α-Synuclein kinetically regulates the nascent fusion pore dynamics

α-Synuclein (α-syn_FL) is central to the pathogenesis of Parkinson's disease (PD), in which its nonfunctional oligomers accumulate and result in abnormal neurotransmission. The normal physiological function of this intrinsically disordered protein is still unclear. Although several previous studies demonstrated α-syn_FL's role in various membrane fusion steps, they produced conflicting outcomes regarding vesicular secretion. Here, we assess α-syn_FL's role in directly regulating individual exocytotic release events. We studied the micromillisecond dynamics of single recombinant fusion pores, the crucial kinetic intermediate of membrane fusion that tightly regulates the vesicular secretion in different cell types. α-Syn_FL accessed v-SNARE within the trans-SNARE complex to form an inhibitory complex. This activity was dependent on negatively charged phospholipids and resulted in decreased open probability of individual pores. The number of trans-SNARE complexes influenced α-syn_FL's inhibitory action. Regulatory factors that arrest SNARE complexes in different assembly states differentially modulate α-syn_FL's ability to alter fusion pore dynamics. α-Syn_FL regulates pore properties in the presence of Munc13-1 and Munc18, which stimulate α-SNAP/NSF-resistant SNARE complex formation. In the presence of synaptotagmin1(syt1), α-syn_FL contributes with apo-syt1 to act as a membrane fusion clamp, whereas Ca²⁺•syt1 triggered α-syn_FL-resistant SNARE complex formation that rendered α-syn_FL inactive in modulating pore properties. This study reveals a key role of α-syn_FL in controlling vesicular secretion.