane side where the shorter (stem) part of the α-HL heptamer is exposed and (ii) the applied potential is lower on the side of α-syn application. While the on-rate of α-syn binding to the channel exponentially increases with the applied field, the off-rate displays a crossover behavior. Statistical analysis of the blockage events indicates that at voltages >80 mV a significant portion of α-HL-bound α-syn undergoes subsequent translocation. The observed on-rate varies by more than 100 times, depending on bilayer lipid composition. To delineate the role of specific domains, N-terminal membrane-interacting and acidic C-terminal tail, we produced and examined 1) a C-terminal truncation variant lacking the last 25 amino acids (α-syn115) and 2) a synthetic peptide composed of the last 23 residues (C23). In the case of α-syn115, the absence of the highly negatively charged C-terminal tail leads to a significant decrease in the rates of both binding and translocation events. For C23, no time-resolvable blockades of the channel current could be detected, suggesting that membrane binding of α-syn plays an important role in the translocation process. Surprisingly, we found that α-syn can also effectively block mitochondrial channel VDAC, an endogenous β-barrel protein, suggesting that “large” membrane pores may serve as gateways for α-synuclein transport in vivo.