The Role of Membrane Affinity and Binding Modes in Alpha-Synuclein Regulation of Vesicle Release and Trafficking

Alpha-synuclein is a presynaptic protein linked to Parkinson’s disease with a poorly characterized physiological role in regulating the synaptic vesicle cycle. Using RBL-2H3 cells as a model system, we earlier reported that wild-type alpha-synuclein can act as both an inhibitor and a potentiator of stimulated exocytosis in a concentration-dependent manner. The inhibitory function is constitutive and depends on membrane binding by the helix-2 region of the lipid-binding domain, while potentiation becomes apparent only at high concentrations. Using structural and functional characterization of conformationally selective mutants via a combination of spectroscopic and cellular assays, we show here that binding affinity for isolated vesicles similar in size to synaptic vesicles is a primary determinant of alpha-synuclein-mediated potentiation of vesicle release. Inhibition of release is sensitive to changes in the region linking the helix-1 and helix-2 regions of the N-terminal lipid-binding domain and may require some degree of coupling between these regions. Potentiation of release likely occurs as a result of alpha-synuclein interactions with undocked vesicles isolated away from the active zone in internal pools. Consistent with this, we observe that alpha-synuclein can disperse vesicles from in vitro clusters organized by condensates of the presynaptic protein synapsin-1.